UNIVERSITY OF CALGARY

Optoelectronic Systems and Applications for In Vivo Fiber Photometry

by

Kathryn Ronayne

A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN BIOMEDICAL ENGINEERING

CALGARY, ALBERTA
SEPTEMBER, 2016

Kathryn Ronayne 2016

Abstract
The emergence of fluorescent reporters has enabled neuroscientists to image neurons in action in
live mice. Conventional imaging setups, however, cannot interrogate structures below the surface
of the brain, and the animal must be restrained.
This work advances fiber photometry, a simple method to monitor the average activity of a
population of cells, to enable experiments in freely-moving mice and in deep brain structures.
By targeting a small-diameter probe to the structure of interest, single fiber optoelectronic systems
may be designed that monitor changes in calcium-mediated fluorescence intensity. The systems are
optimized for an application in a CRH-Cre transgenic mouse, with the calcium reporter GCaMP6s,
for minimal damage to tissue.
This thesis covers: (a) optoelectronic system design, characterization, and refinement (b) application and results in awake and freely-moving mice, (c) investigation into potential artifacts and
(d) method and results of an improved targeting protocol.

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Acknowledgements
I am extraordinarily grateful to have had the experience of being a student in Dr. Kartikeya Muraris lab. Id like to thank my supervisor for helping me discover many passions, as well as for
his patience, for making the time for thoughtful discussions, and for passing on his wealth of
knowledge and experience.
I would also like to thank Dr. Jaideep Bains for the opportunity to collaborate with his lab on
this project. From their lab: Tamas Fuzesi, David Rosenegger, Dina Baimoukhametova, and Mio
Tsutsui have been extremely helpful with in vivo and slice experiments. This experience gave me
perspective on how research happens outside of the engineering world.
From my lab and those across the hall: Linhui, Isaac, Daniel, Nikhil, Yuting, Ulian - thank you
for making the last two years fun and for pushing me to do my best.
Finally, to my husband, my family and friends back home, for your constant support and encouragement, and for making the effort to stay in touch across timezones and busy lives.

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Table of Contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
List of Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 Specific Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Organization of the Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1 Neural Recording in Awake and Freely-Moving Animals . . . . . . . . . . . . . .
2.2 Survey of Existing Fiber Photometry Systems . . . . . . . . . . . . . . . . . . . .
2.3 Problem Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
FIBER PHOTOMETRY SYSTEM DESIGN, CHARACTERIZATION, AND VALIDATION IN AN ANIMAL MODEL . . . . . . . . . . . . . . . . . . . . . . . .
3.1 Design Constraints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 Optical Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1 Light Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2 Optical Tether . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3 In situ Characterization of GCaMP6 . . . . . . . . . . . . . . . . . . . . .
3.2.4 Photodetector Selection . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.5 Optomechanics and Alignment . . . . . . . . . . . . . . . . . . . . . . . .
3.3 Electronic Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1 Signal Conditioning Circuitry . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2 Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4 Implementation and Characterization . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 In vivo Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.1 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
FIBER PHOTOMETRY SYSTEM REFINEMENT AND EXPERIMENTAL APPLICATION IN AN ANIMAL MODEL . . . . . . . . . . . . . . . . . . . . . . .
4.1 Lock-In Amplifier Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1 Block Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2 Modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3 Demodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.4 Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 In vivo Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1 Fluorescence Intensity Changes due to Calcium Dynamics in Response to
Conditioned Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2 Measurement Immunity to Neurovascular and Hemodynamic Coupling . .
4.3 Discussion and System Comparisons . . . . . . . . . . . . . . . . . . . . . . . . .
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5
OPTICAL GUIDANCE FOR IMPROVED STEREOTACTIC TARGETING
5.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2 Optical System Design . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Application and Evaluation Procedure . . . . . . . . . . . . . . . . . . . .
5.4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
CONCLUSION AND FUTURE DIRECTIONS . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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List of Tables
3.1

3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.1
4.2
4.3
4.4

4.5
4.6
4.7

4.8

4.9

Constraints that guide the design of this fiber photometry system and acknowledge
the compromise between the systems ability to detect small signals (defined by its
signal-to-noise ratio, SNR) and tissue damage caused by the optoelectronic system
(either through exposure to high light irradiances or large-fiber optic probes). . . .
Spectral Maxima for GCaMP6s . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Estimated or predicted contributions of collected fluorescence from various sources
at several levels of excitation light . . . . . . . . . . . . . . . . . . . . . . . . . .
Components selected to realize 2nd order stages in this design. . . . . . . . . . . .
Protection from ambient room lighting provided by the enclosure for the optical
system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Resulting CNR, where A is the peak to peak amplitude of the modulated sine wave.
Detecting changes in fluorescence intensity from CRH cells in freely-moving mice
in response to stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
in vivo Photobleaching rates for control animals. . . . . . . . . . . . . . . . . . . .
Summary of fiber photometry system optical and electrical characteristics. . . . . .
Design criteria for Lock-in Amplifier . . . . . . . . . . . . . . . . . . . . . . . . .
The OPA171 is a suitable choice for use in the LED driver circuit. . . . . . . . . .
Components selected to realize 2nd order stages in this design. . . . . . . . . . . .
Comparison of systems sensitivity to ambient light, with measurements due to fluorescence (FAF ) and fluorescence plus light from an incoherent source (FAF + L).
/ / SNR Signal mean/ standard deviation/ signal-to-noise ratio (SNR = ). A
515/30 nm filter was used to set the emission band for the PMT in all cases. The
LIA design exhibits improved signal-to-noise ratio over the system designed in
Chapter 3, and is able to detect signals in the presence of incoherent sources
(SNRLIA 1) where the DC system cannot (SNRDC 1). *SNR was calculated
using the mean of the measurement with fluorescence FAF , and the standard deviation of the measurement with fluorescence and light, FAF+L . . . . . . . . . . . . .
Detecting changes in fluorescence intensity from CRH cells in freely-moving mice
in response to stress with the LIA system. G = GCaMP6s animal, E = EYFP animal.
Experimentally-determined mean sensitivity to changes in GCaMP6s fluorescence,
for given emission bands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimentally-determined sensitivity to physiological changes in optical tissue
properties with a 505/10 nm filter, normalized to sensitivities determined using a
515/30 nm filter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experimentally-determined sensitivity to physiological changes in optical tissue
properties with a 535/30 nm filter, normalized to sensitivities determined using a
515/30 nm filter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparison of performance specifications between the LIA and DC systems. . . .

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5.1

5.2

5.3
6.1

Approximate results from the theoretical model of detection of fluorescence in

brain tissue from structures separated from the fiber probe, and the resulting baselinereferred changes in fluorescence, F/Fo , when background sources of fluorescence (from the fiber and co-expressed markers) are taken into account. . . . . . . 93
Increases in fluorescence intensity with depth of the optical fiber probe in six CRHCre animals with GCaMP6s. If an increase was found at presumed coodinatres, the
surgery was deemed a success. If no increase was found, alternate coordinates were
attempted until a structure was found, up to a maximum of five attempts. Increases
in fluorescence intensity of 10% at 515 nm were considered successful. The endeavour to find the PVN in animal #5 was halted early (only three attempts were
made) due to time constraints. In animal #3, alternate coordinates were attempted
beyond the 10% threshold defined as surgical success, in an effort to find a higher
region of fluorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Increases in fluorescence intensity with depth of the optical fiber probe in six CRHCre animals with GCaMP6s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Comparison of the contributions of the optoelectronic systems designed in this
research to those proposed by others. . . . . . . . . . . . . . . . . . . . . . . . . . 106

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List of Figures and Illustrations

2.1

Schematic of a typical Single Fiber Photometry System . . . . . . . . . . . . . . . 11

3.1
3.2

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The optics employed in a typical Single Fiber Photometry System . . . . . . . .

The numerical aperture of an optical fiber describes the range of angles for impinging rays that are accepted. Ray 2, shown in red, impinges the core at an angle
greater than the numerical aperture, and therefore leaks through the outer layers of
the fiber. Ray 1, by contrast, is accepted as it undergoes total internal reflection at
the core-cladding interface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3 The coupling efficiency of a pigtailed LED depends on the emitter size, fiber diameter, and acceptance angle of the fiber. . . . . . . . . . . . . . . . . . . . . .
3.4 Normalized excitation spectra for fluorescent calcium reporters in the GCaMP
family [1]. The proposed excitation and emission filters are marked in blue and
green, respectively. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5 Schematic for LED driver circuit . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6 The optical tether couples light to and from fiber optic implant that is in permanent
contact with brain tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7 Two optical fibers, of the same length, are exposed to the same light level, and
produce the fluorescence spectra depicted here. 200/240/900-0.48 (from Doric
Lenses) is employed in the system due to its extremely low autofluorescence in the
emission spectrum for GCaMP6s. Both curves are normalized to the peak detected
fluorescence, in counts, from FT-200EMT (from Thorlabs). . . . . . . . . . . . .
3.8 The spectral dynamics of tissue with GCaMP6s expression were characterized in
fresh tissue slices, with simultaneous epifluorescence imaging and fiber photometry. a) In situ characterization was performed with the aid of an epifluorescence
microscope b) The arrangement enabled optical access to the sample for both an
optical fiber and a camera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9 The optical system employed for spectral characterization of GCaMP6s . . . . .
3.10 a) A representative spectrum collected from the interaction of laser light with the
brain slice, normalized to the maximum intensity from backreflection of the laser
at 48 nm, and b) at wavelengths near GCaMP6s emission. . . . . . . . . . . . .
3.11 Results of simultaneous microscopy (left) and fiber photometry (right) on fresh,
250 m slices containing CRH-Cre cells expressing the GCaMP6 fluorescent calcium reporter. Additions of Potassium Chloride (KCl), activated cells. An addition
of Artificial Cerebrospinal Fluid (aCSF), used as a control, had no effect. Imaging
and spectroscopy data were synchronized, and a selection of common time points
are denoted. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.12 Spectral characterization informs decisions to optimize detector sensitivity. a)
Spectra acquired when cells were inactive (dark green) and active (bright green)
b) Spectral sensitivity to GCaMP6s (averaged, n = 3) c) Of four possible filters
considered, the 535/30 nm filter (for which the transmission profile is denoted by
(center wavelength)/(bandwidth)) will enable greatest sensitivity to modulations in
fluorescence due to GCaMP6. . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.13 Structure and operation of a PMT. A PMT consists of a light-sensitive photocathode held at a large negative potential, and a configuration of amplifying dynodes
held at successively higher potentials leading to the devices anode held at ground
potential. The electric fields in each PMT stage result in a cascade of secondary
emission processes that give the photodetector a large internal gain and high sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.14 The optomechanics involved in launching excitation light from the light source into
optical tether, and focusing fluorescence carried by the tether to the photodetector
(PMT). M: alignment mirror, DBS: dichroic beam splitter, EX: excitation filter,
EM: emission filter, SH: shutter. L1 = L2: collimating or focusing lenses, L3:
focusing lens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.15 Schematic drawing of the coupling optics in the collection path (All dimensions are
in millimeters). The distance between the focusing lens and the shutter was varied
in a Zemax simulation to achieve a 3.6 mm diameter spot at the photocathode of
the PMT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.16 Block-diagram of 5th -order low-pass Butterworth filter. The first stage was realized
with a band-limited transimpedance amplifier (TIA). . . . . . . . . . . . . . . .
3.17 TIA schematic used in this design . . . . . . . . . . . . . . . . . . . . . . . . .
3.18 a) Concept of a background-zeroing current source to increase SNR on the fly
with increased delivered excitation light and b) implementation with an field-effect
transistor (FET) operating in subthreshold mode when Vgs <Vth . . . . . . . . . .
3.19 Sallen-Key circuit configured as a low-pass filter . . . . . . . . . . . . . . . . .
3.20 Simulated frequency response of signal conditioning circuitry design, showing
magnitude and phase characteristic. . . . . . . . . . . . . . . . . . . . . . . . .
3.21 a) Stability of delivery path and light source.b) Range of output power enabled by
the light source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.22 a) Measured magnitude response of TIA, as measured at output of amplifier. b)
Measured noise spectrum of TIA . . . . . . . . . . . . . . . . . . . . . . . . . .
3.23 Investigation of the systems ability to detect small changes. a) Use of the bleed
transistor allows for optical measurements to be made with increased CNR by allowing for greater optical power to be used. However, large modulations near DC
are added, resulting in a slow drift over time. b) Frequency domain representation
of the traces in a) shows the low-frequency noise added by the transistor. . . . . .
3.24 Investigating the response of CRH cells of the hypothalamic PVN to conditioned
stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.25 a) Traces of detected fluorescence intensity from CRH-Cre mice expressing either GCaMP6s (green) or EYFP (orange). Red lines denote the transfer to/from
the shockcage. b) The response to conditioned stress was stable as measured by
repeating the experiment on three consecutive days. . . . . . . . . . . . . . . . .
3.26 Recordings under ambient lighting resulted in a 90.3% increase in noise power
compared to the recording in darkness. Sensitivity is reduced further from the
induced offset. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.27 Representative fluorescence trace from an EYFP mouse and line of best fit, showing little photobleaching. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4.1

4.2
4.3
4.4
4.5
4.6

4.7

4.8

4.9
4.10
4.11

4.12

4.13

4.14
4.15

Lock-in amplifiers use coherent detection to enable low-level optical measurements. The numbers at each of the points in the signal path refer to example
frequency-domain representations of signals in Figure 4.2 . . . . . . . . . . . . . .
Frequency domain representations of the signal at points a) 2, b) 3, and c) 4 in the
lock-in amplifier circuit depicted in Figure 4.1 . . . . . . . . . . . . . . . . . . . .
LED driver circuit for the modulated light source in LIA design. To maximize
vmod , the amplitude of VMOD , this design operates off of e = 15 V. . . . . . . . .
Spectral analysis of the modulation scheme at 571 Hz . . . . . . . . . . . . . . . .
The demodulation circuit consists of a wideband transimpedance amplifier, a multiplication stage, and a low-pass filter. . . . . . . . . . . . . . . . . . . . . . . . .
The transimpedance amplifier design for this wideband application must take into
account finite input resistance, and input capacitances at the opamp terminals. Zi
and Z f denote the parallel combinations that determine the frequency-dependent
input and feedback impedances. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bode plot showing the the open-loop gain magnitude curve of the op amp, Ao (s);
the reciprocal of feedback fraction, 1/ (s); and the gain due to the feedback network Z f (s). Z f (s) achieves circuit stability by cancelling out the zero due to
1/ (s). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Simulated Magnitude and Phase response of TIA in demodulation subcircuit. f3dB
is predicted to be 14.93 kHz with Ci = 20 pF, C f = .15 pF, RF = 100 M and Ri =
1 T, and GBW = 4 MHz (opamp used for simulation was LF411CN.) . . . . . .
Minimal component Sallen-Key circuit configured as a low-pass filter with gain = 1
a) Magnitude and Phase response of TIA in demodulation subcircuit. f3dB was
measured at 5.5 kHz. b) LIAs input-referred noise spectrum. . . . . . . . . . . . .
a) The systems response to b) a step input, characterizes the systems bandwidth.
The output of the LIA changes from 10% to 90% of its final value in 340 ms. A
step input was emulated by rapidly opening the shutter of the light source for the
optical system, and quantified by measuring the resulting light launched into an
optical fiber with a wide bandwidth photodiode (PDA36A, Thorlabs) . . . . . . . .
a) A thick application of dental cement mixed with red food coloring reduces the
number of photons in the emission band that can be collected by the optical fiber
implant during in vivo study. b) A 515/30 nm emission filter blocks a large peak
in fluorescent lighting near 543 nm that would be relayed to the detector with the
535/30 nm filter. The addition of food coloring to dental cement (red) blocks 70%
of the photons that from ambient lighting that would otherwise penetrate the skull
and brain tissue, compared to the uncolored dye (pink). . . . . . . . . . . . . . . .
Representative traces from a GCaMP6s (green) and EYFP (orange) CRH-Cre mouse.
Reintroduction of the animal to the conditioned stress at t = 5 minutes resulted in
changes in detected fluorescence intensity (GCaMP6s: F/F = 31 % 39 %, n =
4; EYFP: F/F = 2 %, n = 1). Red lines denote transfer to and from the shock
cage that provides the conditioned stimulus. . . . . . . . . . . . . . . . . . . . . .
Performance comparison of both optoelectronic designs in vivo. . . . . . . . . . .
a) Ratio of absorption coefficient of Oxyhemoglobin (HbO2 ) to Hemoglobin (Hb).
b) Absorption spectrum of blood, assuming 50% oxygen saturation and 1M concentration [2] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.16 Detected fluorescence intensity changes in response to re-exposure to conditioned

stress in two CRH-Cre mice, with three emission filters. a) 535/30 nm filter and
515/30 nm filter, and in another animal b) 505/10 nm filter and 515/30 nm filter. . . 81
4.17 a) Changes in reflectance as measured at 515 nm, over time in response to exposure
to innate stress. b) Change in spectral reflectance between baseline (0 - 100 s) and
exposure to stress (120 - 240 s). . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.18 Noise spectra from LIA and DC systems. RMS noise computer for 0.1-10 Hz. . . . 86
5.1

5.2
5.3

5.4
5.5

Monitoring fluorescence in tissue with separation between the probe tip and the target structure. The target structure (a population of cells expressing a fluorescence
reporter) is modelled as a plane whose brightness is modulated homogenously. r f :
radius of the optical fiber; Ix,o : excitation intensity at the plane of the probe tip;
Im,o : intensity of emission photons at the plane of the probe tip; z distance separating the probe tip from the fluorescent plane; n: refractive index of tissue, S:
scattering coefficient per unit thickness; NA: numerical aperture of optical fiber;
Ix,z : excitation intensity at the fluorescent plane; Im,z : emission intensity at the
fluorescent plane; e f c : fluorescence conversion efficiency. . . . . . . . . . . . . .
Theoretical results for detection of fluorescence from distant structures in brain
tissue in response to delivery of Pex = 1 W. . . . . . . . . . . . . . . . . . . . .
Proposed optical system for surgical guidance. EX: 470/40 excitation filter; ND:
Neutral density filter; DBS: Dichroic beam splitter; N: Notch filter; L1/L2: Coupling/collimation lenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spectra due to collected fluorescence at 500 m from the fluorescent structure
(black) and at the final location (green). . . . . . . . . . . . . . . . . . . . . . .
Identification of CRH neurons expressing GCaMP6s in the hypothalamic paraventricular nucleus of the hypothalamus of a mouse, and location of implanted fiber
optical probe. The PVN is the cluster of fluorescent cells outlined in the micrograph for # 1. Proper expression and probe placement were confirmed in # 1, 4,
and 6. Probes in # 2,3, and 5 were found to be far from the structure in the sagittal
plane: (#2: 200 m caudal, #3: 200 m anterior, #5: 240 m anterior). . . . . . .

xi

. 90
. 93

. 94
. 97

. 99

List of Symbols, Abbreviations and Nomenclature

Abbreviations

Definition

UofC

University of Calgary

TIA

Transimpedance Amplifier

LIA

Lock-in Amplifier

GECI

Genetically-Encoded Calcium Indicators

CRH

Corticotropin-Releasing Hormone

PVN

Paraventricular Nucleus of the Hypothalamus

FRET

Forster-Resonance Energy Transfer

GFP

Green Fluorescent Protein

EYFP

Enhanced Yellow Fluorescent Protein

2PE

Two-Photon Excitation

PMT

Photomultiplier Tube

LED

Light-Emitting Diode

NA

Numerical Aperture

FET

Field Effect Transistor

RMS

Root Mean Square

GBW

Gain-Bandwidth Product

QE

Quantum Efficiency

WT

Wild Type

xii

Symbols

Definition

SNR

Signal-to-noise ratio

F/F

Baseline-referred changes in fluorescence intensity

Pex

Delivered excitation power for fluorescence

Ftotal ( )

Total collected fluorescence

FGCaMP6 ( )

Reporter-mediated fluorescence due to calcium dynamics

Ftissue ( )

Fluorescence due to tissue (background)

Ff iber ( )

Fluorescence due to fiber

R( )

Spectral sensitivity of a system to changes in fluorescence

Rf

TIA feedback resistor

H( j)

Transfer function

Complex frequency

f3dB

Filter cut-off frequency

Filter pole quality factor

Cf

TIA feedback capacitance

Ao (s)

Opamp open-loop gain function

(s)

Voltage feedback fraction at inverting input

vT IA

Output of transimpedance amplifier in LIA

vMULT

Output of mutliplier in LIA

vMOD

Input to multiplier in LIA

vLIA

Output of LIA

xiii

Chapter 1
INTRODUCTION
Due to its involvement in membrane and synaptic dynamics, the intracellular concentration of
the free calcium ion, Ca2+ , is an indicator of neuronal activity. The emergence of fluorescent
calcium reporter proteins has made recording calcium dynamics with techniques such as imaging
a powerful tool in neuroscience. Whereas most imagers cannot provide optical access below the
surface of the brain due to the scattering of light by tissue, optical fibers provide a minimally
invasive way to monitor cells at arbitrary depths. As such, employing optical fibers is one of the
few strategies that supports measurement from awake and freely-moving animals. When used
in conjunction with sophisticated genetic targeting strategies, a single multimode fiber provides a
cost-effective solution to sense the average activity of a specific population of cells. This technique
is referred to as fiber photometry in neuroscience, and the signal it yields is a powerful behavioural
correlate.
This thesis presents prototype benchtop optoelectronic systems for monitoring qualitative dynamics of calcium concentration in cells as reported by a fluorescent, genetically encoded calcium
indicator. This thesis covers the design, characterization, and refinement of a fiber photometry
system, including in situ spectral characterization of the fluorescent calcium reporter GCaMP6s,
and results from the intact brains of awake and freely-moving animals subjected to standard behavioural protocols. An investigation into the efficacy of applying optical systems as an aide to
the standard stereotactic surgeries is also presented. This chapter describes the specific aims of the
research and the organization of the thesis.

1.1

Specific Aims

Neuroscientists are interested in the behavioural correlates of specific cellular activity, as recorded
from the intact brains of awake and freely moving animals. While electrophysiology is a commonly
applied technique in this paradigm, it records indiscriminately from all cells in the vicinity of the
electrode tip. The emergence of genetically-encoded fluorescent proteins, by contrast, has provided
a family of methods that yields insight into the activity of specific cell types. Due to their superior
brightness and dynamic range, proteins that modulate their fluorescence with intracellular calcium
concentration (as compared to those that modulated their fluorescence with membrane potential,
or with the presence of neurotransmitters) are currently dominating the study of cellular dynamics
in behaving animals. These Genetically-Encoded Calcium Indicators (GECIs) have been critical
to a number of discoveries about the brains structure and function of its circuits [35].
Several photometry systems have been built by others, where feature addition has been a major
focus, wherein multichannel recording [6], artifact-correction [5], [7], or simultaneous optogenetic photostimulation [8] are examples. However, improving the sensitivity of such systems has
received little attention. A second challenge remains in ensuring the optical probe is positioned correctly near the structure in question. To meet this challenge, optical systems have been developed
and employed as a means to guide the surgery along gradients of fluorescence intensity [3], [9]
The goal of this research is to improve optical monitoring of brain structures expressing fluorescent reporters with multimode fibers, or fiber photometry, by building novel optoelectronic
systems that will extend our ability to detect fluorescent signals. This will be done by building more sensitive optical systems, and developing a protocol that results in more accurate probe
placement. Improving such optical monitoring should be associated with a simplification of subsequent data analysis from long-term and repeated recordings of neuronal populations of awake and
freely-moving animals.
The systems developed in this research are tested using GCaMP6s in a CRH-Cre transgenic
mouse model. GCaMP6s is a fluorescent protein, to which the binding of calcium ions causes

an increase in brightness. Its expression is targeted to corticotropin-releasing hormone (CRH)

cells of the brain via the Cre-recombinase enzyme. In the mouse, CRH cells are clustered in the
paraventricular nucleus of the hypothalamus (PVN), neighbouring cells of distinct lineages. The
PVN is involved in the stress response, but the role of CRH cells is unknown. By enabling highlyspecific interrogation of the activity of neuronal subtypes, fiber photometry is a suitable method to
employ for investigation.
Damage to brain tissue is minimized by employing a single optical fiber of diameter less than
300 m (the upper limit considered safe for mice [10]) to both deliver excitation light to, and
collect emitted photons from, the desired tissue. The damage of light to tissue (phototoxicity)
is mitigated by employing a highly sensitive photomultiplier tube as a detector such that lower
excitation powers can be used.
Spectral characterization of the dynamics of GCaMP6s, the calcium reporter, in tissue slices
informed the selection of optical filters to optimize sensitivity to changes in fluorescence intensity. The system is redesigned for modularity and signal-to-noise ratio by refining the selection of
optical filters, and redesigning the electronics for a modulation/demodulation configuration.
The Specific Aims (SA) of this research are:
SA1 Design, build, and characterize a highly sensitive single-fiber optoelectronic system.
SA2 Characterize the spectral properties of GCaMP6s and surrounding tissues when expressed
in a CRH-Cre mouse.
SA3 Test the performance of the optoelectronic system in vivo.
SA4 Identify artifacts associated with optical recording from awake and freely moving mice.
SA5 Determine the viability of applying optical systems as stereotactic guidance tools.

1.2

Organization of the Thesis

Chapter 1 gives a brief justification for the research, provides an overview of the approach taken,
and enumerates the specific aims of the research. Chapter 2 provides a review of the literature:
fiber photometry is placed in context alongside other methods of neural recording, and a survey of
reported fiber photometry systems is given. The limitations of existing systems as well as gaps in
knowledge is highlighted, providing motivation for the specific aims of the research. Chapters 3
and 4 each describe the design, characterization, and experimental application of fiber photometry
systems. Chapter 3 presents the initial design, characterization, and experimental application in
vivo. The performance of the optoelectronic system in this context is discussed, and justification
for the systems refinement is given. Chapter 4 then presents how the system was redesigned for
simplicity, to mitigate the effect of various artifacts, and to further improve the signal-to-noise ratio. The experimental results obtained through application of the refined system in vivo are also
presented and discussed. Chapter 5 addresses the challenge of targeting small structures located
deep in the brain for optical interrogation with fibers: it describes the investigation into whether
an optical system can improve the rate of successful surgeries compared to using stereotactic coordinates alone. Chapter 5 describes the methodology (including the design of a suitable optical
system) employed, supplies the evaluation procedure used to rate the methods effectiveness, and
finally provides the results of surgeries conducted with the method. Chapter 6 concludes the thesis with a summary, considers the contributions of the research, and what challenges remain in
applying fiber photometry to behavioural studies on awake and freely-moving animals.

Chapter 2
BACKGROUND
2.1

Neural Recording in Awake and Freely-Moving Animals

The behavioural correlates of cellular activity is of interest in neuroscience. Such investigations

are undertaken to clarify the role played by certain cells in the realization and management of,
or response to, a certain behaviour. Because electricity underlies the function of the nervous system, electrophysiology enables a direct recording of the fundamental cells of which the system is
composed [11].
In awake and freely-moving animals, electrophysiology is usually accomplished by introducing an electrode in the animals intact brain. Depending on the size of the electrode, the detected
signal will have been mediated by a single cell (Single-Unit recording with probe size on the order
of 10 m), or the aggregate activity of a brain region (multi-unit recording with probe diameter
on the order of 100 m). Recordings from large arrays of such electrodes enables the mapping of
activity between brain regions [12]. As of this writing, advances in electrophysiology technology
have enabled simultaneous recording from hundreds of channels [12] and thousands of cells or
neuronal features. Significant progress has been made towards providing wireless and real-time
data acquisition for multi-electrode recordings [13]. Such hardware solutions support investigation in close-to-natural settings, which enables the most organic expression of behaviour. While
electrophysiology is a natural choice to interrogate the brain in vivo, the method results in an indiscriminate recording from all cells in the vicinity of electrode tip. This precludes the study of
subpopulations of neurons within a brain region, whose neighbors likely have distinct cell lineages
and functions.
The development of optical reporters, together with their expression in transgenic animals
through genetic encoding technology, has enabled interrogation of brain behaviour with this level
5

of precision [14]. These reporters transduce the physiological dynamics of cells, such as membrane potential [15], the intracellular concentration of ions such as free Calcium [16], as well as
neurotransmitters such as acetylcholine [17], serotonin [18] and, most notably, glutamate [19]. A
wide range of such proteins have been engineered in recent years, based on principles such as
bioluminescence [20] and Forster-Resonance Energy Transfer (FRET) [21]. These advances have
been reviewed by others [22], and reporters based on fluorescence have come to dominate optical
recording due to their ability to provide contrast on a background, of great use to imaging. Another
advance of fluorescence reporters is that they can be detected coherently. Fluorescent indicators
absorb a photon and then emit a photon at a longer wavelength. However, the spectrum of the
photons emitted by the molecule is modulated in some way by the changing environment inside
the cell, such that relative changes can be measured.
The free calcium ion, Ca2+ , is involved in several intracellular processes. In the cells of brain
tissue, intracellular calcium concentration is an indicator of neuronal activity. This is largely due to
the behaviour of voltage-gated, calcium-permeable ion channels, whose conformational changes in
response to action potentials lead to an influx in free calcium into the excitable cell. In presynaptic
cells, action potential events are often accompanied by the release of calcium from organelles
within the cell, such as the endoplasmic reticulum, to provoke exocytosis of synaptic vesicles for
release of neurotransmitters into the synapse. Calcium transients in postsynaptic cells, by contrast,
are involved in the mediation of activity-dependent synaptic plasticity. For these reasons, the ability
to monitor calcium in neurons has proven to be a powerful tool for neuroscience [23].
Due to their superior brightness and dynamic range, proteins that modulate their fluorescence
with calcium concentration have emerged as the most viable and popular tool to optically study
cellular dynamics in behaving animals. Progress in the development of calcium indicators has been
reviewed by others [22], [24], [25]. Today, a broad palette is available; competing protein engineering efforts have now come close to optimizing the green fluorescent protein (GFP) molecule
discovered in 1997 [26] for brightness, kinetics and targeted genetic expression. When applied

with methods that enable targeting the proteins expression to specific cell types, these reporters
are known as Genetically-Encoded Calcium Indicators (GECIs). The GCaMP family of calcium
reporters [1] is a prevalent example of GECIs.
GECIs are a powerful tool in neuroscience, as an influx of calcium in the cell is correlated
with an action potential in the neuron. GECIs enable imaging when the camera has line-of-sight
access to the structure of interest, and studying only specific cells through genetic targeting techniques. Common applications of this modality include studying transparent specimens such as the
zebrafish [27], or the cortex of non-transparent specimens such as rats and mice [28]. Studying
deeper structures in small mammals is more difficult. This is because these indicators are excited
by and emit visible light which is strongly scattered by brain tissue [29], resulting in attenuation as
it passes through tissue. Imaging with visible light is restricted to the most superficial regions of
the brain, up to depths of approximately 50 m [11].
To improve penetration depth, several approaches have been proposed. On the instrumentation
side, they include employing nonlinear excitation strategies, endoscopes, imaging with a multimode fiber, and single fiber photometry. These solutions preclude the most natural expression of
the animals behaviour by either requiring that the animal be restrained during the experiment, or
being too invasive to ensure proper brain function.
Equation 2.1 relates the energy carried by a photon, E, to the Plank constant, h (6.626 x 10-34
J/s), the photons wavelength, , and the speed of light, c (2.99 x 108 m/s).

E=

hc

(2.1)

A prominent example of nonlinear excitation is 2-photon excitation (2PE). As the name would
suggest, 2PE involves exciting the fluorescent protein with two simultaneously absorbed photons
of approximately twice the wavelength, (of which each carries half the required energy) that would
be required for single-photon excitation, such that the same energy is absorbed, as demonstrated

in Equation 2.2.

E=

hc hc
+
2 2

(2.2)

Because red-shifted photons can penetrate more deeply than their visible counterparts, imaging
is possible at planes beneath the surface. Depths of approximately 1 mm [30] have been achieved
with this modality. This represents a two hundred-fold improvement over one-photon methods,
and would enable imaging regions such as the majority of the cortex in a mouse model. Another
advantage of this modality is its ability to avoid exciting fluorophores outside of where the laser
beam is tightly focused, providing out of focus rejection and background reduction. This is because
the probability of exciting a fluorophore increases with the square of the excitation intensity at a
given plane.
However, imaging deep brain structures in popular animal models such as the mouse and the
rat are inaccessible to two-photon microscopy, as they have thicknesses of approximately 5 mm
and 10 mm, respectively [31], [32]. Furthermore, as two-photon imaging requires scanning a
tight focal point over the specimen to produce a single image, there is an upper limit on both the
temporal resolution achievable and the practical field of view to approximately 1 mm2 . These
difficulties are somewhat alleviated by light sheet microscopy, wherein the tissue that is irradiated
at any one time is limited to that falling within a plane, and not a cone, as in the conventional
nonlinear techniques [33]. While two-photon microscopy has been adapted for freely-moving
animals [34], light-sheet microscopy is currently limited to transparent, restrained animal models
such as zebrafish [35], or fixed brains processed with an optical clearant such as CLARITY [36].
Endoscopes, a second approach to interrogating the activity of deeper brain structures, involve
implanting a segment of optical fiber in the brain such that the distal tip is flush with the tissue
of interest. There are two basic types of endoscopes: flexible and rigid. Flexible endoscopes are
composed of a bundle of tens of thousand single-mode fibers each with a diameter of approximately
50 m. A single-mode fiber is an optical fiber wherein light travels in only one direction along the

fiber. This contrasts with multimode fibers, where light may travel through the fiber with several
distinct, simultaneous spatial distributions. As such, the use of single-mode fibers in the bundle
permits an image of the tissue to emerge at the fiber bundles proximal end undistorted. The
resolution of the image is approximately the diameter of a single fiber probe [37]. Flexibility is
conferred by the mechanical properties of a bundle of thin wires. Rigid endoscopes, on the other
hand, employ gradient index (GRIN) fibers wherein light is constrained to a certain path along the
fibers length. Similar to flexible endoscopes, the image appears at the detector undistorted, but
flexibility is compromised.
Neither flexible nor rigid endoscopes are viable solutions for exploring brain dynamics in deep
brain structures of small animal models such as mice. Their large diameter (>1 mm) exceed the
300 m determined to be safe for use on mice [10]. Their invasiveness will likely damage the
complex organ beyond the ability to properly interpret experimental results. While imaging with
multimode fibers has been achieved, with diameters sufficiently small for safe use on animals, expensive spatial light modulators (SLMs) are needed to solve the inverse problem and transpose the
image of the tissue at the fibers proximal end [38]. In freely-moving animals, the deformation in
the optical fiber tether to the desktop-mounted optics undoes the action of the spatial light modulator, requiring complex image-recovery algorithms [39]. The complexity and expense represent
barriers to adoption of multi-mode, fiber-based imaging in freely-moving animals.
While the aforementioned approaches enable recording precise spatiotemporal patterns of several neurons simultaneously with imaging, for some applications it suffices to monitor the average
dynamic activity from a population of neurons. In these cases, multimode fibers provide another
option for interrogating deep brain structures with fluorescence. This is known as single fiber
photometry, and optical system configurations are analogous to that used in epifluorescence microscopes [8], [40] where a photomultiplier tube (PMT) is used as a detector instead of a camera.
The light source, optical components, and PMT detector sit on the benchtop while the optical fiber
follows the animal about its course and delivers excitation light, and carries emitted light back. The

metric reported is baseline-referred changes in fluorescence intensity, F/F, on the order of 5% to

100%. Genetic targeting techniques using combinations of viruses, promoters, and/or transgenic
animals, enable cell-type specific, connectivity-dependent, and projection-based expression of the
fluorescent proteins such that researchers can study only those cells and connections in which they
are interested [14]. A wide variety of Optrodes have built on this idea, assembling electrodes and
multimode fibers to provide simultaneous temporal resolution and cellular speicificity [41], [42].
Single fiber photometry is rapidly gaining popularity in neuroscience as a tool to interrogate neural
circuits in freely-moving animals [3], [4], [5], and is the focus of this research.

2.2

Survey of Existing Fiber Photometry Systems

Single fiber photometry involves launching visible light into an optical fiber for which the distal
tip is in contact with brain tissue. In order to see fluorescence, the tissue, and hence the reporter,
is first supplied with this light, which causes the fluorescent reporter to enter an excited state. A
few hundred nanoseconds the molecule returns to its ground state, and the absorbed light energy is
released as a photon. Because some energy is lost to vibration, the photon that is emitted has less
energy than the photon that was absorbed: the radiated photon therefore has a longer wavelength.
This energy difference, expressed in nm, is referred to as the molecules Stokes Shift, and enables
the separation of the excitation and emission photons with optical filters.
A typical fiber photometry system is depicted in Figure 2.1. It consists of a delivery path,
highlighted in blue, which launches excitation light into the optical fiber, and a collection path,
highlighted in green, that relays the photons from fluorescence to a sensitive photodetector. A
dichroic mirror separates the excitation and emission photons after they have interacted with the
sample. The diameter of the optical fiber must be small enough for safe use on the animal to be
studied. In mice, this is approximately 300 m in diameter. Not shown in the figure are excitation
and emission filters, that are often placed at the output of the light source and the input to the
photodetector, respectively, to reduce the leakage of photons from the light source and improve

10

Optical System

Figure 2.1: Schematic of a typical Single Fiber Photometry System

system sensitivity. The transmission profiles of these bandpass filters are often described with two
numbers, separated by a forward slash, that denote the filters center wavelength and bandwidth.
For example, a 535/30 nm filter cuts on at 520 nm and cuts off at 550 nm.
Despite the simplicity of the concept, there are difficulties in repeatability and in data interpretation that make in vivo fiber photometry an expensive endeavour. For example, exciting a fluorophore in living tissue with light can have undesirable consequences, including photobleaching
of the reporter, exciting endogenous fluorophores, and even inducing cell death due to phototoxicity. As these effects are irreversible, the specimen may be essentially destroyed before meaningful
results can be extracted. Secondly, because the path travelled by photons in tissue is dynamic, and
often wavelength selective, the fluorescence trace is distorted. These artifacts may be attributed to
transients in fresnel reflections, absorption, and optical path length, in turn caused by combinations
of movement by the animal and its normal physiologic processes. Finally, because brain tissue is
such a highly scattering medium, the tip of the optical fiber implant must be very close to the
structure that expresses the fluorescent reporters. Otherwise, no signal will be detected. Currently,
these three sources of variability are overcome by doing a large number of experiments.
The instrumentation has progressed to address critical problems. To enable measurement under
low-light conditions, and hence mitigate the risk of phototoxicity and photobleaching, the detection
scheme must be highly sensitive to fluorescence. This is because the number of emission photons
11

is proportional to excitation photons delivered to the tissue. While employing larger-diameter

fibers would improve the systems collection efficiency, there is a safe limit of 300 W for use in
mice. Photomultiplier tubes are therefore most often employed, together with a coherent detection
scheme [40]. Falk and Pashaie [8] improved signal-to-noise ratio further through study of system
noise statistics and their exploitation in receiver design.
Progress in artifact-correction schemes borrow from techniques in spectroscopy. Thompson
and the Deisseroth group propose a system that excites tissue at two wavelengths [5] with distinct
modulation properties. One source is near the indicators peak excitation, and the other, at an
isosbestic point (the fluorescence that results is not mediated by bound Ca2+ ). This configuration
yields a calcium signal and a control fluorescence signal. Immunity from artifacts that affect both
signals idnetically is granted by subtracting the control from the calcium signal, after applying a
fitting function. One drawback to this approach is that the control signal must be generated with
violet excitation at 405 nm. These short wavelengths cause strong tissue autofluorescence, and
stronger photobleaching. A similar approach that overcomes this challenge is a dual emission
band configuration, employed Goltstein [7], that enables collecting both the fluorescence due to
a reporter and a fluorescent marker (although deviations in photostability (bleaching) properties
between the two fluorophores induces a second artifact). Either of these approaches are suitable to
correct for wavelength-indiscriminate artifacts such as modulations in Fresnel reflections, but their
immunity to wavelength-selective events (such as changes in optical path length and absorption)
has yet to be demonstrated. Wavelength-selective artifacts are particularly concerning when the
artifact is correlated with the signal, as is the case for certain hemodynamic processes [43] such as
changes in blood volume and oxygen saturation. Gamm developed a means to extract the shape of
the intrinsic fluorescence spectrum from turbid media, at the expense of lost amplitude information
[44], the quantity of interest in fiber photometry. Moroever, the broad-spectrum source required
would be unsuitable for a fiber photometry application as the backscattered photons cannot be
separated from the fluorescence, and would overwhelm the detector.

12

In addition to ensuring the measurement does not damage the tissue with excitation light, and
that the measurement does not contain false signals caused by artifacts, investigators must ensure
the optical fiber is implanted in the correct location. Stereotactic coordinates and landmarks guide
the surgery, but variability in between animals yield a typicaly success rate of approximately 20%.
Grienberger [3] used a fiber photometry system to search for the region of highest fluorescence during surgery. This approach assumes that the cells are active during surgery. For reporters with large
dynamic range, such as GCaMP6s, inactive cells fluoresce very weakly if at all. Heterogeneity in
the brain due to structures such as vesicles, as well as co-expressed fluorophores with overlapping
emission spectra, further complicate the task of using a single emission band to correctly locate the
structure of interest. [9] uses an array of PMTs, each with a distinct emission band in the visible
spectrum, and use it to improve specificity.

2.3

Problem Statement

Avoiding phototoxicity and photobleaching are major concerns when performing optical measurements in tissue, and so an optoelectronic system capable of performing measurements at ultra-low
light levels will be designed (SA1) and validated in vivo (SA2) as part of this research. This will
include a spectral characterization of the reporter and its dynamics (SA3), such that the systems
sensitivity to changes in GCaMP6s fluorescence is optimized. An improved sensitivity to changes
also means that for the same delivered light level, the system can detect smaller changes in fluorescence intensity. Calcium dynamics can be detected when the fiber probe is far away from the
population of fluorescent reporters, a probable case given surgery success rates.
In order to correct for artifacts, their causes must first be identified (SA4). Despite the fact
that blood volume and oxygen saturation are often correlated with neural activity [43], and that
the changes in absorption they would cause would result in a distortion of the fluorescence signal,
hemodynamic or vascular-related changes have yet to be identified in the context of fiber photometry. To our knowledge, there is no photometry system that can correct for artifacts related to

13

changes in blood parameters. However, the spectral characterization of the reporter (SA2) may
suggest a solution that could later be adapted to imaging modalities, such as multi-mode fiberbased imaging.
Due to individual variation between experimental subjects, it is unlikely that, using stereotactic coordinates alone, a fiber optic probe will be implanted in the brain such that it can detect
calcium-mediated modulations in fluorescence. Optical systems may be applied to improve this
outcome, by enabling the surgeon to search for and follow gradients in fluorescence intensity near
desired structures. This research will therefore design a suitable fiber photometry system, develop
a protocol for its application, and investigate its effect on surgical success rates (SA5).

14

Chapter 3
FIBER PHOTOMETRY SYSTEM DESIGN,
CHARACTERIZATION, AND VALIDATION IN AN
ANIMAL MODEL
This chapter describes a fiber photometry system for monitoring calcium dynamics in the paraventricular nucleus of the hypothalamus of a CRH-Cre mouse as reported by the genetically-encoded
calcium reporter GCaMP6. The system described and characterized here is an initial design, whose
application in behavioural experiments on freely-moving animals informs the refined system described in Chapter 4.
The fiber photometry system is an optoelectronic instrument. Section 3.1 presents the constraints of the design. The appropriate optics employed to provide sufficient delivery and collection
of photons are first discussed in Section 3.2, and then the custom-designed electronics is described
in Section 3.3. Section 3.4 presents the systems bench-top characterization as well as results from
in vivo application. The signals of interest in photometry are often very small changes in fluorescence intensity, so the designs noise and stability are important parameters for characterization.
Section3.6 reflects on the initial design and results, and considers areas for improvement.

3.1

Design Constraints

A fiber photometry system has several opportunities to alter the signals that would otherwise be
recorded from a biological system, especially when that system is the intact brain of a freely moving mouse. For example, the presence of the fiber in the brain can damage tissue and vasculature,
and the light used to excite the fluorophore may cause cell death due to phototoxicity. These causes
of irreversible biological disruption are problematic as they encroach on the methods repeatability
15

and the possibility of extracting meaning from the data.

To address the issue of tissue damage due to the implant, a set of limits for implanting optical
fibers in animal models has emerged in the literature. An optical fiber less than 300 m is accepted
as safe for use in behavioural experiments in mice [10]. To avoid photobleaching for GFP and its
derivatives, the delivered level of excitation optical power must be below 1.2 W [40].
The goal of this design is to build a highly sensitive system, capable of detecting Ca2+ -mediated
changes in fluorescence under ultra-low light levels in a minimally invasive fashion. A common
reported figure in literature is the baseline-referred change in fluorescence, given by

F/Fo =

F Fo
100%
Fo

(3.1)

Where Fo is the average fluorescence intensity during some defined baseline, and F is the
fluorescence intensity, or average fluorescence intensity at some other time, usually at a time correlated with meaningful behaviour exhibited by the animal. Typical values for F/Fo vary widely,
but values smaller than F/Fo of 1% are never reported [5] [4].
A useful performance specification for the systems that will be developed in this research is
contrast-to-noise ratio or CNR. This is a concept borrowed from imaging, and in this context is
the ratio of the change in detected fluorescence, F to the quadrature-summed standard deviation
of the noise N, described by Equation 3.2.

1
N=
2

q
12 + 22

(3.2)

Where 12 and 22 are the standard deviations of the measured signals during the experiment
observation periods that result in measurement of Fo and F, respectively. The CNR, then, is given
by Equation 3.3.

CNR =

F Fo
N

(3.3)

A CNR greater than one, therefore, would indicate that a signal was detected. Together with
16

the assumption that reported fiber photometric signals are greater than 1%, the design constraints
were set, which are presented in Table 3.1.
Parameter
CNR
Probe diameter

Conditions
Pex =1 W; F/Fo >1%
Implanted

Design Constraint
>0 dB
300 m

Table 3.1: Constraints that guide the design of this fiber photometry system and acknowledge the
compromise between the systems ability to detect small signals (defined by its signal-to-noise
ratio, SNR) and tissue damage caused by the optoelectronic system (either through exposure to
high light irradiances or large-fiber optic probes).

17

3.2

Optical Design

dichroic mirror
emission filter

Figure 3.1: The optics employed in a typical Single Fiber Photometry System

3.2.1

Light Source

GCaMP6s is a fluorescent protein. In order to generate fluorescence, the tissue must first be supplied with light by the optical system. A light-emitting diode (LED) was chosen for this purpose,
as they are inexpensive, and their light output is very stable over time. However, coupling LEDs to
optical fibers is fundamentally inefficient, as depicted in Figure 3.3. This is first because an LED
light source has a large emitting area compared to the effective core diameter of an optical fiber.
And second, because LEDs are not directional light sources, but optical fibers accept only those
rays that fall within narrow range of angles.
The rays that an optical fiber can accept are described by its Numerical Aperture (NA), as
depicted in Figure 3.2. Optical fibers constrain the path of light to within its core through a process
called total internal reflection. However, this result occurs only for rays that impinge the fibers
core at an angle less than the fibers numerical aperture. NA is determined by the relative refractive
indices of the medium, n, and the materials used to make the optical fiber: the core, ncore , and the
cladding, ncladding .

18

NA = n sin(max )
q
= n2core n2cladding

(3.4)
(3.5)

ray 2
core, ncore

ray 1

numerical aperture

cladding, ncladding

Figure 3.2: The numerical aperture of an optical fiber describes the range of angles for impinging
rays that are accepted. Ray 2, shown in red, impinges the core at an angle greater than the numerical
aperture, and therefore leaks through the outer layers of the fiber. Ray 1, by contrast, is accepted
as it undergoes total internal reflection at the core-cladding interface.
Although lenses and mirrors change the direction of rays, these inefficiencies cannot be overcome as e tendue (the product of the area of the source and the solid angle it subtends) is conserved
as rays are refracted and reflected. The highest theoretical coupling efficiency may therefore be
achieved by butt-coupling an optical fiber to an LED.
Neglecting the distance between the tip of the optical fiber and the emitting surface of the LED,
and the difference in refractive indices at the interface, the coupling efficiency is determined by the
product of angular and geometric coupling efficiencies [45]:

= geo ang

(3.6)

r2f iber
A f iber
= 2
geo =
Asource
wLED

(3.7)

ang = 1 cos(arcsin(NA f iber ))

(3.8)

where

and

19

2rfiber
NA=sin
WLED

Figure 3.3: The coupling efficiency of a pigtailed LED depends on the emitter size, fiber diameter,
and acceptance angle of the fiber.
Where the light source design employs a multimode fiber (FC-200/230-0.48-5mm-MF2.5,
Doric Lenses) with r f iber = 100 m, NA f iber = 0.22, and the LED (LXML-PB02, Philips) has
wLED = 1.5 mm, the maximum achievable coupling efficiency is 0.067%. Assuming an LXMLPB02 LED, (One of the brightest LEDs available), driven at the maximum 100 mA, a theoretical
maximum of 160 W will be coupled into the optical fiber, that will later be delivered to tissue
downstream in the optical system. This is well above the level of light exposure that induces
phototoxicity [6]. The pigtailed LED light source is therefore sufficient for this design.
The lens of the aforementioned 465 nm LED (LXML-PB02, Lumileds) was removed and buttcoupled to a cleaved and polished 200 m, 0.22 NA fiber with an FC/PC termination (200/240/9000.22, Doric Lenses) with a multi-purpose, silicone rubber sealant (732, Dow Corning). The output
of the light source is collimated by a 0.51 NA fixed focus collimation package (F240FC-A, Thorlabs).
An excitation filter restricts the spectral content of the beam to a band between 450 nm and
490 nm. This attenuates out-of-band energies originating from the broad band LED, centered at
465 nm, and photons generated through excitation of endogenous fluorophores in the optical fiber,
by a factor of at least one million. As depicted in Figure 3.4, the spectrum of the excitation source
overlaps well with the excitation spectrum for GCaMP6s in its excited state. This provides a

20

Figure 3.4: Normalized excitation spectra for fluorescent calcium reporters in the GCaMP family [1]. The proposed excitation and emission filters are marked in blue and green, respectively.
balance between efficiency in exciting the fluorophore and the ability to launch a sufficient amount
of power into the optical fiber.
The schematic of the LED driver circuit is presented in Figure 3.5. A 15 V wall adapter
(WSU150-1200, Triad) supplies power to the driver circuit. An adjustible positive voltage regulator (LM317, ON Semiconductor) steps the voltage down to 12 V and filters out noise from
the switching circuitry. The LED is driven in constant current mode by an operational amplifier
(TCA0372, ON Semiconductor), supplied by the 12 V regulated supply. The op amp is biased at
half this supply potential with the resistors R1 = R2 = 10 k. The current through the LED is
determined by:

ILED =

VREF
RLIMIT + RPOT

(3.9)

2
Where VREF = VSUPPLY R1R+R
, and R1 = R2 = 10 k. The current through the LED is adjusted
2

with the potentiometer, RPOT . When the potentiometer is at its minimal resistance, the current
through the LED, and hence the light output, is maximal. The fixed resistance, RLIMIT , limits this
to 82 mA. RLIMIT is a parallel combination of three 220 resistors.
21

+12V

R1
VREF
R2

RLIMIT

LED

RPOT

Figure 3.5: Schematic for LED driver circuit

The power dissipated in a branch, Pbranch , is determined by the current through a single branch,
Ibranch , and the resistance of that branch, Rbranch

2
Pbranch = Ibranch
Rbranch


ILED 2
Rbranch
=
3

= 0.168W

(3.10)
(3.11)
(3.12)

This configuration ensures that the power dissipation ratings of neither the operational amplifier
nor the

1
4

3.2.2

Optical Tether

Watt resistors are exceeded.

The optical tether delivers excitation light to, and collects emitted photons from, the tissue of interest. A 200 m optical fiber (200/240/900-0.48, Doric Lenses) is employed here. This size of fiber
is acceptable for implanting in mice [10], and the large numerical aperture of the fiber improves the
systems ability to collect photons emitted from the fluorophores. During experiments, the tether
is coupled to a permanent fiber implant through a zirconium sleeve as depicted in Figure 3.6.
The core and the cladding of the optical fiber, while composed primarily of silica, contains
impurities that alter the silicas refractive indices and set the numerical aperture of the fiber. These
22

Figure 3.6: The optical tether couples light to and from fiber optic implant that is in permanent
contact with brain tissue.
impurities will often fluoresce when exposed to light at various wavelengths. In this design, this
fluorescence will contribute to a background in the overall signal, making ineffective use of the
photoreceivers dynamic range and resulting in reduced sensitivity to changes in GCaMP6 fluorescence.
Using a spectrometer and a dichroic mirror, the autofluorescence of several optical fibers were
characterized. The excitation source was a 485 nm laser, whose center wavelength is near that of
the 465 nm LED to be employed in the fiber photometry system. The results of this experiment
are therefore application to the photometry system that will be developed. As shown in Figure 3.7,
the selected fiber from Doric Lenses has a relatively low contribution of fluorescence due to endogenous fluorophores as compared to another optical fiber considered. The large difference in
the autofluorescence produced is likely due to differing compositions of the core and/or cladding.
However, the specific materials used are proprietary information and therefore unknown.

3.2.3

In situ Characterization of GCaMP6

Reporter State
Ca2+ -free
Ca2+ -bound

Excitation
505 nm
497 nm

Emission
517 nm
515 nm

Table 3.2: Spectral Maxima for GCaMP6s

23

Normalized Fluorescence Intensity

200/240/900-0.48, Doric Lenses

FT-200EMT, Thorlabs

0.8
0.6
0.4
0.2
0
500

550

600
650
Wavelength (nm)

700

Figure 3.7: Two optical fibers, of the same length, are exposed to the same light level, and produce
the fluorescence spectra depicted here. 200/240/900-0.48 (from Doric Lenses) is employed in
the system due to its extremely low autofluorescence in the emission spectrum for GCaMP6s.
Both curves are normalized to the peak detected fluorescence, in counts, from FT-200EMT (from
Thorlabs).
Table 3.2 presents the peak wavelengths of the proteins excitation and emission spectra, for
both its calcium-free and calcium-bound states. The spectral properties of the calcium reporter,
GCaMP6s, have been chacterized by others [16].
Equation 3.13 describes the various sources of fluorescence that are collected by a fiber photometry system, for a total signal Ftotal ( ). The systems sensitivity to the signal of interest,
FGCaMP6s ( ), is reduced by the presence of endogenous fluorophores in tissue, such as lipofuscin [46], and in this application, co-expressed fluorophores, that cause a background Ftissue ( ).
The autofluorescence of the fiber, Ff iber ( ), is a second source of background.

Ftotal ( ) = FGCaMP6s ( ) + Ftissue ( ) + Ff iber ( )

(3.13)

In order to maximize the systems sensitivity to changes in GCaMP6s fluorescence, the contribution of these additional sources of fluorescence must be minimized. One way to do this would be
by exploiting differences in spectral properties between these substances. To determine these characteristics, the spectral characteristics of GCaMP6s dynamics, in tissue slices, was interrogated
with the optical fiber probe.

24

Camera
Brain slice

To single
fiber
system
(a)

(b)

Figure 3.8: The spectral dynamics of tissue with GCaMP6s expression were characterized in fresh
tissue slices, with simultaneous epifluorescence imaging and fiber photometry. a) In situ characterization was performed with the aid of an epifluorescence microscope b) The arrangement enabled
optical access to the sample for both an optical fiber and a camera.
Figure 3.8 depicts the experimental setup used to characterize the spectral dynamics of the
GCaMP6s calcium reporter, and serve as a proof of concept for the design. Simultaneous images were acquired from above the sample with an epifluorescence microscope and digital camera
(Chameleon Monochrome USB 2.0, Point Grey) as in Figure 3.8a. The fiber provides a nearby
spectroscopy system with optical access to the sample from below the microscope stage, and provides the only source of excitation light for the experiment. The resulting micrographs contain
fluorescence in localized areas, enabling recovery of the area interrogated with the fiber. A bare
optical fiber (BFH-48, Thorlabs) was used in place of the optical tether due to its flexibility. This
is a valid substitution in this context as it was determined that its fluorescence profile is identical
to that of the tether. A neutral density filter was used to attenuate the power from a 488 nm laser
(TECBL-488, World Star Tech), such that 520 W of excitation light was delivered from below the
sample by the fiber. Although a LED will be used in the eventual design, a laser was used here as
it can more easily provided the elevated levels of excitation light required.
The optical system used for these experiments is depicted in Figure 3.9. It is similar to the typical fiber photometry configuration. A spectrometer is used as the photoreceiver, and the emission
filter is omitted. An excitation filter (470/40), and a dichroic mirror (510LP, Chroma Technology)
25

Fluorescence Intensity (Counts)

Figure 3.9: The optical system employed for spectral characterization of GCaMP6s

Normalized Intensity

1
0.8
0.6
0.4
0.2
0
400

500
600
Wavelength (nm)

700

(a)

500
400
300
200
100
0
500

510
520
530
540
Wavelength (nm)

550

(b)

Figure 3.10: a) A representative spectrum collected from the interaction of laser light with the
brain slice, normalized to the maximum intensity from backreflection of the laser at 48 nm, and b)
at wavelengths near GCaMP6s emission.
enable the collection of both excitation and emission photons on the same device with a limited
dynamic range. The excitation filter, positioned at the output of the laser, attenuates out of band
photons from the light source to improve sensitivity to fluorescence. Because the input power of
the laser is known, by monitoring both of these quantities simultaneously the arbitrary units read
out by the spectrometer can be converted to absolute quantities of optical power. This will enable an estimation of the optical power produced from fluorescence, and inform the selection of a
suitable photodetector. Figure 3.10a shows a representative spectrum collected from the sample.
Activation of cells was achieved by additions of potassium chloride (KCl) to the tissue slices
in a bath of artificial cerebrospinal fluid. A cell normally maintains a resting potential of -70 mV
across its membrane, referred to the inside of the cell. This potential is largely established by the
sodium-potassium pumps, that drive three sodium ions from the cell for every two potassium ions

26

Figure 3.11: Results of simultaneous microscopy (left) and fiber photometry (right) on fresh,
250 m slices containing CRH-Cre cells expressing the GCaMP6 fluorescent calcium reporter.
Additions of Potassium Chloride (KCl), activated cells. An addition of Artificial Cerebrospinal
Fluid (aCSF), used as a control, had no effect. Imaging and spectroscopy data were synchronized,
and a selection of common time points are denoted.
drawn into the cell. When the KCl is added to the solution outside of the cell, the potassium ions are
drawn into the cell by the electrical gradient, passing through passive leak channels. This causes
the cell to increase from its negative resting potential. In other words, the cell depolarizes. [47]
Access to and preparation of brain slices from transgenic animals was provided by Dr. Jaideep
Bains lab in the department of Physiology and Pharmacology at the University of Calgary. Tissue
samples were prepared by placing 250 m-thick coronal slices of the brain in a petri dish with a
glass-slide bottom containing 2 mL of artificial cerebrospinal fluid (aCSF). Stimulation to the cells
was provided through 200 L additions of 1.0 M KCl to the sample, delivered by dropping into the
aCSFs tissue bath with a pipette.
Additions of KCl resulted in increased fluorescence intensity from CRH-Cre cells with GCaMP6
as shown in Figure 3.11. These increases in fluorescence are wavelength-dependent. Because we
are interested in detecting changes in fluorescence intensity with time, referred to baseline intensity,
this spectral sensitivity, R( ), will be defined as the wavelength-dependant change in fluorescence
intensity from a baseline intensity, divided by the same baseline intensity:

R( ) =

Fexcited ( ) Fbaseline ( )
100%
Fbaseline ( )

(3.14)

Where Fexcited is the collected spectrum when the population is in an excited state and Fbaseline
is the collected spectrum when the population is in a baseline state.
27

300

30 %

200

40%

F/F (%)

400

40 %

60%

Before KCl addition

After KCl addition
F/F

Fluorescence Intensity (Counts)

500

20%

10 %

100
0
500

20 %

0
510
520
530
540
Wavelength (nm)

550

(a)

480

500
520
540
560
Wavelength (nm)

(b)

580

505/10515/30535/30525/50

(c)

Figure 3.12: Spectral characterization informs decisions to optimize detector sensitivity. a) Spectra
acquired when cells were inactive (dark green) and active (bright green) b) Spectral sensitivity
to GCaMP6s (averaged, n = 3) c) Of four possible filters considered, the 535/30 nm filter (for
which the transmission profile is denoted by (center wavelength)/(bandwidth)) will enable greatest
sensitivity to modulations in fluorescence due to GCaMP6.
Although the emission maximum for GCaMP6s in its calcium-bound state is 515 nm [16],
Figure 3.12a reveals that fiber autofluorescence is strong near this wavelength. By adding a large
background, these endogenous fluorophores reduce the baseline-referred changes and push the
sensitivity maximum to the longer wavelength of 521 nm. Few photons with wavelengths shorter
than 510 nm were collected, as the dichroic mirror employed cuts on at this wavelength.
Knowledge of GCaMP6s dynamics in situ facilitated an optimal selection of optical filters.
Four possible, off-the-shelf emission filters were considered, as shown in Figure 3.12c. The emission filter centered at 535 nm was selected for this application, as it offers superior sensitivity to
baseline-referred changes in GCaMP6s fluorescence even compared to that with the widest band.
This in situ characterization of the calcium reporter comprised gathering knowledge on its
spectral dynamics and absolute optical quantities. The next subsection will explain how this information was used to make decisions on the design of the optoelectronic fiber photometry system.

3.2.4

Photodetector Selection

As photodetectors vary in sensitivity and minimum detectable power, the optical power due to
fluorescence that can be collected were estimated from the experiments described in Section 3.2.3.
Neglecting interactions of the excitation light beyond the first interface, the total optical power

28

detected at the detector due to the laser in the optical system in Figure 3.9 is given by

Pdet,laser = Plaser R f iberair T ( )dichroic

= (520 W)(0.04)(

1
)
460829

= 45.1pW

(3.15)
(3.16)
(3.17)

Where Pdet,laser is the theoretical optical power detected at spectrometer, Plaser is the optical
power launched into the fiber (520 W), R f iberair is the reflection at the fiber-air interface (0.04),
and T ( )dichroic is the transmission ratio of the dichroic mirror at the center wavelength of the laser
1
at 485 nm).
( 460829

Pdet,laser can be related to the dark-current corrected spectrum due to the laser, S( )det,laser , as
the area underneath this spectrum, in counts, determined by the bandwidth of the laser, B, and the
center wavelength of the laser, p :

+B/2

Pdet,laser p B/2 S( )det,laser

p

(3.18)

A proportionality constant, , was therefore introduced to enable conversion between relative

quantities provided by the spectrometer (counts) and absolute quantities (optical power, in W).

=
=

Pdet,laser
p +B/2
p B/2 S( )DET,LASER

(3.19)

45.1pW
275640

(3.20)

= 0.163 fW /count

(3.21)

The absolute powers from various sources of fluorescence intensity, were then estimated using
Equation 3.22. These quantities were then scaled linearly with excitation power to predict relevant
quantities such as that due to signal from GCaMP6s, and background sources such as tissue and

29

fiber autofluorescence, over the range of the design criteria. The results of these computations are
presented in Table 3.3

Pdet, f luorescence = 520 S( )det, f luorescence

Source of Fluorescence
GCaMP6s
Optical Fiber
Tissue

Contribution to Total Signal

Signal
Background
Background

at 520 W excitation
1.16 pW
0.51 pW
2.82 pW

(3.22)
at 1 W excitation
2.23 fW
0.98 fW
5.42 fW

Table 3.3: Estimated or predicted contributions of collected fluorescence from various sources at
several levels of excitation light
A photomultiplier tube was selected for this application, as devices in this family of photodetectors can provide acceptable signal-to-noise ratios at light levels on the order of 1 fW.
PMTs have a very large internal gain, making them extremely sensitive to light that impinges
on the sensor. PMTs have the ability to amplify photoelectrons through secondary emission. This
phenomenon is caused by the devices construction, as depicted in Figure 3.13, and the high voltage
applied in a step-wise manner across the tube. This configuration gives rise to a cascade effect that
results in a very large number of ejected electrons for a small number of incident photons.
The PMT works through photoemission [48]: photons impinge on the devices photocathode,
releasing electrons through the photoelectric effect. These photoelectrons are accelerated towards
the neighboring dynode by a large potential difference (approximately 100 V) that exists between
the two surfaces. When these photoelectrons reach the dynode, the acceleration from the electric
field causes them to have more energy (momentum) than when they were first ejected by the
incident photon. This increased energy results in several electrons ejected from the dynode for a
single incident electron. A typical PMT may have a dozen such dynodes arranged in series, each
with a slightly more positive voltage than the last, resulting in an overall internal gain on the order
of 104 to 106 .
The PMT (H10771-40P, Hamamatsu) is now considered for use as the photodetector. For an

30

GND
-HV
photon

primary
photoelectron

secondary
electrons
IPMT

photocathode

dynodes

focusing
electrode

anode

Figure 3.13: Structure and operation of a PMT. A PMT consists of a light-sensitive photocathode
held at a large negative potential, and a configuration of amplifying dynodes held at successively
higher potentials leading to the devices anode held at ground potential. The electric fields in each
PMT stage result in a cascade of secondary emission processes that give the photodetector a large
internal gain and high sensitivity.
excitation power of 1 W, the signal-to-noise ratio achieved with this device is given by [48]

I ph
SNR = p
2eFB[I ph + 2(Ib + Id )]

(3.23)

Where
I ph is the signal current at the photocathode, = 0.446 fA
Ib is the background current at the photocathode, = 1.28 fA
Id is the equivalent dark current at the photocathode, = 0.966 fA (equal to the product of dark
counts per second and electron charge, e)
e is the electron charge, = 1.602 10-19 C
F is the PMTs noise figure, = 1.44
B is the bandwidth of the measurement, assumed to be 10 Hz
I ph , Ib , Id were computed from values in Table 3.3 and the anode sensitivity of 0.198 mA/W
at 550 nm. Equation 3.23 yields a signal-to-noise ratio of 2.94. The PMT is therefore a suitable
31

EM SH

DBS

OPTICAL TETHER

L3

L2

BUTT-COUPLED
LED SOURCE

PMT

EX
L1

Figure 3.14: The optomechanics involved in launching excitation light from the light source into
optical tether, and focusing fluorescence carried by the tether to the photodetector (PMT). M: alignment mirror, DBS: dichroic beam splitter, EX: excitation filter, EM: emission filter, SH: shutter.
L1 = L2: collimating or focusing lenses, L3: focusing lens.
choice, as GCaMP6s-mediated changes in fluorescence intensity produce optical power above the
devices minimum detectable power. Equation 3.23 reveals that SNR improves with detected optical power from the signal, and degrades with sources of background optical power and dark
current. Because this figure was obtained by estimating the optical power collected by the spectrometer, but the PMT does not suffer from losses associated with gratings, this calculation is likely
an underestimation of the optical power that will be collected by the PMT.
The PMT in question (H10771-40P, Hamamatsu) is most sensitive to green light in the range
500 - 600 nm, which overlaps with the spectral sensitivity of the fluorescent reporter in question,
and the selected emission filter centered at 535 nm.

3.2.5

Optomechanics and Alignment

The optical components employed in a fiber photometry system are shown in Figure 3.14. The
optomechanics selected for the design are now described in terms of the delivery path, highlighted
in blue, and the collection path, highlighted in green.
From Subsection 3.2.1, the purpose of the delivery path is to launch sufficient excitation power
into an optical fiber. The delivery path consists of the light source and its collimation package, a

32

mirror, a dichroic beamsplitter, and the collimation package for the optical tether. The beam from
the light source is reflected by 45 in the plane of the mounting surface, at both the mirror and
dichroic mirror, and then impinges on the center of the coupling lens for the optical tether in the
direction of this lens optical axis.
The selected optomechanics provide adequate translational and angular adjustment to correct
for deviations between the optical axis of the system and the optical axes of its components, in
order to ensure proper alignment and good coupling of light into the optical fiber. The angle of
the beam is corrected through coordination between two kinematic mounts (KM100T, Thorlabs),
at the source and alignment mirror. The fixed-focus collimation packages (F240FC-532, Thorlabs)
are matched on both the sending and receiving ends of the delivery path, ensuring that the focused
spot size at the tether is equal to that of the butt-coupled fiber.
Excluding the optical filters, the collection path, depicted in green in Figure 3.14 consists of the
tether and its collimation package, a focusing lens, a shutter, and the PMT. The collimation package
for the optical tether is housed in an adapter that does not have tip/tilt capability. Alignment is
achieved by translating the base of the PMT in the x- and y- axes, and through slight rotation of
the PMT about on its base in the y-axis.
The PMTs photocathode exhibits both spatial and angular variance in sensitivity [48]. By
ensuring that the spot at the photocathode is similar in size to the photocathodes effective area
(5 mm), and also that the beam is minimally divergent at that plane, the collection path will be
optimized for sensitivity. A Zemax simulation was used to find the position of L2 relative to the
photocathode that achieves this, while ensuring that no light is lost as the beam travels through
the 7.6 mm diameter aperture of the shutter (SM1SH1, Thorlabs). Models of the lenses used with
7.86 mm and 40 mm focal lengths from (F240FC-532, Thorlabs and AC240, Thorlabs, respectively) were imported into the simulation. The height of the source was set to the radius of the fiber
tether (0.1 mm), and the distance of the source to the first lens was determined by the model of the
collimation package. The distance between the two convex surfaces of the lenses was set to 250

33

mm. A 12.2 mm thick shutter is placed in front of the PMT, whose photocathode lies at a depth of
4 mm. The distance between the lens and the shutter was then varied to achieve a 3.5 mm spot at

Photocathode

the photocathode. The simulation of the collection path is depicted in Figure 3.15.

L3

L2

5.7

250

12.2
4

Figure 3.15: Schematic drawing of the coupling optics in the collection path (All dimensions are
in millimeters). The distance between the focusing lens and the shutter was varied in a Zemax
simulation to achieve a 3.6 mm diameter spot at the photocathode of the PMT.

3.3

Electronic Design

This section describes the design of the electrical modules of this fiber photometry system. The
signal conditioning circuitry is discussed in Subsection 3.3.1, and the software used to acquire data
in behavioural experiments presented in Subsection 3.3.2.

3.3.1

Signal Conditioning Circuitry

The output of the PMT is transformed by signal conditioning circuitry such that good use is made
of the data acquisition cards dynamic range, and that the measured signal contains limited noise.
Changes in fluorescence intensity due to GCaMP6s dynamics are very slow, and so a low-pass
filter is needed. This type of electronic filter allows signal components of low frequency to pass,

34

while attenuating those at high frequencies. The design of this circuitry involves shaping its frequency response through pass band and stop band definition, setting the gain, and selecting the
components.
The design began with defining the passband and stop band characteristics, as this determined
the circuits complexity. The Butterworth response was be used in this design as it provides the
most constant gain in the passband: the response is maximally flat. This is a common need in data
acquisition applications such as this one [49].
15 Hz was selected for the edge of the passband; components having a higher frequency than
this will be attenuated. As coupling from the power line is likely, components near 60 Hz must
be strongly attenuated. It was decided that components at 60 Hz must contribute no more than
0.1% to the detected signal. While a notch filter (or bandstop filter; this type of filter attenuates
signal components in a narrow band about some center frequency) could be used to attenuate this
component strongly, the Butterworth filter approach provides a solution with the same performance
but with less stages and components.
To achieve sufficient attenuation at 60 Hz, the filter must have a steep roll-off in the stop band.
This is the rate at which attenuation increases with frequency, and is determined solely by the
order of the filter. A first order Butterworth filter, for example, has a roll-off of 20 dB/decade.
Higher order filters of this type have roll-offs determined by 20n dB/decade, where n is the filter
order. Equation 3.24 gives the required order of a Butterworth filter to achieve 60 dB attenuation
at 60 Hz [49], .

n=

Ho 2
log ( 12 ( H(
) 1)
s)

2 log ps

(3.24)

Where is determined by 2 = 1 + A2MAX , AMAX = 0.5 dB is the maximum attenuation in

the passband, whose edge is defined by p = 2(15Hz). H(s ) = 1/AMIN , where AMIN is the
minimum attenuation in the stopband, whose edge is defined by s = 2(60Hz). Ho is normalized
to 1.

35

Substituting these values yields the required filter order of 4.44. Rounding up to the nearest
whole number, n = 5 was selected. A fifth-order, low pass filter was therefore designed. This was
realized with one first-order filter, in cascade with two second-order filter stages, as depicted in
Figure 3.16.

IPMT

T1 (TIA)

T2

T3

n=1

n=2

n=2

VOUT
Figure 3.16: Block-diagram of 5th -order low-pass Butterworth filter. The first stage was realized
with a band-limited transimpedance amplifier (TIA).

The design of the filter will now be presented, beginning first with Stage 1. Figure 3.17 depicts
a typical TIA circuit that allows the output current from a photomultiplier tube, IPMT , to be accurately measured. IPMT flows through the parallel combination of R f and C f , and is transformed to
a voltage VT IA . In this design, R f is first selected to set the gain of the filter, and then C f is selected
to set the pole of the filter and shape its frequency response.

Cf
Rf

IPMT
VTIA

PMT
-HV

Figure 3.17: TIA schematic used in this design

Because the charge carriers that generate IPMT are electrons, IPMT is negative. Its application
36

to the inverting terminal of the op amp causes the TIA output to be positive. The input impedance
looking into the input terminals of the op amp, ZIN,opamp , is very high (approximately 10 T). And,
because the PMT can be modelled here as an ideal current source with infinite output resistance,
IPMT is blocked from flowing into any circuit element other than the feedback resistor. However,
this requires that the input impedance of the opamp is much larger than the transimpedance gain.
From Ohms law, the mean value of VT IA is determined by:

VT IA, = IPMT R f

(3.25)

The measured signal value is proportional to the size of this feedback resistor. However, the
dominant source of noise in this circuit is the Johnson Noise caused by this feedback resistor.
Because the selected transimpedance gain is so large, all other sources of noise are negligible. As
p
Equation 3.26 reveals, the Johnson noise increases with R f [50].

VT IA, =

p
4kT BR f

(3.26)

Where k is Boltzmans constant: 1.38 10-23 J/K, T is Temperature (K), and B is Bandwidth (Hz).
The signal-to-noise ratio is then

VT IA,
VT IA,
IPMT R f
=p
4kT BR f
p
IPMT R f
=
4kT B

SNR =

From 3.27, SNR

(3.27)
(3.28)
(3.29)

R f , so the feedback resistance should be as large as possible to provide

the best signal-to-noise ratio. However, the condition

ZIN,opamp  R f

37

(3.30)

places an upper limit on R f . Assuming a FET opamp will be employed, ZIN,opamp will be on
the order of 1 T. A resistance of 22 M was selected for R f . This gives a signal value of 220
mV for an input of 10 nA, which is a measureable signal. For this pre-amplification stage, an op
amp with a rated bias current lower than 10 pA (this represents 1% of the total signal detected),
and rail-to-rail output was desired as the opamps output range can be more easily matched to that
of a data acquisition device. The OPA140 was selected as it offers rail-to-rail output, has a bias

current of 10 pA, and has low input current noise (0.8 fA/ Hz) for frequencies lower than 10 Hz,
and can work off a large range of power supplies.
The required feedback capacitor, C f , to realize the f3dB = 15 Hz is then determined through
3.32

Cf =

1
R f 2 f3dB

= 480pF

(3.31)
(3.32)

As signal to noise ratio is proportional to the current generated by the PMT, SNR can be increased by elevating the delivered excitation light (resulting in more detected photons). To avoid
saturating the transimpedance amplifier, a current source may be added to bleed off a constant
current of magnitude equal to the current resulting from the background.
Figure 3.18 presents the concept of using a transistor to effectively zero the background current
seen by the TIA. This enables the use of higher light power, or greater PMT gain, such that smaller
modulations may be quantified.
The transistor enables on the fly reconfiguration of the instrumentations dynamic range. This
is useful when the modulations are small compared to a large background of tissue fluorescence.
When the fiber optic implant is far from the population of cells that expresses the reporter, for
example, the signal may fall below the dynamic range of the instrumentation. Redefining the
dynamic range solves this problem. Of course, the use of increased excitation light has adverse
effects: on tissue, in the form of photobleaching and phototoxicity, and on the signal itself, as the
38

Vdd

Isignal
Isignal+Ibackground

Ibackground

Vgs

Ibackground

Isignal+Ibackground

(a)

Isignal
(b)

Figure 3.18: a) Concept of a background-zeroing current source to increase SNR on the fly with
increased delivered excitation light and b) implementation with an field-effect transistor (FET)
operating in subthreshold mode when Vgs <Vth .
sensitivity of the PMT decreases with light exposure, particularly beyond levels that result in 100
nA of current or more [48].
As depicted in Figure 3.18, the current source was realized with a FET. As the transistor will
generate at most 100 nA, it is operated in its subthreshold region. The AO3162 from Alpha &
Omega Semiconductor was selected for its high threshold voltage of 4V, enabling finer control
over its drain current, Id , by controlling Vgs , as related by

Id eq

Vgs Vth
nkT

(3.33)

where Vgs is the potential applied across the devices gate and source terminals, and Vth is the
devices threshold voltage, or the gate source potential at which it turns on. q, n, k, and T are
constants.
The design of stages 2 and 3, the two second-order stages in Figure 3.16 will now be described.
j)
The general transfer function, H( j) = VVoi (( j)
of one of these second-order circuits, representing

a single stage in the overall cascade design is given by [51]:

H( j) =

K
2
c2

j
+ Q
+1
c

(3.34)

Where j is the complex frequency, c describes the pole at 2 f3dB , and Q is the Pole quality
39

factor.
The Sallen-Key circuit is a second-order, active filter design capable of realizing low pass
electronic filters [52]. Its topology, configured as a low pass filter, is depicted in Figure 3.19. The
transfer function it realizes is given in Equation 3.35.

C2
R1

R2
C2
R4

Figure 3.19: Sallen-Key circuit configured as a low-pass filter

R3 +R4
R3



H( j) =
R4
2
(R1 R2C1C2 ) + j R1C1 + R2C1 + R1C2 R3 + 1

(3.35)

By comparing Equations 3.34 and 3.35 the following may be recognized:

R4
R3
1
o =
R1 R2C1C2

R1 R2C1C2
Q=
R1C1 + R2C1 + R1C2 (1 K)
K = 1+

(3.36)
(3.37)
(3.38)

By selecting resistance and capacitance values, Ri and C j , the DC gain, K, cut-off frequency,
o , and pole quality factor, Q, of an individual stage are defined. K2 = 3 and K3 = 1 were selected
for stages 2 and 3, respectively. The total gain of the signal conditioning circuit is now 66 M.
This selection of gain means that as the PMT is operated near the edge of its linear range (at 75
nA), an analog-to-digital converter with 5 V range will be close to saturating. The gain therefore
40

enables the dynamic range of the PMT to be matched to that of the converter. The following design
procedure was used to determine values for Ri and C j .
1. Assume R1 = mR and R2 = R
2. Assume C1 = C and C2 = nC
3. Select standard values for R and C
4. Solve for mn using Equation 3.37
5. Solve for m using Equation 3.38 given Q for that stage
6. Solve for n
7. Determine R1 , R2 , C1 , C2
8. Check availability of components for R1 and C2 , and readjust, if necessary.
This procedure generated the selection of components in Table 3.4.
Component

R1 ,

R2 ,

R3 ,

R4 ,

C1 , F

C1 , F

Stage 1

576 k

50 k

1k

2k

100 n

39 n

Stage 2

604 k

576 k

open

short

10 n

330 n

Table 3.4: Components selected to realize 2nd order stages in this design.

The frequency response of the designed circuit was validated through simulation, and Figure 3.20 characterizes the filters magnitude and frequency response. The f3dB frequency is at
14.83 Hz, compared to the designed 15 Hz.

3.3.2

Data Acquisition

The analog signal from Vout was digitized at 100 Hz by a data acquisition card (USB-6351, National
Instruments), and values spanning the range of - 1 V to 1 V were mapped to signed, 16-bit binary
41

-200

-500

10

Phase (degree)

Gain (dB)

200

-1000

10
Frequency (Hz)

Figure 3.20: Simulated frequency response of signal conditioning circuitry design, showing magnitude and phase characteristic.
values and saved in .bin files with user-defined filenames. Data acquisition control was provided
by custom programs written in C++ with the niDAQ-mx libraries from National Instruments. Prior
to data analysis, the binary values are converted to analog values with a custom Matlab script. The
incoming data is displayed in real-time, by tracing a sequence of average values obtained. This
was enabled through application of the open-source, low-level graphics library Allegro. Control
over the vertical scale of the display was achieved with keystrokes.

3.4

Implementation and Characterization

The optomechanics for the fiber photometry system were secured on a 5 x 15, 1/2-thick aluminum slab, and a light-tight box with removable top built around the frame. A slit for the optical
fibers (for both the adjustible light source and the tether) were cut in the box. The signal conditioning circuitry was implemented on a 1.25 x 0.75 printed circuit board (PCB). Using spacers, the
PCB was fixed inside an aluminum enclosure to shield it from electromagnetic coupling. Coaxial
cables provided the PCB inputs from the PMT and the controlling voltage for the bleed transistor
from the data acquisition card, and the PCB output from the TIA, Vout .
The construction of the enclosure for the optical system was tested for leakage due to ambient
light. Measurements were taken with the fiber tether removed from the system, the light source
42

off, and the system optics enclosed in the light-tight box. Measurements were taken both with and
without the shutter open. Table 3.5 shows that very little ambient light is leaking into the housing.
The leakage of ambient light consumes 0.18 % of the instrumentations dynamic range.
The systems light source and amplifier will first be characterized, followed by the
PMT shutter state
CLOSED
OPEN

textbfTIA output
0.7 mV
1.6 mV

Table 3.5: Protection from ambient room lighting provided by the enclosure for the optical system.
The systems ability to launch sufficient excitation light and its adjustibility was next tested. To
determine the light sources stability, the excitation light power was measured with a power meter
(S130C, Thorlabs) sampling at 0.1 Hz, at the output of the distal tip of the optical fiber tether. The
light source was stable to 0.2% its output. The delivered excitation power may be adjustible from
approximately 350 nW to 75 W. Figure 3.21 characterizes the adjustibility and stability of the
light source.
2

Light Source Output (uW)

Optical Power (uW)

10
0.5
0.4
0.3
0.2
0.1

10

10

-1

0
0

10
20
Time (minutes)

10

30

(a)

5
10
15
Potentiometer Turns

20

(b)

Figure 3.21: a) Stability of delivery path and light source.b) Range of output power enabled by the
light source.

The electronics were then characterized. The TIA and low pass filters frequency response
43

were investigated with a frequency-swept, modulated current source, and the output of the 5-th order
Butterworth filter was measured. The resulting magnitude response VItia
is depicted in Figure 3.22a.
in
The amplifiers noise spectrum, acquired with a spectrum analyzer (SR760, Stanford Research
Systems) is shown in Figure 3.22b. The noise spectrum reveals that the energy at 60 Hz to be
negligible, as designed.
-2

10
Magnitude (Vrms per rtHz)

160
150

Gain (dB)

140
130
120
110 -1
10

-4

10
-6

10
-8

10
-10

10
10
Frequency (Hz)

10

10

-2

10

(a)

10
10
Frequency (Hz)

10

(b)

Figure 3.22: a) Measured magnitude response of TIA, as measured at output of amplifier. b)

Measured noise spectrum of TIA

The TIAs corner frequency, f3dB was measured at 14.7 Hz, compared to 15 Hz for which
it was designed. Its gain differed from the designed value by 0.2 dB. The noise in a 0.1-10 Hz
bandwidth was found to be 8.26 V(RMS). Referring this quantity to the input, the circuit adds
noise equivalent to 125 fA (RMS).
The system was then characterized for its ability to detect changes that would mimic those
seen in vivo. In this context, the ability of a system to detect signals at a certain frequency in the
presence of noise is given by

CNR =

A f und,pkpk
noise

Where
44

(3.39)

A f und,pkpk is the peak-to-peak amplitude of the modulating sine wave, and

noise is the standard deviation of the noise.
A 505 nm LED was modulated with a 0.1 Hz sine wave superimposed on a background and
fed to the distal tip of the fiber tether, for relay to the optical system and PMT. The mean optical
power to deliver to the PMT was set such that the resulting signal value was identical to the autofluorescence produced from the fiber tether in response to Pex = 1 W. This was found to be 50
fW of fluorescence. Because autofluorescence is a background contribution that is, for the sake of
this discussion, a constant in all measurements, the average power collected from the LED was set
to 50 fW using neutral density filters.
In response to this input light, the gain of the PMT was adjusted such that the output of the TIA,
Vout , was 3.5 V, or close to saturating the amplifier (it was found that the electronic instrumentation had a functional output range of 4.7V). This condition provides the worst-case CNR. The
resulting signal was sampled at 100 samples per second, and the acquisition time was 10 seconds.
This experiment was repeated at successively lower amplitudes of the sine wave, until a signal
with a CNR 1 was measured. Measurements were performed both with and without the bleed
transistor active. Vgs of the bleed transistor was set such that twice the optical power could be
detected; it was generating approximately 53 nA. Increased light, for the case when the transistor
is ON, was then delivered by modifying the alignment between the LED and the optical fiber
tether. Signal-to-noise ratio, SNR, was computed using Matlabs snr() function, which performs
an approximation of

A2f und,rms
2
noise

. SNR was then converted to CNR using

A f und,pkpk
noise



A f und,rms 2
2
CNR = 2 2
noise
q

CNR = 2 2SNR
CNR =

The results are summarized in Table 3.6.

45

(3.40)
(3.41)
(3.42)

A f und,pkpk
CNR, Transistor OFF,
CNR, Transistor ON,

0.7 V
31.5
42.9

0.35 V
16.7
20.9

0.175 V
8.43
10.68

0.07 V
3.16
4.13

0.035 V
1.66
2.09

0.018 V
1.28

Table 3.6: Resulting CNR, where A is the peak to peak amplitude of the modulated sine wave.
Because we are limited only by shot noise, the bleed transistor enables measurements at a
higher CNR. However, the bleed transistor is adding noise, as depicted in Figure 3.23
0

0.2

Magnitude (dB)

Amplifier Output (V)

0.4

-50

-100

-0.2
-0.4
0

without transistor
with transistor
20
40
60

80

-150 -2
10

100

Time (s)

without transistor
with transistor
0

10
Frequency (Hz)

(a)

(b)

Figure 3.23: Investigation of the systems ability to detect small changes. a) Use of the bleed
transistor allows for optical measurements to be made with increased CNR by allowing for greater
optical power to be used. However, large modulations near DC are added, resulting in a slow drift
over time. b) Frequency domain representation of the traces in a) shows the low-frequency noise
added by the transistor.

3.5

In vivo Application

The CRH cells of the PVN of the hypothalamus are known to play a role in the neuroendocrine
response of the body to stress, acting on the order of minutes [53]. However, recent studies have
found evidence that these cells also play a role in mediating complex, rapid behavioural responses
following exposure to stress [54]. As fiber photometry can yield insight into the activity of these
cells, as reported by the intracellular calcium dynamics, with adequate temporal resolution, this
method is therefore applicable to study the behavioural correlates of CRH neuron activity.

46

3.5.1

Methods

The photometry system was applied to study behaviour in a freely-moving animal in response
to conditioned stress. The conditioned stress, in this context, is the brains response to a previous
training period, in which an innocuous stimulus was paired with a stressful unconditioned stimulus.
The result of this training period is a change in behaviour in response to the conditioned stimulus,
without the presence of the stressor [55].
The pairing of conditioned and unconditioned stimuli enable investigation into the formation
of emotional memories. This protocol is applicable here to help understand how the brain, and
in this context how CRH neurons, react to a conditioned stress. The conditioned stimulus was
the visual and environmental cues presented by a shockcage, a cage where the animal can be
electrically shocked, and the unconditioned stimulus is a series of footshocks, the application of
such an electrical shock delivered in said cage. After several pairings of these two stimuli, it is
expected that upon reintroduction to the shockcage, the animal will exhibit changes in behaviour
due to memory of stress.
Stress responses, measured in CRH-Cre mice transfected with either GCaMP6s (experimental)
or Enhanced Yellow Fluorescent Protein, (EYFP, control) through viral injection, were implanted
with fiberoptic cannula implants (MFC-200/230-0.48-5mm-MF2.5-FLT, Doric Lenses). As a targeting procedure that exploits fluorescence was used to guide surgery, using a control group that
expresses a fluorophore was required.

47

Day 0
Implantation

Days 1-9
Recovery,
Handling, and
Habituation

Day 10
Stress
Conditioning

Day 11
Re-exposure
to conditioned
stress

Figure 3.24: Investigating the response of CRH cells of the hypothalamic PVN to conditioned
stress.

The procedure used to investigate the neural correlates of conditioned stress is depicted in Figure 3.24. The animal was implanted with the probe on day zero, and then 9 days were allowed for
recovery, handling, and habituating. On day ten, the animal was subjected to a series of footshocks
((0.5 mA for 2 s, ten times in 5 min) [54]) delivered in the shockcage. On day eleven, fiber photometry measurements were taken, to see fluorescence intensity changes in response to reintroduction
to the environment in which the footshocks were given (the shockcage).
The photometry recording consisted of a 3-minute baseline recording, a 5-minute exposure to
stress, and concluded by a 3-minute post recording. Animals were moved from their home cage
to the environment in which the stress was conditioned at t = 3 minutes. Because the animal had
previously been subjected to a stress in the environment, upon reintroduction to that environment,
it is expected that the animal experience the memory of the stress. It is therefore also expected
that this memory will be associated with increased CRH neuron activity in the form of increased
intracellular calcium concentration in those cells. This will be seen as increased fluorescence
intensity in GCaMP6s animals, but no change in fluorescence detected in EYFP animals.
The changes in fluorescence intensity were quantified using a measure of contrast to noise
ratio, defined as the ratio of the difference in mean signal values between two time periods, to the

48

quadrature-averaged standard deviation of the traces from the two time periods.
2 1
CNR = q
1
2
2
2 1 + 2

(3.43)

Where 1 and 1 are the mean and standard deviation of the fluorescence signal during the first
time period, and 2 and 2 are the mean and standard deviation of the fluorescence signal during a
second time period. In this research, the first time period is the baseline recording, and the second
time period is that during exposure to conditioned stress.
Possible tissue damage due to light was quantified by determining the photobleaching characteristic in EYFP mice. The photobleaching characteristic describes how the fluorescence intensity
decreases over time. During the design of the photometry system, it was determined that photobleaching occurs at light levels above 1 W for GCaMP6s, whereas photodamage, wherein cells
are destroyed by the light, occurs at much higher intensities.
As photobleaching is expected to be minimal, it was quantified by fitting a line of the form
y = mt + b to the fluorescence trace using Matlabs Curve Fitting Toolbox, where y is the transimpedance amplifier output (measured in Volts), m and b are measured in V/s and V, respectively,
and t is time. m is the rate of bleaching.

49

3.5.2

Results

6%

2.5%

4%
F/F (%)

F/F (%)

3%

GCaMP6s
EYFP

2%
0%

1.5%
1%
0.5%

-2%
0

2%

5
Time (minutes)

10

(a)

Day 1 Day 2 Day 3

(b)

Figure 3.25: a) Traces of detected fluorescence intensity from CRH-Cre mice expressing either
GCaMP6s (green) or EYFP (orange). Red lines denote the transfer to/from the shockcage. b)
The response to conditioned stress was stable as measured by repeating the experiment on three
consecutive days.

Figure 3.25 depicts a representative trace from an animal expressing the GCaMP6s reporter and
the EYFP indicator. Reintroducing the animal to the shock cage, and hence providing the environmental cues that collectively serve as the conditioned stimulus, resulted in a 2.55 % increase in
detected fluorescence in the GCaMP6s animal, but no increase in the EYFP animal. Figure 3.25b
shows that subsequent re-exposure to the conditioned stress produced similar F/Fo on successive days. Importantly, no photobleaching effects were noticed, corroborating the evidence from
literature that Pex = 1 W eliminates this effect.

50

Signal Parameter

GCaMP 1

EYFP 1

EYFP 2

EYFP 3

EYFP 4

2 1
q
1
2
2
2 1 + 2

0.05

-0.020

-0.045

-0.036

-0.0004

0.017

0.013

0.030

0.021

0.010

CNR

2.968

-1.598

-1.518

-1.734

-0.0353

Table 3.7: Detecting changes in fluorescence intensity from CRH cells in freely-moving mice in
response to stress.

Table 3.7 depicts the results for monitoring changes in fluorescence of CRH cells due to conditioned stress. A measureable signal was produced only in the GCaMP mouse, as it produced the

Amplifier Output (V)

only trace with CNR >1.

Room in darkness
With room lighting

1.9
1.85
1.8
1.75
1.7
0

20

40

60
80
Time (seconds)

100

120

Figure 3.26: Recordings under ambient lighting resulted in a 90.3% increase in noise power compared to the recording in darkness. Sensitivity is reduced further from the induced offset.

Figure 3.26 depicts baseline recordings performed under ambient fluorescent lighting and darkness, in the same animal. This reveals that the system is sensitive to ambient lighting, reducing
the systems sensitivity to changes in calcium-mediated changes in fluorescence by increasing the
background, which results in a direct increase in shot noise. It was found that photons from ambient lighting are penetrating the skull and brain tissue, collected by the fiber probe and relayed to
the optical system with the reporter fluorescence.
51

TIA Output (V)

2.5
y = - 3.9e-05*x + 2.6
2

1.5
0

100

200

300
400
Time (s)

500

600

Figure 3.27: Representative fluorescence trace from an EYFP mouse and line of best fit, showing
little photobleaching.

Figure 3.27 depicts a representative trace from an EYFP mouse, showing little photobleaching,
as the fluorescence intensity dims as approximately 39 V s1 . This translates to a loss of approximately 1.5% of fluorescence intensity in a typical fifteen-minute experiment. Table 3.8 presents the
photobleaching rates observed for the control population, showing similar, and negligible, bleaching characteristics.
#

Photobleaching rate, m (V/s)

#1

-0.0019

#2

-0.000039

#3

-0.0000081

#4

-0.000048

Table 3.8: in vivo Photobleaching rates for control animals.

52

3.6

Discussion
Parameter

Design Constraint

Measured Specification

Probe diameter

<300 m

240 m

Light source output

N/A

Adjustible; 0.35 W - 75 W (450 nm - 490 nm)

Emission band

N/A

520 - 550 nm

Offset from light leak

0.1% of DR

2% of DR

CNR, F/F = 0.5%

1.28 (with bleed transistor)

TIA Bandwidth

14.7 Hz

TIA Noise

125 fA rms

TIA Output Range

-4.72 V to 4.65 V

Power Supply

-5 V to 5 V

Table 3.9: Summary of fiber photometry system optical and electrical characteristics.

Through the design of the fiber photometry system, described in this chapter, Specific Aims 1-3
of this research were met. The system is highly sensitive, enabling experiments at low-levels of
excitation light. Signals can be detected with high fidelity without risking the negative effects
of photobleaching or phototoxicity. Indeed, the traces obtained did not show any evidence of
photobleaching. Damage to the intact brain is mitigated by using a small-diameter optical probe.
Characterizing the optical properties of GCaMP6s in situ were necessary to accomplish these goals.
While the system met the design constraints, the instrumentation could be made more sensitive by reducing the bandwidth of the TIA and by assigning the gains of the stages in the Butterworth filter differently to maximize its output characteristic, and hence the instrumentations
dynamic range. Even though the bleed transistor enables measurements at increased CNR, the
low-frequency noise it adds is undesirable, and will impede the ability to measure small signals.
The systems application in vivo to study the effects of a conditioned stress resulted in the
detection of modulations in fluorescence intensity for only the GCaMP6s animal. It is therefore
53

likely that the action of GCaMP6s produced these modulations, although a sustained decrease in
blood flow to the area would have produced similar results. The potential effect of neurovascular
artifacts on the signal trace are investigated in Chapter 4.
If this response is indeed due to calcium dynamics in CRH cells, then it would add evidence to
the hypothesis that CRH cells are not only involved in slow, neuroendocrine mediations, but that
their activity is also strongly correlated with behaviour due to remembered stress, as Figure 3.25a
shows that the increase in fluorescence intensity occurs immediately upon reintroduction to the
shock cage, and an decrease in that same intensity is seen immediately upon removal.
Use of the system in typical protocols revealed that the ambient lighting is an artifact in the
trace. Its presence increases the noise floor and makes it difficult to interpret the results of an
experiment, possibly restricting the type of experiments that can be performed. Chapter 4 will
present the design of a refined system with improved immunity to this artifact, and the investigation
into other possible sources of signal-disruption.

54

Chapter 4
FIBER PHOTOMETRY SYSTEM REFINEMENT AND
EXPERIMENTAL APPLICATION IN AN ANIMAL MODEL
In Chapter 3 we presented the design, characterization, and experimental application of a benchtop
optoelectronic system for fiber photometry in freely-moving mice. The system was designed to
maximize sensitivity to changes in GCaMP6s fluorescence such that minimal optical power could
be used, and photobleaching would therefore be avoided. We found that when the animal was
reintroduced to a conditioned stress, an optical fiber implanted near its PVN collected increased
levels of fluorescence. The room lighting, however, causes an artifact.
This chapter describes the refinements to the optoelectronic system presented in Chapter 3
required to eliminate or correct for the artifacts discovered through the application of the first prototype in standard behavioural protocols. A modification to the instrumentation and experimental
protocol to provide immunity to ambient lighting is presented in Section 4.1. The systems performance when applied in vivo and an investigation into whether neurovascular or hemodynamic
coupling is distorting the detected trace is presented in Section 4.2. Section 4.3 compares the two
system designs and considers areas of further improvement and investigation.

4.1

Lock-In Amplifier Design

A lock-in amplifier (LIA) is a concept borrowed from communication theory to perform low-level
measurements in optics. It works by exciting the sample with a modulated light source (the
carrier) such that the optical signal that has interacted with the sample is correlated with the
source at the photodetector. Because the carrier is known, the signal of interest (the baseband)
can be recovered through demodulation. The measurement is taken with a limited bandwidth,

55

lowering the noise floor. Importantly, any extraneous contributions that are not correlated with the
source will be ignored. This last property of lock-in amplifiers motivates its application to provide
immunity to ambient lighting for fiber photometry. The lock-in amplifier is commonly applied in
fiber photometry systems for behavioural applications [40], [8].
This section describes how the fiber photometry system presented in Chapter 3 was adapted to
a lock-in amplifier configuration to provide immunity to ambient lighting from the environment.
Henceforth, the optoelectronic system designed in Chapter 3 will be referred to as the DC configuration, and the design in this chapter will be referred to as the LIA configuration. The design
criteria that must be met are presented in Table 4.1. Because the DC system was designed to
reliably detect changes larger than 1%, we decided the LIA must reject >99% of ambient light.
The primary motivation for pursuing this design is to provide signal immunity to ambient light, an
artifact that appears due to photons due to room lighting penetrating the skull, tissue, and being
collected by the optical fiber probe. However, the design should not result in compromised performance from the DC system. The photodetector, light source, and data acquisition software are the
same as that described in Chapter 3.
Design Criteria
Ambient Light Rejected
CNR
Probe diameter

Conditions
at 515 nm
F/F = 1%; Pex =1 W
Implanted

Specification
>99%
>1
300 m

Table 4.1: Design criteria for Lock-in Amplifier

56

4.1.1

Block Diagram
VTIA

photodetector

VMULT

VLIA
Low
Pass
Filter

TIA

modulated
optical signal

DAQ to
Computer

demodulation
VMOD

sample

modulated
light source

LED Driver
modulation

Figure 4.1: Lock-in amplifiers use coherent detection to enable low-level optical measurements.
The numbers at each of the points in the signal path refer to example frequency-domain represen-

20

-20

-20

-40
-60
-80
-100
0

Signal Value (dB)

Signal Value (dB)

Signal Value (V)

tations of signals in Figure 4.2

-20
-40
-60

100
200
300
Frequency (Hz)

400

-80
0

-40
-60
-80

100
200
300
Frequency (Hz)

(a)

(b)

400

-100
0

100
200
300
Frequency (Hz)

400

(c)

Figure 4.2: Frequency domain representations of the signal at points a) 2, b) 3, and c) 4 in the
lock-in amplifier circuit depicted in Figure 4.1

Figure 4.1 shows the signal path for an optical system for fluorescence that employs a lock-in
amplifier, similar to that employed in Stanford Research Systems analog instruments [56]. A light
57

source driven with some modulation delivers excitation light to the sample, whose fluorescence is
detected by a photodetector. In this example, the modulation frequency of the light source, and
hence the collected fluorescence, is 200 Hz. The electronic signal transduced by the photodetector,
represented in Figure 4.2a, contains the signal of interest at 200 Hz. Harmonics from the power line
frequency at 60 Hz and its multiples are also present, as well as random noise. This signal is then
multiplied with the reference signal that modulated the light source. As depicted in Figure 4.2b, this
multiplication results in a signal for which each of its components have been shifted by the carrier
frequency fc ; the baseband, true fluorescence signal is reflected to DC. The large component
at 200 Hz represents 1/ f noise that is strongest at frequencies close to DC. A low-pass amplifier
attenuates these and other high-frequency components, and the signal in Figure 4.2c is then relayed
for data acquisition. While other configurations for lock-in amplifiers have been employed by
others [8], [40], the instrument designed for this thesis will be based off of this topology.
The design of the lock-in amplifier will be described in two parts: The design decisions related
to modulation will first be presented in Subsection 4.1.2, and then the demodulation scheme will
be described in Subsection 4.1.3.

4.1.2

Modulation

A lock-in amplifier provides immunity to uncorrelated noise sources by modulating the excitation
of an experiment at some carrier frequency, fc . Similar to the DC system designed in Chapter 3,
however, measurements from the lock-in amplifier configuration are also taken at DC, due to the
multiplication of the result of the experiment with a reference signal having the same frequency
and phase as the source in a process called demodulation.
Ideally, the output of the lock-in amplifier would represent solely the result of the experiment,
and extraneous contributions from the environment would be made negligible. The amplifiers
ability to do this depends on the nature of the modulation, and its relation to environmental components in the frequency domain. Assuming that the reference is in phase with the signal, the result

58

of the demodulation, VMULT is determined by the product of the reference and the signal:
VMULT (t) = (vtia sint +VT IA )(vmod sint +VMOD )

(4.1)

= vtia vmod sin(t)sin(t) + (vtiaVMOD +VT IA vmod )sint +VT IAVMOD

(4.2)

vtia vmod
[1 cos2t] + (vtiaVMOD +VT IA vmod )sint +VT IAVMOD
2

(4.3)

Where vtia sint +VT IA is the signal from the photodetector, transformed to a voltage, representing
VT IA in Figure 4.1 having some amplitude vtia and offset VT IA . Because detected light cannot be
negative, VT IA the average light detected, must always be greater than vtia , the modulation about
that mean:

VT IA vtia

(4.4)

vmod sint +VMOD describes the reference, VMOD (t) in Figure 4.1, with its own amplitude vmod
and offset VMOD .
VLIA (t) is then what remains of VMULT (t) after removing the AC components with a low-pass
filter, and follows from Equation 4.3. VLIA (t) is relayed for data acquisition.
VLIA (t) =
vtia vmod
2

vtia vmod
+VT IAVMOD
2

(4.5)

represents the fluorescence due to modulation, and VT IAVMOD is the fluorescence due to the

dc component of excitation light. VT IAVMOD is an undesirable term that carries the uncorrelated
sources of noise through to the lock-in amplifiers output, including ambient light artifacts. To
eliminate it, the reference signal can be modulated with zero mean, for VMOD = 0. The instantaneous value of VLIA is then determined solely by vtia and vmod .
From Figure 4.1, VMOD (t) = vmod sint acts as the reference signal during both modulation
and demodulation. The schematic of the light source that realizes this concept, wherein an LED
is employed again, is shown in Figure 4.3. An LED is a suitable choice in this context because
its light output is linear with the current that flows from its anode to its cathode. By operating
off a bi-polar supply, the modulation can take on both positive and negative values, all the while
59

ensuring the current through the LED is always positive. It was decided that the LED driver will
operate off of 15 V.

e
VMOD
-e

RLED

LED
-15V = -e

Figure 4.3: LED driver circuit for the modulated light source in LIA design. To maximize vmod ,
the amplitude of VMOD , this design operates off of e = 15 V.
As VMOD has zero mean, the average current through LED, ILED,AV G , is then given by

ILED,AV G =

e
RLED

(4.6)

Where e is the 15 V supply, and RL ED is the current setting resistor shown in Figure 4.3. It
was found that ILED,AV G = 150 A launches 1 W into the optical fiber tether (as measured at the
output of the distal fiber tip) and so RLED was set to 10 k.
This configuration enables flexibility over the selected modulation frequency. Four guiding
principles may be drawn will help select this parameter:
1. The modulation frequency should be less than one tenth the bandwidth of the transimpedance
amplifier.
2. The frequency should be far away from power-line harmonics.
3. The sampling frequency at which data is acquired should not be a multiple of the modulation
frequency. [57].

60

4. Higher frequencies are preferred as the strength of both 1/ f noise and power-line harmonics
fall off with frequency.
Assuming the bandwidth of the TIA is 10 kHz, the highest permissible modulation frequency
is 1 kHz. As power line harmonics occur at 60n Hz, item 2 dictates that the selected frequency be
near 60n + 30 Hz. Anticipated sampling frequencies are likely multiples of 10 or 100 samples per
second. Some possible modulation frequencies that satisfy these conditions are 209 Hz and 571
Hz.
To enable the highest possible modulation frequency, the operational amplifier must have an
adequate Slew Rate. This is the maximum possible rate of change of the op amp output, expressed
in V s1 . If this specification is inadequate for the intended application, the output will be distorted. The required Slew Rate of an the op amp is determined by

Slew Rate = 2 fcVpeak

(4.7)

Where fc , is the expected modulation frequency, in Hz and Vpeak is the amplitude of the modulation signal, which is also its peak value . Which results in a requirement of 0.1 V s1 for
Vpeak = 15 V (the modulated waveform cannot exceed the power supply rail of the LED driver
circuit) and fc = 1 kHz. The op amp in the driver should furthermore have rail-to-rail output. This
is because the LED requires a higher potential at its anode (at the output of the op amp) than at
this cathode (at the inverting input terminal of the of amp), when it is on. This potential, VLED ,
is usually 2 to 3 V, which is often the required headroom on op amps that are not specified as
rail-to-rail. By ensuring the op amp can operate to each of the supply rails, this constraint will
not impose a limit on the modulation amplitude. To mitigate the appearance of switching noise
in the modulation, the driver is operated off of batteries. The op amp should therefore have low
quiescent current such that the battery capacity can be used efficiently. An upper limit of 0.5 mA
was selected for this purpose. The OPA 171 from Texas Instruments exceeds the design constraints
as summarized in Table 4.2
61

Parameter
Quiescent Current
Output Range
SLEW RATE

Design Constraint
0.5 mA
Rail-to-Rail
0.1 V s1

OPA171 Specification
0.475 mA
Rail-to-Rail
1.5 V s1

Table 4.2: The OPA171 is a suitable choice for use in the LED driver circuit.
With this choice, the light source can be modulated with arbitary c e VLED , where VLED 3
V [58].
In this context, the modulation index will be defined as the ratio of the amplitude of modulated
light to the average light delivered:

IMOD =

c
e

(4.8)

The output of the LIA is determined solely by the modulated light; the mean delivered light
power has no impact on the signal acquired and is essentially a waste in the signal. Imod should
therefore be as large as possible such that the excitation light is converted efficiently to the signal
that will be acquired. We found that this was limited only by the output range of the function
generator: The data acquisition card (USB-6361, National Instruments) running a custom c++
program has an output range of 10 V. A conservative amplitude of 7.5 V was selected.

Magnitude (dBc)

1142 Hz;
-33.93 dBc

-10
-20

2284 Hz;
-38.95 dBc

-30
-40
-50 2
10

10
Frequency (Hz)

Figure 4.4: Spectral analysis of the modulation scheme at 571 Hz

The design of the modulation scheme was tested by coupling the light source to a wide62

bandwidth photodiode (PDA36A, Thorlabs) and measuring the photodiodes output with a spectrum analyzer (SR760, Stanford Research Systems). Figure 4.4 depicts representative results of a
spectral analysis of the output. Because the modulation scheme introduces minimal distortion in
the light source, fluorescence may be detected efficiently after demodulation.

4.1.3

Demodulation

In a lock-in amplifier configuration, the PMT outputs a modulated current signal on the order of
a few tens of nanoamps, and contains both AC and DC components. This section describes the
circuitry that recovers the baseband signal by first converting the PMT output current signal to a
voltage signal, multiplying it by the reference, and finally removing high-frequency components
and noise with a low-pass filter. The schematic of the lock-in amplifier demodulation subcircuit is
presented in Figure 4.5.

i(t)PMT

v(t)LIA
v(t)TIA

v(t)MULT
v(t)MOD

-HV

Figure 4.5: The demodulation circuit consists of a wideband transimpedance amplifier, a multiplication stage, and a low-pass filter.
As established in Chapter 3, the SNR of the output of a TIA increases with the size of the
feedback resistance, R f . A resistance of 100 M was selected for this application. This will
produce VT IA = 10 V for an input current of IPMT = 100 nA, which uses the TIAs output range
efficiently, and is also at the limit of the PMTs linear range.
In Chapter 3, the model for an ideal transimpedance amplifier for a PMT was given. Because
this is a wideband application, however, a more nuanced model that takes into account the nonidealities of the photodetector is required. Figure 4.6 depicts the transimpedance amplifier circuit
63

model, where the photodetector has been replaced with an ideal current source, in parallel with
its finite and non-zero equivalent resistance and capacitance. This design will also account for the
gain-bandwidth product of the opamp to be employed.

Zf

Cf
Rf

i(t)PMT

Zi

v(t)TIA
Ri

Ci
-HV

Figure 4.6: The transimpedance amplifier design for this wideband application must take into
account finite input resistance, and input capacitances at the opamp terminals. Zi and Z f denote
the parallel combinations that determine the frequency-dependent input and feedback impedances.
The transfer function of the TIA depicted in Figure 4.6 may be described by

vT IA (s)
iPMT (s)
Z f (s)
=
1
1 + Ao (s)
(s)

Tz (s) =

(4.9)
(4.10)

Where
Z f (s) is the impedance of the feedback network Z f (s) =

Rf
1+sR f C f

(s) is the voltage feedback fraction at the inverting input, (s) =

Ao (s) is the op amp open-loop gain function Ao (s) =

Aoo
s
1+ GBW

Zi
Zi +Z f

where Aoo is the opamps open-loop

gain and GBW is its gain-bandwidth product.

The Ao (s) (s) term is referred to as the loop gain, L(s), of the closed-loop. The manner in
which L(s) varies with frequency (in both magnitude and phase) determines the stability of the
64

circuit. When its phase is 180, the feedback will be positive as this negative signal is applied to
the inverting terminal of the opamp. If at this frequency the magnitude is also equal to unity, the
denominator of Tz (s) will be zero and the circuit will be unstable: it will oscillate at this frequency.
The phase of L(s) when |L(s)| = 1 is therefore of interest. This occurs when

|Ao (s) (s)| = 1

|Ao (s)| =

gain

(4.11)

1
| (s)|

(4.12)

unstable configuration

Ao(s)
Zf(s)
stable configuration

1/ (s)
fz

fp

fo

fc

Figure 4.7: Bode plot showing the the open-loop gain magnitude curve of the op amp, Ao (s); the
reciprocal of feedback fraction, 1/ (s); and the gain due to the feedback network Z f (s). Z f (s)
achieves circuit stability by cancelling out the zero due to 1/ (s).
Figure 4.7 depicts a simplification of how the gain magnitude curves for the opamp openloop gain (described by fo ), the feedback network (characterized by f p ), and the noise gain,

(determined by fz ) behave in the frequency domain. Because poles and zeros are each associated
with 90 phase shifts (in opposite directions), if the relative rate of approach is -40 dB/decade at the
intersection point, the circuit will be unstable as this means that the phase of L(s) will be -180 at
this point. The frequency of intersection, fc , is determined by the relative locations of the feedback

65

pole, f p , the noise gain zero fz [59], and the gain bandwidth product (GBW ) of the opamp:
"
fc4 + fc2 fz2

BW
1
fp

2 #

(BW fz )2 = 0

(4.13)

Where
fp =
fz =

1
2R f C f

(4.14)
1

(4.15)

R R

f i
2 R f +R
(Ci +C f )
i

BW = GBW

Ri
R f + Ri

(4.16)
(4.17)

Ci is the sum of the equivalent circuit capacitances of the photomultiplier tube (approximately
20 pF [60], [48]) and the common-mode and differential input of the opamp (7 and 10 pF, for the
OPA140 respectively, [61]) for a total of C f = 37 pF. Ri , the equivalent resistance of the PMT is 1
T. C f is then the sum of the parasitic capacitance due to non-idealities in R f and traces on the
PCB for a total of 0.15 pF [62]. Solving Equation 4.21 then yields the fc = 45.6 kHz. Ao ( fc ) (s).
The phase of the loop gain is then given by [59].

"
1+
GBW Ri
Ao (s) (s) = 6
j f c Ri + R f 1 +

j fc
fp
j fp
j fz

#
(4.18)

Which yields a phase margin of 76 when the OPA140 is employed, which has a nominal
GBW of 11 MHz. As a phase margin greater than 45is recommended for circuit stability [62],
this design is already overcompensated and it is not expected to oscillate. The OPA140 is therefore
appropriate for this wideband, high gain application.
The f3dB bandwidth of the amplifier, that will inform the decision on modulation frequency,
is then given by [59]:

66

s
f3dB =

GBW
2R f (Ci +C f )

s
1+


1 p
1 4Q2 + 8Q4 1
2Q

where

(4.19)
(4.20)

Q=

GBW
fz
1 + GBW
Fp

(4.21)

Which yields a bandwidth of 13.5 kHz.

This design was then simulated using Altium Designer software. Figure 4.8 depicts the magnitude and phase responses. The results are in good agreement with theoretical analysis, with f3db
= 14.93 kHz.
0

-200

-200

10

Phase (degree)

Gain (dB)

200

-400

10
Frequency (Hz)

Figure 4.8: Simulated Magnitude and Phase response of TIA in demodulation subcircuit. f3dB is
predicted to be 14.93 kHz with Ci = 20 pF, C f = .15 pF, RF = 100 M and Ri = 1 T, and GBW =
4 MHz (opamp used for simulation was LF411CN.)

The output of the transimpedance amplifier, VT IA , is then multiplied with a reference signal
having the same frequency and phase as the source. In this design, this signal is VMOD , the input
to the LED driver circuit described in Subsection 4.1.2. This process is realized with a multiplier
integrated circuit (AD734, Analog Devices), connected to realize the transfer function given in
Equation 4.22, where W is the output of the multiplier, and X and Y are the inputs to undergo
multiplication (VT IA and VMOD ). The scaling factor of 10 in the denominator was selected as it

67

offers the lowest noise [63].

W=

XY
10

(4.22)

The limiting factor in the LIAs output range is the input to the multiplier. When working off
the 15 V supply, its input common mode range is 12.5 V, meaning that a maximum of 125 nA
may be converted to a voltage by the TIA with the gain of 100 as designed. This is above the linear
range of the PMT, above which its sensitivity decreases with output current. Whereas this may
be acceptable in the DC configuration, in the LIA configuration, this non-linearity results in major
distortion of the modulated current sine wave prior to multiplication with the reference, resulting
in a decrease in VLIA for an increase in iPMT . It is therefore imperative that the PMT be operated
in its linear range. This bars the possibility of combining the bleed transistor employed in the DC
system design with this LIA design.
After multiplication, the components nearest to DC in the frequency domain are located at 29
Hz and 31 Hz (These are components from the power line harmonics reflected from 600 Hz and
540 Hz, respectively), and were found to be present with a magnitude of approximately -30 dB
from the magnitude at DC. Applying again Equation 3.24, from Chapter 3, the required filter order
for a pass band edge of 10 Hz such that these components are attenuated to -60 dB was found to
be n = 4.
As in Chapter 3, Sallen-Key circuits will again be applied to realize the low pass active filter.
This time, however, the topology depicted in Figure 4.9 will be used, for which the transfer function
is given in Equation 4.24. This circuit realizes a DC gain of zero dB, which minimizes the number
of components on the board.

H( j) =

H( j) =

2 (R

1
2
c2

j
+1
+ Q
c

1
1 R2C1C2 ) + j(R1C1 + R2C1 ) + 1
68

(4.23)

(4.24)

C2
v(t)MULT R1

R2
v(t)LIA

C2

Figure 4.9: Minimal component Sallen-Key circuit configured as a low-pass filter with gain = 1
By comparing Equations 4.23 and 4.24, and assuming R1 = mR, R2 = R, C1 = C, and C2 = nC
1

RC mn

mn
Q=
1+m

o =

(4.25)
(4.26)

The correct values of Q in the two second-order stages to realize a 4th -order Butterworth low
pass filter were obtained from filter tables [49] (0.55 and 1.31 for Stages 1 and 2, respectively).
In order to realize o = 2 f3dB (for 10 Hz) and Qdesigned , resistance and capacitance values for
components Ri and C j were then selected following this design procedure [51]:
1. Select a standard value for C
4. Select m and n such that Qactual Qdesigned
5. Solve for m using Equation 4.26 given Q for that stage
6. Solve for R using Equation 4.25
7. Determine R1 , R2 , C1 , C2
8. Check availability of components, and readjust, if necessary.
This procedure generated the selection of components in 4.3.
69

Component

R1 ,

R2 ,

C1 , F

C1 , F

Stage 1 (Q = 0.55)

270 k

220 k

100 n

390 n

Stage 2 (Q = 1.31)

57.6 k

57.6 k

100 n

680 n

Table 4.3: Components selected to realize 2nd order stages in this design.

Using spacers, the PCB was mounted inside a 1.625 x 1.5 x 1.5 enclosure. BNC connecters
were secured for the LIA inputs (v(t)MOD and i(t)PMT ) and output (v(t)LIA ) A custom c++ program
running a data acquisition card (USB-6361, National Instruments) supplies v(t)MOD and saves
samples of v(t)LIA to .bin files. As in Chapter 3, these files are read by custom Matlab scripts.
The modulation and demodulation circuits were fabricated on the same PCB. 15 V was supplied by bipolar, low-dropout (LDO) regulators (MC7815 (+15 V) and MC7915 (-15 V), ON
Semiconductor) each in turn supplied by 3- 9 V batteries connected in series. The decision to operate off batteries mitigated the risk of coupling switching noise and/or power line harmonics into
the measurement that might otherwise be the case with a wall adapter.

4.1.4

Characterization

The primary motivation for redesigning the fiber photometry system described in Chapter 3 was
to eliminate the contribution of ambient lighting to the measurement. To investigate the new instrumentation designs ability to reject ambient light, the PMT was exposed to autofluorescence
from the tether (FAF ), and autofluorescence plus light emitted by a green LED positioned near the
tethers distal tip (FAF + L). Table 4.4 presents the results of this comparison. Signal-to-noise ratio
was calculated as

SNR =

(4.27)

The LIAs ability to reject light, RLIA , as compared to the DC system was then determined. This
was quantified by the difference in mean () signal values at the output of both systems (DC and
70

Signal
no input (PMT
is shuttered)
FAF
FAF + L

DC
-1.5 mV

DC
-0.33 mV

SNRDC

LIA
-0.23 mV

LIA
0.19 mV

SNRLIA

21.3 mV
961 mV

11 mV
82 mV*

6.33 dB
-11.3 dB

12.3 mV
11.9 mV

0.38 mV
0.70 mV

31.6 dB
24.8 dB*

Table 4.4: Comparison of systems sensitivity to ambient light, with measurements due to fluorescence (FAF ) and fluorescence plus light from an incoherent source (FAF + L). / / SNR Signal
mean/ standard deviation/ signal-to-noise ratio (SNR = ). A 515/30 nm filter was used to set the
emission band for the PMT in all cases. The LIA design exhibits improved signal-to-noise ratio
over the system designed in Chapter 3, and is able to detect signals in the presence of incoherent
sources (SNRLIA 1) where the DC system cannot (SNRDC 1). *SNR was calculated using the
mean of the measurement with fluorescence FAF , and the standard deviation of the measurement
with fluorescence and light, FAF+L .
LIA), with (FAF+L ) and without (FAF ) the PMT collecting light from the incoherent LED source,
as a fraction of the difference in mean signal values incurred on the DC system:

RLIA =

|FAF+L,DC FAF,DC | |FAF+L,LIA FAF,LIA |

FAF+L,DC FAF,DC

(4.28)

Equation 4.28 yields RLIA = 99.96% with the values presented in Table 4.4. When the uncorrelated source (the LED at the distal tip, in this case) is introduced, VLIA decreases, as does its SNR.
This can be explained by the nonlinearity of the PMTs output characteristic, as sensitivity reduces
with increasing output current [48]. For the best measurements in vivo, therefore, the number of
photons that are collected by the PMT from incoherent sources, such as ambient lighting, should
be minimized, even when the LIA is used. The LIA is more sensitive than the DC system as it
enables measurements at increased SNR to their counterparts, taken with the DC system.
The frequency-domain characteristics of the LIA were then determined. Figure 4.10 shows the
results of frequency response and noise spectrum analysis on the LIA. The noise spectrum was
obtained using the same procedure presented for the characterization of the TIA in the DC system.
The modulation signal was a 209 Hz, 7.5 V amplitude sine wave provided by a function generator
(33522A, Agilent).
Characterization of the TIAs magnitude response, in Figure 4.10a, reveals a lower f3dB band-

71

100

0.5

0 0
10

10
Frequency (Hz)

-100
4
10

Magnitude (A per rt Hz)

200
Phase (degree)

Gain (dB)

-10

1.5

10

-12

10

-14

10

-16

10

0.01

0.1

1
10
100
Frequency (Hz)

(a)

1000

(b)

Figure 4.10: a) Magnitude and Phase response of TIA in demodulation subcircuit. f3dB was
measured at 5.5 kHz. b) LIAs input-referred noise spectrum.
width than expected (at 5.5 kHz compared to 14.93 kHz from simulation) and slight (0.86 dB)
peaking near 3 kHz despite anticipated overcompensation. From Section 4.1.3, this is likely due to
a mismatch in expected and actual input capacitance Ci , and feedback capacitance due to parasitics
on the PCB C f . This can be corrected by using a larger bandwidth opamp, such as the OPA657
(GBW = 1.6 GHz), to shift the intersection point of Ao (s) and
that the effect of

1
(s)

1
(s)

to a higher frequency, such

is attenuated at that point. Figure 4.10b shows the phase shift of the TIA.

As expected for an inverting amplifier configuration, the phase shift at dc is 180. Because the
PMT generates a negative current, this effect shifts the signal back into phase for multiplication
with the reference. A small phase shift is induced at several of the higher possible modulation
frequencies generated in Subsection 4.1.2. At 571 Hz, for example, the TIA induces a phase shift,
of 8.6 (from 180). At the output of the multiplier, this phase shift will result in a reduction of
VLIA by [56]

VLIA, = cos VLIA, =0

(4.29)

= 0.986VLIA, =0

(4.30)

This represents a reduction in VLIA by 1%, and so it was decided fc = 571 Hz will be the highest
72

frequency at which the LIA should be operated.

The input-referred noise in a 0.1-10 Hz bandwidth was found to be 5.5 fA(RMS). Compared
to the TIA designed for the DC system, for which this figure was found to be 125 fA rms, this
represents close to a 20-fold improvement in noise performance in the frequency band of interest.
However, noise is elevated from the floor at higher frequencies, consistent with the peaking in the
frequency response.
The bandwidth of the LIA was then characterized by measuring the systems rise time in response to a step input. For a second-order system, the bandwidth is related to rise time according
to
#
"
p
1

1
p
)
o =
tan1 (
2

RISE 1

(4.31)

Where o is the natural frequency of the filter, RISE is the time is takes for the output of the
filter to change from 10% to 90% of its final value, and is the filters damping ratio, related to
pole quality factor, Q, through =

1
2Q .

In a cascade filter topology, the overall rise time of the system is determined by the rise times
of its stages:

q
2 + 2 + + 2
r0 = r1
rn
r2

(4.32)

Equation 4.32 reveals that the rise time of the overall system cannot be faster than its slowest
stage. In this 4th -order filter design, this is the stage with the lowest pole quality factor, Q = 0.55,
for = 0.91.
The systems response to a step input is shown in Figure 4.11. The input is the modulated
light source, and the output is VLIA . The bandwidth of the overall system is then computed from
Equation 4.31 to be 8.5 Hz, compared to the 10 Hz designed frequency of the low-pass filter.

73

2
Modulated Light Input

Normalized LIA Output

1.5

1.5

1
0.5

RISE TIME

0.5

-0.5
-1

-0.5
-1

Time (s)

Time (s)

(a)

(b)

Figure 4.11: a) The systems response to b) a step input, characterizes the systems bandwidth.
The output of the LIA changes from 10% to 90% of its final value in 340 ms. A step input was
emulated by rapidly opening the shutter of the light source for the optical system, and quantified
by measuring the resulting light launched into an optical fiber with a wide bandwidth photodiode
(PDA36A, Thorlabs)

4.2

In vivo Application

In this section, the new optoelectronic design is applied to study the dynamics of intracellular
calcium concentration in the PVN of a mouse, as reported by GCaMP6s in a CRH-Cre mouse.
All experiments were once again performed in Dr. Bains lab. The results of this application
are presented in Subsection 4.2.1, and subsection 4.2.2 investigates the contribution of correlated
artifacts to these recordings of fluorescence intensity changes.

4.2.1

Fluorescence Intensity Changes due to Calcium Dynamics in Response to Conditioned

Stress

This section presents the results obtained through the application of the refined optoelectronic
design to study calcium-mediated changes in fluorescence in a CRH-Cre mouse. The parameter of
interest in this experiment is F/Fo , the baseline-referred change in fluorescence, where F is the
mean fluorescence intensity during the first 100 seconds of exposure to conditioned stress, and Fo

74

is the fluorescence intensity during the first 100 seconds of baseline recording.

F/Fo =

F Fo
100%
Fo

(4.33)

To minimize the number of photons from uncorrelated sources that are collected by the PMT,
the dental cement used to secure the optical fiber implant was mixed with red food coloring, as
shown in Figure 4.12. The food coloring strongly absorbs green light, preventing those photons
from penetrating further. Furthermore, the experiments were conducted under fluorescent lighting
only. Fluorescent lighting is characterized by discrete peaks in the visible spectrum, with litte
energy in between those peaks. This contrasts with incandescent or LED lighting, that have broad
and relatively flat spectra. This enabled the application of another emission filter (515/30 nm,
Chroma Technology) to avoid the largest peaks in the spectrum. While a colored light source, such
as one whose energy is concentrated in red would have also helped solve this problem, the mouse
retina has no sensitivity in this band, and it would essentially be blind during the experiment.
As the experiment relies on visual environmental cues to provoke the conditioned response, this
solution is unacceptable.
The population of cells expressing the fluorophore were targeted using stereotactic coordinates
as well as the optical guidance system to be described in Chapter 5.
The protocol to investigate calcium transients in CRH-Cre mice with GCaMP6s was described
in Chapter 3. Briefly, CRH-Cre mice transfected with either GCaMP6s (experimental) or EYFP
(control) through viral injection were implanted with fiberoptic cannula implants. After one week
of recovery, they were allowed to habituate to an environment for three days. On day four, the
animals were subjected to a series of footshocks. On day five, fluorescence intensity changes were
measured with the fiber photometry system.
Figure 4.13 depicts representative traces of fluorescence intensity that resulted when animals
were reintroduced to a conditioned stress. A 5-minute baseline recording (t = 0 to 5 minutes) while
the animal was in its home cage, followed by a 6-minute exposure to the preconditioned stress (t =

75

Transmission of Fluorescent Lighting

(a)

regular cement
colored cement
emission filter cut on/off

0
450

500

550

600

650

Wavelength (nm)

(b)

Figure 4.12: a) A thick application of dental cement mixed with red food coloring reduces the
number of photons in the emission band that can be collected by the optical fiber implant during
in vivo study. b) A 515/30 nm emission filter blocks a large peak in fluorescent lighting near 543
nm that would be relayed to the detector with the 535/30 nm filter. The addition of food coloring
to dental cement (red) blocks 70% of the photons that from ambient lighting that would otherwise
penetrate the skull and brain tissue, compared to the uncolored dye (pink).
5-11 min), and finishing with a 4 minute post-recording.
Signal Parameter
F(
q 2 1 )
N( 12

12 + 22 )
CNR

G1
0.559

G2
-0.010

G3
0.007

G4
0.142

G5
-0.025

G6
0.071

E1
-0.009

0.068
8.171

0.004
-2.795

0.008
0.891

0.019
7.337

0.064
-0.383

0.012
5.759

0.036
-0.249

Table 4.5: Detecting changes in fluorescence intensity from CRH cells in freely-moving mice in
response to stress with the LIA system. G = GCaMP6s animal, E = EYFP animal.
As in Chapter 3, the systems ability to measure changes in fluorescence are quantified with
CNR. The results are presented in Table 4.5. Three of the six GCaMP6s animals tested exhibited
elevated levels of fluorescence while it was under stress.
The two optoelectronic designs were then compared for their ability to detect changes in vivo
in a single animal. From the results of Chapter 3, it was found that, in the same animal, repeated exposures to a conditioned stress produces approximately equal baseline-referred changes
in fluorescence intensity. It is therefore reasonable to assume that two identical experiments, done
in succession, will produce the same results. Employing again the aforementioned behavioural
76

 F/F (%)

100%

GCaMP6s
EYFP

50%

0%
0

10

15

Time (minutes)

Figure 4.13: Representative traces from a GCaMP6s (green) and EYFP (orange) CRH-Cre mouse.
Reintroduction of the animal to the conditioned stress at t = 5 minutes resulted in changes in
detected fluorescence intensity (GCaMP6s: F/F = 31 % 39 %, n = 4; EYFP: F/F = 2 %, n =
1). Red lines denote transfer to and from the shock cage that provides the conditioned stimulus.
protocol (5 minute baseline, 6 minute exposure to conditioned stress, 4 minute post-recording),
Figure 4.14 shows that while both systems recorded similar changes in fluorescence intensity (DC
system (EM: 535/30 nm, dark green): CNR = 2.46 with F/Fo = 9.5% and LIA (515/30 nm, light
green): CNR = 3.43 with F/Fo = 7.3%),the LIA system detected signals with greater fidelity. In
both cases, Pex = 1 W.

10%

F/F

20%

-10%
0

5
10
Time (minutes)

15

Figure 4.14: Performance comparison of both optoelectronic designs in vivo.

The results acquired with the LIA configuration are similar to those obtained in Section 3.4,
as in both cases, reintroduction of the GCaMP6s animals to an environment for which they have
77

a conditioned stress results in increased levels of detected fluorescence intensity for the duration
of the stress. Signals were detected in 50% of the experimental group, and no signal was detected
from the experimental group. Both optoelectronic designs presented in this thesis recorded elevated
levels of fluorescence intensity upon reintroduction of a GCaMP6s animal to a conditioned stress.
These elevated levels of fluorescence were not seen in the control group.
Histology, the data for which is presented in Chapter 5, confirmed correct probe placement and
expression of GCaMP6s in CRH cells for mice # 1, 4, and 6. The probe was found to be misplaced
in animals #2, 3, and 5. Signals with CNR >1 were therefore detected only in those animals where
the probe was placed correctly. We conclude that the application of this fiber photometry system
design resulted in the successful detection of calcium-mediated changes in fluorescence intensity.

4.2.2

Measurement Immunity to Neurovascular and Hemodynamic Coupling

It is known that changes in neuronal physiology that result in increased calcium concentration
(transduced to fluorescence by GCaMP6s in this application) may require a response from hemodynamic or vascular processes. Reexposure to conditioned fear in freely-moving rats, for example,
results in changes in cerebral blood flow patterns [64]. In all regions of the hypothalamus studied,
increases in blood flow were found.
Because blood has dynamic optical absorption properties, hemodynamic and/or vascular responses such as the aforementioned may be modulating the light that is delivered, and that which
is collected from, fluorescent reporters in fiber photometry measurements. In this context, hemodynamics will refer to processes that change the blood oxygen saturation, and vascular response
refer to changes in blood flow. Shen [43] found that fluorescence detected from neurons underlying arterioles decreased with arteriole dilation during imaging. Because the contribution of this
artifact would be highly dependent on the placement of the probe in relation to a nearby artery,
identification of this artifact may help explain the large variability in the experimental group.
This effect is also potentially problematic because of its expected correlation with neuronal
activity, which may result in exaggerated or underestimated reports for F/Fo . While, as described
78

in Chapter 2, numerous approaches have been taken to correct for artifacts associated with changes
in coupling and fiber bending, there is no existing fiber photometry configuration that can correct
for wavelength-selective artifacts. To our knowledge, the effect of neurovascular coupling has yet
to be investigated in a fiber photometry application. The investigation into whether this effect
can distort the signal trace for a population of cells in the PVN of the hypothalamus will now be
described.
0

Absorption coefficient (1/cm)

2.5

HbO: Hb

2
1.5
1
0.5
0
400

500
600
Wavelength (nm)

700

(a)

10

-1

10

-2

10

-3

10
400

500
600
Wavelength (nm)

700

(b)

Figure 4.15: a) Ratio of absorption coefficient of Oxyhemoglobin (HbO2 ) to Hemoglobin (Hb). b)

Absorption spectrum of blood, assuming 50% oxygen saturation and 1M concentration [2]

Figure 4.15 describes the optical absorption spectrum of blood components. The major absorbers in blood in the visible spectrum are the two states of hemoglobin. In the visible spectrum,
absorption of light by blood is strongly dependent on wavelength [29]. In the range of 418 nm to
484 nm, the absorption of light by blood increases with decreasing wavelength. Beyond 580 nm,
absorption falls off quickly with wavelength.
Dynamics in blood parameters presents a potential artifact for in vivo fiber photometry. To
investigate whether the changes in fluorescence intensity detected in vivo are the sole result of
calcium dynamics, ex vivo and in vivo data may be compared. Brain slices do not have functional
vasculature, and are therefore immune to neurovascular and hemodynamic artifacts.
From the ex vivo spectral characterization done in Chapter 3, the spectral sensitivity of the
reporter is known. By using different emission filters during in vivo recordings, this model can
79

tested. Because it was found that percent changes in fluorescence recorded from repeated exposures to pre-conditioned stress remained constant between measurements in the same animal, it
is reasonable to assume that the calcium-mediated changes in fluorescence shall remain constant
between measurements.
Agreement between ex vivo and in vivo data will indicate that changes in fluorescence intensity
are the sole result of calcium-mediation. Disagreement will suggest a potential artifact, and should
motivate further investigation.
4.2.2.1

Fiber photometry in response to conditioned stress in a CRH-Cre mouse with GCaMP6

The mean expected sensitivity to changes in GCaMP6s, based on ex vivo data, for several filters
are presented in Table 4.6. The values are normalized to the sensitivity granted by the 515/30 nm
filter.
Filter
ex vivo F/Fo

505/10 nm
0.5

515/30 nm
1

535/30 nm
1.12

Table 4.6: Experimentally-determined mean sensitivity to changes in GCaMP6s fluorescence, for

given emission bands.

To determine if there is a wavelength-selective process that could distort the detected fluorescence trace, fiber photometry was performed on two animals. One of which was interrogated with
a 515/30 nm filter followed by a 535/30 nm filter, and the other, a 515/30 nm filter followed by a
505/10 nm filter. The optoelectronic system was configured for the LIA, with Pex = 1 W.
Traces of fluorescence intensity resulting from re-exposure to conditioned stress, taken in succession with different emission filters are shown in Figure 4.16. A 5-minute baseline recording
of the animal in their homecage was followed by a 6 minute recording from the animal under
re-exposure to conditioned stress, followed by a 4-minute post-recording when the animal had
returned to its home cage.
The sensitivity comparison of 515/30 nm filter to the 535/30 nm filter yielded F/Fo = 73.25%
and 44.94%, respectively, and the comparison of 505/10 nm to 515/30 yielded F/Fo = 7.66%

80

100%

50%

F/Fo (%)

F/Fo (%)

20%

0%

-50%
0

EM: 515/30 nm
EM: 535/30 nm
5
10
Time (minutes)

10%
0%

-10%
15

(a)

EM: 505/10 nm
EM: 515/30 nm
5
10
Time (minutes)

15

(b)

Figure 4.16: Detected fluorescence intensity changes in response to re-exposure to conditioned

stress in two CRH-Cre mice, with three emission filters. a) 535/30 nm filter and 515/30 nm filter,
and in another animal b) 505/10 nm filter and 515/30 nm filter.
and 10.85%, respectively. Compared to the mean spectral sensitivities for GCaMP6s presented
in Table 4.6, it is known that the 535/30 nm filter is the most sensitive to F/F. However, the
F/F value was 38.6 % less than that recorded with the 515/30 nm filter. Similarly, a 505/10
nm filter should detect changes of half the magnitude of that produced with the 515/30 nm filter.
Both of these findings are inconsistent with that model, suggesting an additional factor or set of
physiological factors.
4.2.2.2

Reflectance spectroscopy in response to innate stress in a wild-type mouse

Disagreement between predictions based on ex vivo data and in vivo data motivated an investigation into the cause of the disagreement. To determine the nature of a potential artifact, reflectance
spectroscopy was used in a freely-moving mouse that does not express fluorophores (This wild
type animal will henceforth be referred to as WT). A broad-spectrum light source and a spectrometer enabled spectrally-resolved measurements, such that wavelength-selective features may
be discovered.
Reflectance spectroscopy involves delivering a broad light source with a known spectrum to
tissue, and comparing it to the spectrum that returns. Reflectance spectroscopy has been adapted
for in vivo measurement [65]. The system designed by Yu uses a beam splitter to separate the
delivered and collected light, and polarizers are used to reduce the background in the measured
81

spectrum by attenuating reflection at interfaces. This improves sensitivity to changes in scattering

and absorption events in the tissue.
The experiment will result in a series of measured spectra, acquired at some interval, whose
absolute values vary with wavelength, and with the sample number, i. S( , i) is the set of spectra
collected during an experiment.
As experiments will be done on awake and freely-moving mice, fluctuations due to fiber bending are overcome by normalizing each point in a given spectrum to the value measured in the same
acquisition but at 700 nm, to produce a spectrum corrected for fiber-bending losses, Sn ( ):

Sn ( , i) =

S( , i)
S( = 700nm, i)

(4.34)

At longer wavelengths, the polarizers in the optical system lose their efficiency in attenuating
the background contribution to the measured spectrum, and sensitivity to reflectance from tissue
is lost. However, sensitivity to bending losses is preserved. Normalizing the result to the value at
700 nm is therefore a reasonable correction strategy.
After normalization, changes in reflectance may then be quantified in time or across wavelength. These changes will be referred to the average spectrum resulting from the first 100 seconds
of baseline measurement, So ( )

So ( ) =

1 M
Sn( , i)
M i=1

(4.35)

Where M is the number of samples acquired, equal to the sampling rate times the acquisition
period (100 seconds).
The percent change in reflectance over time is then given by R/Ro

R/Ro ( , i) =

Sn ( , i) So ( )
100%
So ( )

(4.36)

An intuitive understanding of the evolution of R/Ro ( , i) may then be gained by analyzing

through index, i (essentially time) by examining a single wavelength, , or for spectral variation by
82

selecting a single spectrum at i, or set of spectra to average, and then referring them to the baseline
measurements.
To interrogate reflectance during exposure to stress in vivo, WT mice (no expressed fluorophores) were implanted with fiberoptic cannula implants (MFC-200/230-0.48-5mm-MF2.5-FLT,
Doric Lenses) using stereotactic coordinates to target the fiber to the expected location of the PVN.
After one week of recovery, they were allowed to habituate to an environment for three days. On
day four, the animals were subjected a stress protocol, during which time reflectance spectra were
recorded. The stress protocol consisted of baseline recording while the animal was in its home
cage, transfer to a footshock cage at 120 seconds, at which time it was subjected to repeated,
2-second footshocks at 1-minute intervals, and then replacement in the home cage at 420 seconds.
4%
3%

4%

R/R (%)
o

R/R (%)
o

6%

2%
0
-2%
4%
0

100

Normalized to R(700 nm)

0.1 Software Hz Filter
200
300
400
500
Time (s)

2%
1%
0
450

(a)

500
550
600
Wavelength (nm)

650

700

(b)

Figure 4.17: a) Changes in reflectance as measured at 515 nm, over time in response to exposure
to innate stress. b) Change in spectral reflectance between baseline (0 - 100 s) and exposure to
stress (120 - 240 s).
Figure 4.17 depicts % changes in reflectance in one animal in response to innate stress. Changes
in time, shown in Figure 4.17a were quantified At 515 nm, according to Equation 4.36:

R/Ro ( = 515 nm, i) =

Sn ( = 515 nm, i) So ( = 515 nm)

100%
So ( = 515nm)

(4.37)

Elevated levels of reflectance were found during exposure to innate stress (120-420 seconds)
(R/Ro = 3.1%, n = 1).
Drawing again on Equation 4.36, the wavelength-dependent change in reflectance between
83

stress and baseline is determined by referring the average of spectra acquired during 120-220
seconds (first 100 seconds of exposure to stress) Sn,avg ( , i = 240 : 480) =

1
200

480

n ( , i) to the
i=240

average baseline spectrum, So ( )

R/Ro ( ) =

Sn,avg ( , i = 240 : 480) So ( )

100%
So ( )

So ( ) =

1 M
n( , i)
M i=1

(4.38)

(4.39)

Figure 4.17b shows the wavelength-dependent elevation in reflectance in response to this innate
stress. This suggests a decrease in blood flow to the area near the implant. The peaks at 538 nm and
578 nm are near to peaks in the absorption spectrum of blood as shown in Figure 4.15b. Although
blood oxygen saturation changes may be a contributing factor in these modulations in reflectance,
the absence of any points in the plot of spectral sensitivity where the change is zero (excluding
the point at 700 nm used for normalization) rules out the possibility of oxygen saturation changes
acting as the sole cause.
The detected changes in reflectance are similar in magnitude to changes in fluorescence intensity measured in animals expressing the GCaMP6s calcium reporter. Because the fiber probe
was implanted near the PVN, it is therefore possible that neurovascular dynamics are distorting the
detected fluorescence trace during fiber photometry.
Tables 4.7 and 4.8 compare ex vivo and in vivo data. The mean sensitivities to modulations in
reflectance in each emission band are shown alongside this data. Discrepancies between the model
(ex vivo data) and the actual result (in vivo data) suggest it is unlikely that changes in detected
fluorescence intensity were the sole result of calcium mediations of GCaMP6s fluorescence.
Filter
515/30 nm
505/10 nm

ex vivo F/Fo
1
0.5

in vivo F/Fo
1
0.72

R/Ro (i)
1
0.89

Table 4.7: Experimentally-determined sensitivity to physiological changes in optical tissue properties with a 505/10 nm filter, normalized to sensitivities determined using a 515/30 nm filter.

84

Filter
515/30 nm
535/30 nm

ex vivo F/Fo
1
1.12

in vivo F/Fo
1
0.61

R/Ro (i)
1
1.40

Table 4.8: Experimentally-determined sensitivity to physiological changes in optical tissue properties with a 535/30 nm filter, normalized to sensitivities determined using a 515/30 nm filter.
Blood volume increases would account for a reduction of F/Fo from predicted for the 535/30
nm filter, and a larger F/Fo from predicted for the 505/10 nm filter. Blood volume increases are
consistent with these results.
Changes in GCaMP6s fluorescence and absorption from blood volume are spectrally distinct.
For example, the 535/30 nm filter is most sensitive to changes in GCaMP6s fluorescence, but also
most sensitive to changes in blood volume due to its overlap with a spectral peak in the optical
absorption curve for blood (Figure 4.15b).
The results of this investigation into the effect of dynamic tissue absorption properties, in an
awake and freely-moving, tethered animal, suggest the presence of a large potential artifact in fiber
photometry measurements taken in the PVN. The results indicate that blood volume changes is the
most likely contributor.
These findings motivate further investigation into the cause and nature of the artifact for recordings from this particular brain structure, as it is correlated with neuronal activity. For example,
recording from each animal, with all three emission filters (505/10, 515/30, 535/30) would have
enabled collection of more conclusive data. Nevertheless, this section has presented evidence that
wavelength-dependent artifacts may be disrupting the fluorescence trace.

4.3

Discussion and System Comparisons

The application of the system described in this chapter resulted in the successful detection of
calcium-mediated changes in fluorescence intensity from CRH cells expressing GCaMP6s. Calcium transients were induced in the PVN of a freely-moving mouse through conditioned stress, and
detected (CNR >1) in GCaMP6 animals #1, 4, and 6. Histology confirmed proper probe placement
85

Parameter
Probe Diameter
Light source output
Excitation Band
Emission Band
Noise, 0.1-10 Hz
SNR, FAF
CNR, in vivo
Bandwidth
Output Range

Specification, DC System
240 m
Adjustible; 0.35 W - 75 W
470/40 nm
535/30 nm
125 A rms
6.33 dB (EM: 515/30 nm)
2.46 (F/Fo = 9.5%)
14.7 Hz
4.7 V

Specification, LIA
240 m
Fixed; 1 W
470/40 nm
515/30 nm
5.5 A rms
31.6 dB (EM: 515/30 nm)
3.43 (F/Fo = 7.3%)
8.5 Hz
0-1.5 V

Table 4.9: Comparison of performance specifications between the LIA and DC systems.

Magnitude (A per rt Hz)

10

10

10

10

-10

LIA system noise; 5.5 fA rms

DC system noise; 125 fA rms
-12

-14

-16

0.01

0.1

1
10
100
Frequency (Hz)

1000

Figure 4.18: Noise spectra from LIA and DC systems. RMS noise computer for 0.1-10 Hz.
for these mice, and inadequate targeting for those in which no signals were detected (CNR <1).
Table 4.9 compares the performance specifications of the two systems. The LIA configuration
can detect changes in fluorescence intensity, and showed a 1.4-fold improvement in CNR over the
DC system in vivo. However, this improvement may be attributed in part to the choice of emission
filters employed in both experiments. From Subsection 4.2.2.1, it was found that when the 535/30
nm filter was used, the change in fluorescence is less than expected, and likely due to an increased
sensitivity to blood volume changes. If this is true, the DC systems CNR may be suffering more
from rapid transients in blood volume, that inflate the measure of noise.
Nevertheless, a side-by-side benchtop characterization, shown in Figure 4.18, reveals the LIA
is the lower-noise instrument, providing a 20-fold improvement in the 0.1-10 Hz band.

86

The measurements taken in the in vivo experiments presented in this chapter were not disrupted
by the leakage of ambient lighting. The addition of food coloring to dental cement, in combination
with a thicker application of the dental cement than is typically employed, as in Figure 4.12a,
was sufficient to block the photons in question. Reflectance measurements in response to innate
stress advocate against using the 535/30 nm on the basis of its vulnerability to changes in blood
volume, and perhaps even moving to the 505/10 nm filter despite its lower sensitivity to changes
in GCaMP6s fluorescence.
Improvements to the LIAs instrumentation can be made by replacing the OPA140 in the TIA
with a wider bandwidth opamp to eliminate the peaking in the TIAs magnitude response, and
enable higher modulation frequencies. In addition, the bandwidth of the LPF of the LIAs demodulation subcircuit can be reduced below 10 Hz, as calcium-mediated changes in fluorescence occur
very slowly. Finally, samples of VLIA should be digitized on a scale closer to its dynamic range,
perhaps 1 V.
In addition to designing a fiber photometry system with improved sensitivity to changes in
GCaMP6s, the in vivo experiments described in this chapter corroborate evidence from others that
neurovascular coupling is a correlated artifact in florescence measurements in vivo. Because of the
wavelength-dependent nature of this suspected contribution, artifact-correction schemes proposed
by others would be ill equipped to recover the true signal.

87

Chapter 5
OPTICAL GUIDANCE FOR IMPROVED STEREOTACTIC
TARGETING
Single fiber photometry involves implanting a fiber optic cannula in the brain, using stereotactic
coordinates to target the probe to the population of cells that express the fluorescent reporter.
However, due to individual variation, this approach has been successful approximately 10% of the
time for targeting the PVN of a mouse. This chapter presents the design and application protocol of
a third optical system developed for this research. Its purpose is to guide the surgery and improve
implant success rate, by detecting fluorescence produced only by the reporter or marker of interest.

5.1

Motivation

The optical guidance system described in this chapter was investigated with the goal of improving
the rate of successful implantation of fiber probes targeted to the paraventricular nucleus of the
hypothalamus (PVN) of a mouse. In mice, this structure is located at approximately 4.5 mm below
the surface of the brain. Its total volume is approximately 0.25 mm3 compared to a total brain
volume of approximately 516 mm3 [66].
Stereotactic coordinates of the mouse brain model [31], together with landmarks on the skull
that serve as a reference, are used to predict the location of the structure in the brain for targeting of
the probe. However, variability between animals cause the predicted coordinates to deviate from
the actual coordinates, leading to improper placement of the probe. The challenge is compounded
by the scattering of visible light by brain tissue. The optical fiber probe must be implanted very
close to the population of cells to ensure delivery of excitation photons, and collection of emission
photons from the structure of interest.

88

Tissue scattering also presents a challenge in the delivery of stimulation light for optogenetics.
Aravanis [10] developed and validated a model for determining the proximity of a probe to a
neuronal population such that sufficient irradiance for stimulation is developed. The challenge of
detecting fluorescence from cells is more difficult, as the total optical path length is twice that
involved with stimulation alone.
Figure 5.1 depicts how optical power due to fluorescence, resulting from excitation delivered
by a fiber probe, is collected by the same probe. This section derives a model to determine the
required proximity of the fiber to the fluorescent structure such that modulations in fluorescence
intensity may be detected. The validity of this model will then be tested using data from in vivo
behavioural study, and histological results.

89

2rf

Ix,o

Im,o

tissue properties n, s

NA

Im,z; efc

Ix,z

Figure 5.1: Monitoring fluorescence in tissue with separation between the probe tip and the target
structure. The target structure (a population of cells expressing a fluorescence reporter) is modelled
as a plane whose brightness is modulated homogenously. r f : radius of the optical fiber; Ix,o :
excitation intensity at the plane of the probe tip; Im,o : intensity of emission photons at the plane
of the probe tip; z distance separating the probe tip from the fluorescent plane; n: refractive index
of tissue, S: scattering coefficient per unit thickness; NA: numerical aperture of optical fiber; Ix,z :
excitation intensity at the fluorescent plane; Im,z : emission intensity at the fluorescent plane; e f c :
fluorescence conversion efficiency.

Assuming brain tissue is planar, homogenous, an ideal diffuser, and that reflection and absorption are constant throughout the volume, and further assuming that transmission loss of visible
light from scattering is much greater than that from absorption, transmission of light through tissue

90

may be modelled using the Kubelka-Monk model for diffuse scattering media [10]:

Tscattering =

1
Sz + 1

(5.1)

Where T is the transmission fraction, S is the scattering coefficient per unit thickness and z is
the distance separating two planes in tissue.
Light exiting an optical fiber will diverge, causing the intensity to decrease with distance:
p2
Tdivergence =
(z + p)2

(5.2)

r
n 2
1
p = rf
NA

(5.3)

Where

and r f and NA are the radius and the numerical aperture of the optical fiber, respectively, and n
is the refractive index of tissue.
When both geometric and scattering losses are taken into account, the expression for transmission of excitation light at some distance from the distal tip of the optical fiber is
Ix,z
p2
=
Ix,o (Sz + 1)(z + p)2

(5.4)

The intensity of the fluorescent plane is proportional to the excitation intensity at that plane:

Im,z = Ix,z e f c

(5.5)

The fluorescence conversion efficiency, e f c , is the proportionality constant that relates these
two quantities. Assuming all light is absorbed by the fluorescent reporter (A valid assumption for
GCaMP6 targeted to a dense population of cells with AAV viral injections, with a Molar Extinction Coefficient of 68 500 M-1 cm-1 and cytosolic expression level of 80 M), this is simply the

91

reporters quantum efficiency in its calcium saturated state.

Im,z = Ix,z QE

(5.6)

Modelling the fluorescent plane at z as a Lambertian source, the intensity falls off with the
square of distance. The emitted light is subjected to scattering losses as it propagates towards the
optical fiber for detection:

Im,o
1
1
= 2
Im,z
z (Sz + 1)

(5.7)

The optical power that is collected is reduced further due to a mismatch between the size of the
emitting area and that of the optical fiber core, and the limited acceptance angle of the fiber:

Pm,collected = r2f Im,o



1
1 cos(sin NA)

(5.8)

For grey matter in mice, the refractive index of tissue, n is 1.36, and scattering coefficient S is
10.9 mm-1 . Quantum efficiency, QE for GCaMP6 in its calcium saturated state is 0.61 [16]. For the
optical system proposed in this thesis, r f is 0.1 mm, NA is 0.48, Px,O is 1 W. Selecting a 5 fW step
change in fluorescence as the minimum detectable signal (corresponding to approximately 1.5%
F/Fo on top of 50 fW from fiber autofluorescence and 250 fW from co-expressed fluorophores
(From Chapter 3)) this analysis reveals that the fiber probe must be placed 350 m from the surface
that defines the fluorescent structure. Figure 5.2 depicts the results of this model with continuous
variation in separation of the probe tip and fluorescent structure. Table 5.1 summarizes this analysis
with expected signal strengths (F/Fo ) and the required distance from the population of cells to
achieve the condition.

92

10000000

Detected Power (fW)

100000
1000
10
0.1

100
200
300
400
500
Distance from optical fiber (micrometer)

Figure 5.2: Theoretical results for detection of fluorescence from distant structures in brain tissue
in response to delivery of Pex = 1 W.

Detected Optical Signal Power

F/F

Separation

5 fW

1.5%

350 m

15 fW

5%

300 m

30 fW

10%

250 m

150 fW

50%

200 m

300 fW

100%

175 m

Table 5.1: Approximate results from the theoretical model of detection of fluorescence in brain
tissue from structures separated from the fiber probe, and the resulting baseline-referred changes
in fluorescence, F/Fo , when background sources of fluorescence (from the fiber and co-expressed
markers) are taken into account.

5.2

Optical System Design

Improving stereotactic targeting for fiber photometry with an optical system has been attempted by
others. Grienberger [3] uses a PMT to detect the region of highest fluorescence, as averaged across
the emission band employed. In our application, however, tdTomato, a fluorescent marker with
93

overlapping excitation and emission spectra to both GCaMP6s and EYFP, is expressed in all CRH
cells of the brain. In addition to a strong concentration of such cells in the PVN, they are found
in the cortex, and densely clustered in the amygdala and the bed nucleus of the stria terminalis
(BNST). An increase in detected fluorescence using this method cannot discriminate between the
two fluorophores. To overcome this challenge, Cui [9], uses a multispectral system with distinct
emission bands to create spectral resolution and target the correct fluorophores.
We propose a simple and cost-effective optical system, the schematic for which is presented
in Figure 5.3. Whereas the other optical systems presented in Chapters 3 and 4 use a PMT as a
detector, a spectrometer (QEPRO452, Ocean Optics) is employed for this purpose. This choice
enables the user to distinguish between fluorophores. However, this detector is less sensitive than
a PMT, and higher light intensities are required. While photobleaching could not be avoided,
photoxicity is mitigated by limiting the delivered light to 100 W [6]. A 488 nm laser (488-TECBL,
World Star Tech) is used as the light source, and the power launched into the fiber is controlled
with a neutral density filter. A notch filter (ZET488NF, Chroma Technologies) blocks laser light
from reaching the spectrometer, while passing the photons of all wavelengths outside of that band.

DBS
L1

FIBER TO
IMPLANT

L2
ND
EX

LASER

TO SPECTROMETER

Figure 5.3: Proposed optical system for surgical guidance. EX: 470/40 excitation filter; ND:
Neutral density filter; DBS: Dichroic beam splitter; N: Notch filter; L1/L2: Coupling/collimation
lenses.

94

5.3

Application and Evaluation Procedure

We hypothesize that using the guidance system to optically target structures will result in an increased proportion of successful implants. To investigate whether this is the case, we will assume
that a successful surgery will result in a wavelength-resolved increase in fluorescence as the probe
is lowered into the brain, as this is the principle on which the guidance system operates. This
assumption will be verified later through histology.
Next, the use of the system must be tested against the accuracy of stereotactic coordinates
and the proficiency of the surgeon. To enable comparison of the two, the surgery begins with the
surgeon attempting to find the structure without access to the information on the guidance system:
the presumed coordinates are used. The probe is lowered in steps of 0.2 mm while a second person
monitors the fluorescence spectrum. If an increase in fluorescence is seen, the surgery is considered
a success. If no increase is seen, alternate coordinates will be attempted until the structure is found.
It was decided that a maximum of five sets of coordinates will be attempted.
The performance of the system against the status quo is based on the relative number of successful surgeries with alternate coordinates versus those with presumed coordinates. If the structure is
found consistently with presumed coordinates, the guidance system will have little effect on the
overall success rate and is not needed. A meaningful performance characteristic is the success
rate improvement conferred by the system, Rs,guidance , determined by the number of successful
implants attributed to the surgical guidance system, Is,guidance , the number of successful implants
attributed to stereotactic coordinates alone, Is,stereotaxy , for a given total of implants, as described
by

Rs =

Is,guidance
Is,stereotaxy

(5.9)

The optical guidance system was applied to target the hypothalamic paraventricular nuclei
(PVN) of the 6 CRH-Cre mice expressing GCaMP6s investigated for changes in fluorescence intensity in response to conditioned stress in Chapter 4. Animals were anesthetized and restrained,
95

and a burr hole drilled into the skull such that brain tissue was exposed. With the light source of
the optical system shuttered, the optical fiber, connected to the probe, was attached to a stereotactic
arm and positioned according to appropriate coordinates. The probe was lowered to 2 mm deep
into the brain, and, after 10 seconds, the light source was turned on and a spectrum acquired (1
second integration time, averaged over ten acquisitions). Successive spectra were then acquired
incrementally every 0.2 mm deeper [9], each time waiting 10 seconds before beginning the acquisition. This waiting period allowed the tissue to settle in response to the probes movement.
The percent increase in fluorescence was defined as the dark-current corrected fluorescence
intensity collected at some coordinates, referred to the fluorescence intensity collected at 500 m
dorsal to the structures presumed coordinates:

F/F =

F(d, = 515nm) F(d + 0.5mm, = 515nm)

F(d + 0.5mm, = 515nm)

(5.10)

Where F(d, = 515nm) is the fluorescence intensity at the final probe location and F(d +
0.5mm, = 515nm) is the fluorescence intensity in tissue 500 m from the presumed coordinates
of the PVN. From the model proposed in Section 5.1, at this distance, there should be no discernible
signal.

5.4

Results

A selection of the spectra recorded are shown in Figure 5.4. The spectra collected at 0.5 mm dorsal
to the presumed depth of the structure (black), and the final location of the probe are presented
(green). The spectra obtained at the final locations for animals #1, 3, 4, and 6 exhibit a peak at 515
nm. This second peak is recognized as the GCaMP6s emission spectrum, introduced in Chapter 3.

96

Fl. Intensity (Counts)

300

300

#1

200

200

200

100

100

100

500

550

600

500

Wavelength (nm)
300

Fl. Intensity (Counts)

300

#2

550

600

500

Wavelength (nm)

#6

200

200

200

100

100

100

550

600

600

300

#5

Wavelength (nm)

550

Wavelength (nm)

300

#4

500

#3

500

550

Wavelength (nm)

600

500

550

600

Wavelength (nm)

Figure 5.4: Spectra due to collected fluorescence at 500 m from the fluorescent structure (black)
and at the final location (green).

While the shape of the spectrum was monitored using real time display of spectral data, the
values presented in Table 5.2 refer to fluorescence intensities at 515 nm. A successful surgery was
defined as one for which at least a 10% increase in fluorescence intensity was seen at the final
coordinates.

97

Animal #

Presumed

Alternate 1

Alternate 2

220%

-15%

-34%

-15%

49%

82%

156%

20%

0%

-4%

9%

24%

Alternate 3

Alternate 4

Final
220%

-14%

-38%

-38%
156%
20%

2%

2%
24%

Table 5.2: Increases in fluorescence intensity with depth of the optical fiber probe in six CRHCre animals with GCaMP6s. If an increase was found at presumed coodinatres, the surgery was
deemed a success. If no increase was found, alternate coordinates were attempted until a structure
was found, up to a maximum of five attempts. Increases in fluorescence intensity of 10% at 515
nm were considered successful. The endeavour to find the PVN in animal #5 was halted early
(only three attempts were made) due to time constraints. In animal #3, alternate coordinates were
attempted beyond the 10% threshold defined as surgical success, in an effort to find a higher region
of fluorescence.

Table 5.2 shows the increases in fluorescence intensity seen during surgery. According to the
definition of success, mice #s 1, 3, 4, and 6 were deemed successful surgeries. Half of these surgeries were then successful due to the employment of alternate coordinates. The optical guidance
system therefore doubled the rate of successful surgeries for this group.
To validate the assumption that a successful surgery is associated with an increase in fluorescence intensity from tissue as the probe approaches coordinates near the presumed coordinates of
the PVN, histology was performed on all six mice to confirm expression and implant placement.
Fluorescence micrographs of these six animals, shown in Figure 5.5, were obtained from 250 m
coronal slices of brain tissue. The probe placements were inferred from tissue scarring. The dotted
rectangles denote the position and angle of the probe, depicting information about its lateral/medial
98

and dorsal/ventral positions. Its position in the sagittal plane is not depicted in these micrographs.
Figure 5.5 shows that probes were placed correctly in animals #1, 4, and 6. Probes in animals
# 2, 3, and 5 were found to be far from the PVN in the sagittal plane (#2: 200 m caudal, #3:
200 m anterior, #5: 240 m anterior). Histological results are consistent with spectra acquired
during surgery, apart from animal #3, where a large increase in fluorescence consistent with the
GCaMP6 emission spectrum was seen, but the probe was incorrectly placed.

#1

#2

ventricle

3rd ventricle

3rd ventricle

#3

#4

3rd

100 m

200 m

3rd ventricle

#6

3rd ventricle

3rd ventricle

#5

200 m

200 m

200 m

200 m

Figure 5.5: Identification of CRH neurons expressing GCaMP6s in the hypothalamic paraventricular nucleus of the hypothalamus of a mouse, and location of implanted fiber optical probe. The
PVN is the cluster of fluorescent cells outlined in the micrograph for # 1. Proper expression and
probe placement were confirmed in # 1, 4, and 6. Probes in # 2,3, and 5 were found to be far from
the structure in the sagittal plane: (#2: 200 m caudal, #3: 200 m anterior, #5: 240 m anterior).

99

5.5

Discussion

This chapter has introduced a model for the detection of changes in fluorescence in tissue, has proposed an optical system and investigated its efficacy in targeting fluorescent deep brain structures.
The model derived in Section 5.1 does not agree with the results obtained during surgery. It
overestimates the fluorescence intensity that is collected. For example, at 100 m from the structure, the detected optical power due to GCaMP6s fluorescence is 2.5 pW, which would represent
an increase from baseline of approximately 900%. This is much higher than what was actually
observed (220 %) for #1. The discrepancy is likely because the cells are distributed sparsely, in
contrast to the fluorescent plane as the structure was modelled. A more conservative model is
therefore needed.
The spectral characteristics of tissue at the final placement of the probe varied widely, however
an increase in fluorescence near 515 nm was often associated with an increase in fluorescence near
581 nm. Because the spectrometer enabled monitoring of fluorescence from all wavelengths in the
visible spectrum, fluorophores could be distinguished by monitoring the shape of the spectrum.
This additional information provided by the array detector justifies its use over the point detector.
In addition to distinguishing between the coexpressed fluorophores in this case, using this detection
scheme would also enable the identification of endogenous fluorophores, such lipofuscin [46],
having a distinct spectrum from both GCaMP6s and tdTomato.
Histology invalidated the assumption that a spectrally-resolved increase in fluorescence during
surgery is a successful surgery: while a 156% increase in fluorescence intensity was seen during
surgery for #3, the probe was found 200 m anterior to the desired target. This false positive is
difficult to explain, as the spectra acquired exhibited similar features to those surgeries that were
deemed successful through histology.
Taking histological data into account, the success rate improvement granted by the optical
guidance system is 50%, as only the structure for #6 could not be located using stereotactic coordinates alone. Modulations in detected fluorescence during re-exposure to conditioned stress, as

100

introduced in Chapter 4, were seen only in those animals for which fluorescence was found during
surgery, as may be seen from Table 5.3. Assuringly, no changes in fluorescence intensity were
observed in those animals for which the surgery was unsuccessful.
Animal #

Increase (Surgery)

CNR (Conditioned Stress)

Probe Placement

220%

8.171

Correct

-38%

-2.795

Inadequate

156%

0.891

Inadequate

20%

7.337

Correct

2%

-0.383

Inadequate

24%

5.759

Correct

Table 5.3: Increases in fluorescence intensity with depth of the optical fiber probe in six CRH-Cre
animals with GCaMP6s

The data shown in Table 5.3 show no obvious relationship between the magnitude of the fluorescence increase seen during surgery and that which was seen during behaviour. This may suggest
that some cells and regions within the PVN play a larger role in the stress response than others.
The optical guidance system provided a modest improvement in surgical success in this small
study. This should motivate further investigation in a larger study. The instrumentation and protocol could be further improved. For example, a modulation/demodulation scheme should be incorporated to provide the measurement immunity to ambient lighting artifacts. A spectrometer should
continue to be used, as it enables the surgeon to distinguish between any number of fluorophores.
The systems specificity may be improved by fitting the acquired spectra to a known reference
spectrum of the desired fluorophore.

101

Chapter 6
CONCLUSION AND FUTURE DIRECTIONS
This thesis described the development of highly sensitive optoelectronic systems for in vivo fiber
photometry, and their application in awake and freely-moving mice to interrogate the behavioural
correlates of the stress response. The system is optimized to detect changes in fluorescence from
GCaMP6s. Validation experiments were performed in CRH-Cre mice expressing the fluorescent
reporter. The LIA system in Chapter 4 improves on the DC system by conferring greater flexibility
in the experimental setup. Immunity to ambient lighting was achieved by altering the selected
filter set and adding food coloring to the dental cement. The ability to produce measurements at
higher signal-to-noise ratio were enabled by realizing a modulation/demodulation scheme, and a
very robust form of immunity from uncorrelated light sources.
Table 6.1 contextualizes the specifications of the design proposed in this research. In contrast
the system proposed here, significant works by others enable some aspect of immunity to artifacts.
For example, A dual-excitation scheme [5] provides immunity to artifacts resulting from movement or fiber bending losses, that affect photons of all wavelengths approximately equally. The
system proposed by Cui [9] improves on this by providing time-resolved spectra from the visible
band. Because this scheme provides wavelength-resolved information, it has the potential to enable correction of wavelength-selective artifacts that result from processes described in Chapter 4.
The tradeoffs include the increased level of excitation power required, and in the case of the multispectral system, the need for an array of 16 PMTs. This last feature massively inflates the cost of
the system, representing a barrier to adoption.
Table 6.1 also reveals a trend toward the incorporation of additional features in fiber photometric systems. The purpose of these features is to provide simultaneous and complementary
information for a more nuanced interrogation of brain function. The availability of both optoge-

102

netic reporters and actuators, with a wide range of spectral characteristics from which to choose, is
driving the development of multichannel excitation and emission systems as in [42], [8]. System
such as these provide a way to monitor and control the activity of several reporters and actuators
simultaneously. However, as excitation spectra for these proteins often have significant overlap,
the stimulation cannot be decoupled from sensing when they are targeting the same volume of
tissue.
Finally, fiber photometric systems need not compromise between between the specificity provided by optical reporters and the information provided by electrical recording. [42] demonstrates
that cells can be investigated with both. This yields insight into the interdependent dynamics of
the ions monitored (in this case, Ca2+ ) and membrane potential. Furthermore, while the small size
of this probe ensures the resulting signal originates from only one cell, the optical fiber, together
with genetically-encoded fluorescent reporters, provides a way to ensure the recordings are done
from a cell of interest by targeting the probe in a manner similar to the approach outlined in this
research in Chapter 5.
The main contribution of this work, therefore, is the development of a device with very high
sensitivity, conferring it the ability to measure signals resulting from a population of neurons at
ultra-low light levels compared to work by others. This enables measurements to be taken without
the nonlinearity induced by photobleaching, which simplifies subsequent data analysis and will
allow for the investigation of behavioural progressions that may take days or weeks to manifest.
In vivo reflectance measurements taken near the PVN of an awake and freely moving mouse
revealed a potential artifact correlated with neuronal activity. To our knowledge, this is the first
time wavelength-selective artifacts have been investigated for their role in distorting the detected
fluorescence trace. The data most likely represents blood volume changes during the exposure to
stress. While the results of the fiber photometry experiments using different filter sets corroborate
the hypothesis of increased blood flow during re-exposure to conditioned stress, as documented
by others [64], the number of animals investigated was very small and therefore serves as weak

103

evidence of the appearance of distortion in the trace.

The histological results suggest that the optical guidance system for surgery found a balance
between required wavelength resolution, mitigation of photodamage, cost efficiency and simplicity.
Such a system could be easily adopted in many labs to spectrally target any fluorescent structure
in the brain, enabled by swapping out a small number of optical components. The number of
successful surgeries was increased by 50% with the guidance system, and successfully identified
surgical failures. This will enable higher n values in experiments, and also lead to less wasted
experiments as the failed implants can be removed from the study. The application of the system
will improve the efficiency of photometry experiments.
More importantly, histological results for the data presented in Chapter 4 confirm that the
changes in fluorescence intensity observed, were in fact from calcium transients in CRH cells of
the PVN. To our knowledge, this is the first time that CRH cells have been investigated with fiber
photometry in freely-moving mice. These transients were detectable despite performing but one
series of pairings of the conditioned and unconditioned stimuli. This is significant as other groups
report requiring several such pairings to induce a conditioned response [55]. Such a result would
not be obtained with any modality other than fiber photometry, that, in this research, provided a
way to interrogate the activity of only CRH neurons.
Another interesting result was the consistency of calcium transients observed across repeated
trials. One would expect a decay in this response over time, as the absence of the unconditioned
stimulus erodes the conditioned response. However, this corroborates the theory that while behavioural responses from emotional memories do adapt with new information such as this, it is
instead due to override by higher cognitive processes [55]. Taken together, these results suggest
that memories of stress are formed quickly, but perhaps never forgotten.
Finally, the immediacy of the response to the conditioned stimulus supports a changing view
of the role of CRH neurons in the stress response. The calcium signal is highly correlated with
the exposure to the conditioned stimulus, indicating that CRH neuronal activity increases rapidly

104

in response to the perceived threat. In addition to its role in mediating the endocrine systems
response to stress that acts on the order of minutes, this research supports findings from others that
CRH neurons are likely implicated directly in regulating behaviour [54].
Future work for this research will involve an investigation into the nature and extent of wavelengthselective artifacts that are correlated with neural activity on the fiber photometry trace. The result of
these experiments will inform the design of a system that can correct for them, while maintaining
sensitivity. By adapting an established design for greater sensitivity, this design enabled interrogation of the behavioural correlates of calcium dynamics in the PVN of a transgenic mouse model.
The benchtop optoelectronic system has enabled discovery of the properties of the fluorescent reporter, and it may now be appropriate to attempt to miniaturize the system such that experiments
can be performed on freely-moving, untethered animals. This would support the interrogation of
the behaving brain in an even closer-to-natural setting.

105

Work Reporter
[5]

GCaMP6

Multiple
[9]

Optical Probe
Power Size
30 W 400 m

#
of
PMTs
1

100 W 2x
125 m

16

Artifact Immunity
Dual excitation of reporter and endogenous
fluorophores:
correction for wavelengthindiscriminate artifacts.
Reduced sensitivity to
fiber bending losses
with excitation power
delivered with a single
mode fiber.

Potential for correction

of wavelength-selective
artifacts.

Crimson
[8]

1.5
mW

230 m

Strong photobleaching

EYFP
OGB-1

1.0
mW

10 m

Restriction to restrained
mice enables immunity
to movement artifacts

[42]

Additional Features
N/A

Spectral information for any

320
nm-wide
band in the 300
nm to 850 nm
range.
Ratiometric
measurements.
Fluorescence
lifetime
measurements
Simultaneous
Optogenetic
Stimulation
Dual Excitation,
Emission
Simultaneous
electrophysiology
Single-unit
recording

This GCaMP6s 1 W
work

230 m

Negligible photobleaching

Simultaneous
optogenetic
stimulation
N/A

Table 6.1: Comparison of the contributions of the optoelectronic systems designed in this research
to those proposed by others.

106

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