A concentrated uorescent dye solution as a standard for use in

quantication of uorescence in a microuidic platform

Ryan J. Dee
Supervisors: Dr Lewis Grin, Dr Nicolas Szita
CoMPLEX Summer Project, August 30th 2011
Word count: 8374
Abstract
A microuidic platform capable of culturing embryonic stem cells has been developed by Reichen
et al. [10] and it is possible to image the cells in culture. On-line analysis of the images has been
proposed. The system is capable of imaging uorescent biosensors but a method of quantitative analysis
has yet to be developed. I propose a system for this analysis that involves the use of a concentrated
uorescent dye as a standard as demonstrated by Model and Burkhardt [27] to reference out spatio-
temporal inhomogeneities in the incident light eld. In a test system where GFP is used as a biosensor for
pluripotency, uorescein dye is shown to decrease the coecient of variation in images of uorescent cells.
It is also shown to uoresce with a relatively stable intensity for 100 minutes. Background uorescence
is found to be a problem with the use of GFP and an alternative biosensor is suggested. Possible dyes
to be used with the biosensor and an approach to integrating the standard with the microuidic system
are suggested.
1
1 Introduction
1.1 Fluorescence in biology
Fluorescence occurs when a molecule, known as a uorophore, in a singlet ground state, S
0
, is excited to
a higher electronic state, usually either S
1
or S
2
, through the absorption of a photon. Through a process
known as internal conversion it relaxes to a lower vibrational state of the excited electronic state very quickly
(usually within 10
12
seconds). After this process has occurred the molecule returns to the ground state by
either emitting a photon or through non-radiative relaxation. It is the emission of the photon at a longer
wavelength than that absorbed which characterises uorescence. Phosphorescence is a similar process which
occurs when the molecule transitions from the S
1
to the T
1
state. The transition from T
1
to S
0
is forbidden
and so occurs much more rarely than the S
1
to S
0
transition.
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"
%

&
%
"
'

())
!#
*+
*+
*+
!#
Figure 1: A Jablonski/Frank-Codon diagram illustrating the processes involved in uorescence and phos-
phorescence including: IC - Internal Conversion; NRR - Non-Radiative Relaxation; and ISC - Inter-System
Crossing. Solid curves S
0
, S
1
and T
1
are electronic states and dashed lines are vibrational states.
Fluorescence has proved very usefully in biology as a biosensor. By targeting uorescent molecules to dif-
ferent cellular species and also by examining the natural uorescence of cells, researchers have been able
to study isolated aspects of cell structure, dynamics and function. For example antibodies conjugated to
uorescent molecule can show the location of a particular membrane protein in a cell and/or which cells
produce the protein. Transgenic cell lines that co-express uorescent proteins with a gene of interest have
also been developed that report on the expression levels genes. The applications of these techniques go
beyond basic biological research, in to clinical and pharmaceutical research.
A large amount of uorescence imaging has been qualitative, with imaging systems being designed for
high imaging quality rather than quantication [1]. There are however some widely used systems that use
uorescence quantitatively. For example, microarrays use a uorescent labelled oligonucleotide probe to
quantify cell RNA levels, allowing the quantication of the level of up or down regulation of many genes
at once. Approaches using uorescence quantitatively allow researchers to probe the subtleties of dierence
between cells or changes in the cellular environment by examining the levels of gene products.
1.2 Platforms for quantication
In Section 1.1 microarrays were mentioned as a uorescence quantication platform. They enjoy wide usage
but require the isolation of RNA before it can be quantied, necessitating the destruction of cells. It also
2
can not be used on a cell by cell basis. Flow cytometry is a widely used technique to quantify uorescence
in labelled cells on a cell by cell basis, though this approach requires the cells to be analysed to be sus-
pended in uid, and measurements on a particular cell cannot be repeated at a later time. As cells prefer
to be attached to a surface, suspension in uid in the ow cytometer leads to a large number of the cells dying.
Approaches where the properties of adhered cells, rather than cells in ow, are measured and quanti-
ed are generally termed image cytometry [2]. Though laser scanning [3] and confocal [4] approaches to
image cytometry can be used, the simplest to implement is wide-eld microscopy image cytometry. Work in
this area of image cytometry has mainly been in slide-based image cytometry (SBC) [5] which can provide
morphological data on top of uorescence intensity data, and also allows further measurements of the same
cells to be made after an initial measurement [6]. However this method also has drawbacks, notably: long
sample preparation and analysis time; harsh xation methods which can change cellular composition; and
a lack of automation that reduces its usefulness in high-throughput applications. An alternative to SBC
is chip-based cytometry (CBC), where cells are immobilised in a microuidic chip instead of on a slide.
Hennig et al. [7] presented an iterative staining CBC method where cells were immobilised on cell-adhesive
surface. Some of these cells were xed as in the SBC methods but others were immobilised in PBS. At room
temperature and without the necessary nutrients that are in culture media these cells slowly died.
To be able to image live, healthy and growing cell populations, the cells need to be immobilised to a
suitable surface, incubated at 37

C and in suitable culture medium. However this is currently done in a

culture ask/dish stored in a large incubator with regular changing of the culture media required to remove
cell waste products and renew nutrient supplies. Imaging from these is dicult as the ask/dish needs to be
removed from the incubator each time it is to be imaged. Changing the constitution of the cell medium is
also a time consuming and invasive process aecting all cells in the ask. For images to be taken at regular
intervals a researcher has to remove and replace the ask/dish from the incubator, making automation
impossible.
Szita et al. [8] showed that bacterial cells could be fermented on a microuidic device made from poly-
dimethylsiloxane (PDMS) and poly-methylmethacrylate (PMMA), with an adequate supply of oxygen, cul-
ture medium continually perfused through the system and a benchtop incubator to house the microuidics.
In further work Reichen et al. [9, 10] showed that human cells could also be cultured in a microuidic chip
with an incubator and hosing small enough to be placed on a microscope and image. Such a system opens
up the possibility of non-invasive and precise control of the cell micro-environment, parallelization of cell
cultures and on-line imaging. With quantitative measurement of the uorescence of cells expressing a uo-
rescent biosensor in such a system it would be possible to automatically change the cell microenvironment
in response to uorescence measurements. A variety of dierent cellular environments could be trialled
automatically cutting down experimental time currently required by a large amount.
1.3 Methods for quantication
Except at a very high concentrations [11] there is a linear relationship between a uorophores concentration
and the intensity of the uorescent signal it produces. However the measured intensity in a microscopy image
is not just dependent on the uorophores concentration but also by spatial and temporal inhomogeneities
in the incident light-eld [12], the response of the camera and the optics used in the microscope. Physical
properties of the uorescence such as its lifetime [12, 13] or its polarisation [11, 14], that are not inuenced
by these eects, have been used instead of intensity for quantitative analysis, as have approaches using the
3
ratio of two dierent signals rather than the absolute value of one [15]. However the signal in these methods
is dependent on the presence of some analyte rather than the concentration of the uorophore itself, making
them unsuitable for transgenic biosensors.
The solution is to use the intensity of the biosensor as a measure of its concentration, but to reference
out the uctuations in the system. To do this requires a suitable uorescent standard. Requirements for
such a standard are that it be: easy to prepare and use; stable over time; cover the whole eld of view of
the microscope; subject to the same optical pathway as the sample; be highly reproducible; and uoresce
at a similar wavelength and intensity as the biosensor of interest. Some approaches for a uorescent stan-
dard that have been implemented include microdroplets of uorescent solutions [16], microcapilaries lled
with uorophore solutions [17, 18, 19], uranyl-containing glasses[20, 21], inorganic ion-doped bres [17] and
uorescent beads [22, 23, 24]. However there are issues with all these standards. Microdroplets require the
measurement of solution volume based on microscope images which is not very accurate. They are also
only single use and many would be required across an image to determine spatial inhomogeneity in the
incident light eld. Similar problems arise with uorescent beads which are not reliably photostable over
time and are not certain to have a reproducible uorescent intensity [4]. Uranyl-containing glasses are more
photostable but are no longer commercially available [25] and their successors the ion-doped bres have
the same imaging problems as beads in that the only cover a small proportion of the eld of view. The
microcapillaries are very much similar to the ion-doped bres although they can be produced in the lab and
have constant photostability over two months [26].
Model and Burkhardt [27] proposed a simple and eective uorescence standard for shading correction
that is also stable enough to be useful in correcting for temporal uctuations in the microscope system.
Their approach uses a concentrated solution of a uorescent dye prepared on a slide as a standard. As long
as the solution is suciently concentrated and the extinction coecient of the dye is high enough then light
entering the solution will only penetrate a small depth, such that the uorescence intensity is not dependent
on the depth of the solution. Brighter parts of the incident light eld will therefore penetrate more deeply
and excite more uorescent molecules and vice versa. In this way the inhomogeneities of the incident light
eld can be imaged and used to correct other images.
The combination of this standard with the microuidic platform described in Section 1.2 opens up the
exciting possibility of imaging and quantifying the uorescence of living cells in real-time while having a
high level of control over the cells micro-environment.
4
2 Physics of a concentrated standard
As stated in Section 1.3, the stability of the Model-Burkhardt standard is dependent on it being highly
optically dense, so that light can only penetrate a short distance in to the solution. The distance the light
travels in to the solution can be determined by the Beer-Lambert law for uids:
I
I
0
= 10
cd
(1)
where I
0
is the incident light intensity, I the intensity at depth d (in cm), c is the concentration (in M) and
is the decadic molar extinction coecient (in M
1
cm
1
). For the Model-Burkhardt standard to work, the
distance d where the intensity is a very small fraction of I
0
must be much smaller than the overall depth of
the uid. For a uorescein solution of 0.25 M concentration and extinction coecient of 76 000 M
1
cm
1
,
as measured by Mota et al. [28], the intensity decays away as it enters the uid as per Figure 2.
0 1 2
0
10
20
30
40
50
60
70
80
90
100
d (m)
P
e
r
c
e
n
t
a
g
e

o
f

I
0
Figure 2: The decay of light intensity as a beam enters a solution of 0.25 M uorescein solution.
Most of the light has been absorbed after 1 m of solution, giving an estimate of the depth of the uorescing
layer and a lower bound on solution depth for the process to work properly for a 0.25 M solution. For a
0.03 M solution most of the light has been absorbed by 10 m of solution.
Inside the uorescing layer molecules will be photobleached. Photobleaching is the process by which uores-
cent molecules in the excited S
1
and T
1
states undergo a (usually) irreversible photochemical reaction and
can no longer uoresce. The reaction is usually with another uorescent molecule or an oxygen molecule.
The rate of photobleaching will depend on the intensity of the light incident on the uorescent molecules as
the higher the intensity of light the more often the uorescent molecule will be in the excited state. It will
also depend on the lifetime of the excited states, especially T
1
as this is much longer lived than S
1
.
The replacement of photobleached uorescent molecules in the uorescing layer of the standard by un-
bleached molecules before an appreciable number have been bleached is ultimately what gives the standard
its stability. This occurs by the Brownian motion of the uorescent molecules. The root mean squared
displacement of Brownian particles in one dimension follows the Einstein equation:
r.m.s =

x
2
=

2Dt (2)
5
where D is the diusion coecient and the factor two indicating that a particle can move in a positive or
negative direction along a line. If we wish to look at the minimum time it would take to go in one direction
across a distance l we can write this as:
t =
l
2
D
(3)
Culbertson et al. [29] determined D for uorescein in aqueous solution at 25

C to be 4.25 10
10
m
2
s
1
,
which means that if we assume the uorescing layer ends roughly when incident intensity drops to 1% then
in a 0.25 M solution of uorescein a uorescent molecule would take a minimum of 2 ms to travel across
a distance of 1 m and leave the uorescing layer. Song et al [30] showed that a 10 nM uorescein sodi-
um/PBS (pH 7.6) saturated with air and constantly exposed to a 100 W mercury arc-lamp took nearly 90
mins to drop to half its original intensity. There should therefore be no appreciable photobleaching in the
time taken for a uorescein molecule to leave the uorescing layer.
Highly concentrated solutions actually show a decrease in uorescence intensity and so a concentrated
standard is much less bright under illumination than a diluted solution. The reason for this is a process
generally termed concentration quenching. Apart from the trivial mechanism of absorption of emitted uo-
rescence light in the optical dense solution, a few other mechanisms are thought to be at play. The formation
of non-uorescent dimers is thought to be one, supported by the nding that uorescence of concentrated
solutions can increase on heating [31], the opposite eect to that which occurs in dilute solutions. This is
presumably because dimer formation is less likely at higher temperature as molecules aggregate less. Other
mechanisms are thought to include Forster resonance energy transfer and collisional interactions.
6
3 Materials and methods
3.1 Fluorescent standard
A solution of 0.1 M NaHCO
3
was made by dissolving 1.7g of dry NaHCO
3
(Sigma) in 200mL of distilled
water. To make the concentrated solution, 3g of dry sodium uorescein (Sigma) was mixed with 30mL of
the 0.1 M NaHCO
3
. This solution was vortexed until no visible undissolved particles remained. It was then
centrifuged at 4000 rpm for 4 minutes, after which the volume of the solution was measured as 32.5mL,
making it 9.2% w/v or 0.25 M. Dilutions of this solution were made by adding 0.5mL, 0.25mL and 0.125mL
of the 0.25 M uorescein solution to 0.5mL, 0.75mL and 0.875 of 0.1 M NaHCO
3
solution. These solutions
were 4.6% w/v (0.12 M), 2.3% w/v (0.06 M) and 1.15% w/v (0.03 M) respectively.
Microscope slides (VWR) were cleaned by soaking in acetone while rubbing the surface of the slide for
several minutes, then soaking in isopropanol for several more minutes before being rinsed thoroughly with
reverse osmosis (RO) water and dried immediately using compressed air. Once cleaned, 25 L (unless oth-
erwise stated) of uorescein solution was pipetted on to the slide and covered with a coverslip (22 22
mm, Menzel-Glaser) such that the entire area under the coverslip was covered with solution. Occasionally,
especially if the slide was not used straight away, it was sealed using nail polish to reduce the drying out of
the solution.
3.2 Cell preparation
For experiments to measure the eectiveness of the technique on live cells, murine embryonic stem cells
(mESC) expressing GFP-tagged Oct-4 were used. Cells were adhered to a T-25 tissue culture ask using
3 mL of 0.1% gelatin solution. Cells were cultured in a medium containing: GMEM, -mercaptoethanol,
FBS, amino acids, L-glutamine, sodium pyruvate and Leukaemia inhibitory factor (LIF).
The mESC are a pluripotent cell type extracted from the inner cell mass of mouse blastocysts [32] and
on reintroduction to a blastocyst they are capable of forming any cell type including germ cells. They can
be used for a variety of purposes including developmental biology research, the creation of specic cell types
for study and as a vector for the creation of transgenic mice [33]. Several transcription factors responsible
for maintaining the pluripotency of mESC have been identied, one of which is Oct-4 (octamer-binding
transcription factor 4) which is only expressed in early embryo and germ cells [34]. This makes it a useful
marker for pluripotency of the mESC when combined with the green uorescent protein (GFP) derived from
aequorea victoria. LIF is a cytokine which suppresses mESC cell dierentiation [35]. Its removal results in
the mESC dierentiation and the loss of Oct-4 expression.
3.3 Image acquisition
Images were acquired using a Eclipse TE2000-U microscope (Nikon); equiped with DAPI, FITC and TRITC
lters; with uorescence being excited by a C-HGFI Intensilight Precentered Fiber Illuminator (Nikon) with
a 130 W C-LHGFI HG lamp (Nikon). Images were captured using 12 bit DS-Fi1 CCD camera (Nikon)
using NIS-Elements BR 3.0 (Nikon) image capture software. Most images were captured using a Plan
Achromat 10/0.25 air objective with a working area of 870653 m. Exposures ranged from 6-30 seconds
and no neutral density lters were used. To make sure the focus of the images was reproducible the images
were focused on to the edge of the coverslip. Images captured on the NIS-Elements BR 3.0 program were
automatically saved as TIFs; the majority at a resolution of 25601920. The images were saved as RGB
images with a total bit depth of 24 bits per pixel, 8 bits per pixel in each channel. For correction of images
7
of cells, both a raw uorescent image of the cells and an image of the standard were captured. A phase
contrast image was also captured and processed as described in Section 3.4.3, to be used as a cell mask for
the corrected uorescent images.
3.4 Image processing
Image processing was carried out using either MATLAB R2010a or MATLAB R2011a.
3.4.1 Image correction
Images were imported in to MATLAB using the imread function. To carry out arithmetic operations on
the images, they were converted from RGB to grayscale, and from 8 bit precision (with pixel values ranging
from 0-255) to double precision (with pixel values ranging from 0-1). To corrected for spatial and temporal
inhomogeneities in illumination, the intensity values, I, of a raw uorescent image were divided by the
intensity values, S, of the standard to produce a corrected image with intensity values I

.
I

i,j
(t) =
I
i,j
(t) B
i,j
(t)
S
i,j
(t) B
i,j
(t)
(4)
The use of the subscripts i and j indicate that the division is done on a pixel by pixel basis. B is a black
image, although if the dark current noise is suciently low then Equation 4 can be simplied to:
I

i,j
(t) =
I
i,j
(t)
S
i,j
(t)
(5)
As both the standard and raw images are illuminated by the same light eld at time t, pixels higher in
intensity in S will be higher in intensity in I and likewise for lower intensity pixels. The result of the
division therefore is the image I

which would be produced under homogenous lighting conditions. Also, at

a later time t + , all intensity values of S may have changed by a certain proportion due to uctuations
from the light source (assuming stability of the standard over the time ). The same change will be true for
all intensity values of I. Unless a cells uorescence levels have changed in the time , the ratio between all
pixels of I and S will be the same from t to t + . In this way I

is corrected for temporal inhomogeneities

as well.
3.4.2 Image statistics
Some simple statistical measure of captured images were taken to characterise the uorescence of the images
as done by Poulin et al. [36]. These included the integrated optical intensity (IOI), mean optical intensity
(OI Mean), variance of the optical intensity (OI Var) and the coecient of variation of the optical intensity
(OI CV). These are dened as:
IOI =
N

i=1
I
i
(6)
OIMean =
1
N
N

i=1
I
i
(7)
OIV ar =
1
N 1
N

i=1
(I
i
OIMean)
2
(8)
OICV =

OIV ar
OIMean
(9)
8
where I
i
is the the intensity level of i
th
pixel and N is the total number of pixels to be summed over.
3.4.3 Determining cell area
Imaging cultured cells rather than xed cells means that cell numbers and conuency change with time.
Determining the cell area by hand is a very time consuming job and is not a plausible option in an on-line
system where cell properties are measured while experiments are taking place. While the automatic identi-
cation of single cells is not yet possible it is possible to determine cell area with reasonable accuracy and
use this as a proxy for cell density.
A system for the automatic on-line determination of cell area has been developed by Jaccard [37]. His
approach uses an integrated LabVIEW, XML and MATLAB platform; where the image processing power
of MATLAB is combined with the exibility of XML and the online image capturing and equipment inte-
gration capabilities of LabVIEW.
Using an XML script a variety of MATLAB image processing routines with varying parameters can be
tested on a phase contrast cell image. The resulting image from a particular combination of image process-
ing routines and input parameters can then be scored against a ground-truth image for similarity using a
measure known as the F-score.
F =
2TP
FP +TP +P
(10)
where TP is true positives
1
, FP is false positives
2
and P is the total amount of positives
3
. The optimal
set of parameters can be found by exploring the parameter space and determining which set of parameters
gives the highest F-score. This set of parameters is then feed back in to the XML script to be used for
online monitoring. A range of pre-processing lters, thresholding techniques and post-processing operations
are used to identify the areas of the image that are covered by cells. Also, as some parameters used in the
processing are scale dependent, the image is automatically resized to a 1M resolution.
This use of phase contrast images to determine the area of the image covered by cells is useful for the
analysis of uorescent images because the level of uorescence per unit of cell area can be calculated. If only
the entire uorescence of the image were used then an increase in the level of the signal could be down to
an increase in the number of uorescent cells rather than an increase in uorescence per cell. As uorescent
cell populations can be heterogenous in their expression of a biosensor and uorescent images can be subject
to background noise a cell mask determined by processing of a phase contrast image is necessary.
3.4.4 Segmenting
While it is possible to determine those areas of an image that are covered by cells it is not possible to follow
how a particular area of cells changes through time. By simple segmentation of an image one can determine
if the statistics of one image segment is behaving dierently from the others or the image as a whole, and
thus whether the cells are changing homogeneously or not.
1
Those pixels identied as a cell by the image processing technique as well as the ground-truth image
2
Those pixels identied as a cell by the image processing technique but not in the ground-truth image
3
Those pixels identied as a cell in the ground-truth image
9
To divide an image of resolution widthheight in to equal segments one needs to nd the greatest common
divisor (GCD) of width and height. The smallest segment size is then:
size =
width
GCD

height
GCD
(11)
The number of segments will be equal to GCD. The segment size can be increased by multiplying by a
prime factor of the GCD (PFGCD). The number of segments will then be GCD/PFGCD. A suitably small
number of segments must be chosen to avoid excessive amounts of data to handle.
3.4.5 Thresholding
The mESC Oct-4 cells cultured in T-25 asks were found to have a high level of background uorescence.
This could be removed using a global threshold after the raw image had been corrected by the standard
image. To do this, normalised intensity level histograms of the entire corrected image and background
uorescence only were calculated. The background uorescence histogram was determined by selecting a
set of pixels known to be background uorescence. Ideally it would be determined by taking an image with
no cells and only background uorescence.
0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
Corrected Intensity
P
r
o
b
a
b
i
l
i
t
y


Background
Cell and background
Figure 3: Normalised histogram of corrected intensity values for a cell image with background uorescence,
and an image of background uorescence only.
As can be seen in Figure 3 the majority of pixels in the corrected image have the same intensity levels
as the background uorescence. The most appropriate method of thresholding was thought to be one
which preserved the highest number of pixels from the corrected image but removed the largest number of
background pixels. This was achieved by calculating the CDFs for both histograms and subtracting the cell
and background CDF from the background CDF. Where the dierence between these two is largest is the
optimal threshold point, as shown in Figure 4.
10
0.6 0.8 1 1.2 1.4 1.6 1.8 2
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Corrected Intensity
C
u
m
u
l
t
a
t
i
v
e

p
r
o
b
a
b
i
l
i
t
y


Background
Cell and background
Difference
Figure 4: Cumulative distribution functions for the corrected image and the background uorescence. The
point where the dierence between the two is largest gives the optimal threshold value.
11
4 Results
4.1 Cell mask error
To determine the uorescence per unit cell area cell masks were constructed using phase contrast images
of the cells and the image processing techniques described in 3.4.3, as can be seen in Figure 5. While the
match of these processed images with human determined ground truth is reasonably good it was not known
what eect any dierence from the ground truth would have on the results for the image statistics.
(a) Phase contrast image (b) Ground truth image (c) Processed image
Figure 5: Images showing the dierence between phase contrast images of the cells and the masks created
(b) by hand and (c) by image processing. Images courtesy of Nicolas Jaccard.
To determine the eect of an increase in the number of either false negatives, false positives or both, a human
determined ground truth image was used as a mask for a corrected image of uorescing Oct-4 mESCs and
noise added to the mask to model the eect of increasing false negatives/false positives. This was achieved
by:
adding salt and pepper noise to the ground truth image, multiplying the resultant image with the
ground truth image before applying it as a mask to the uorescence image to examine the eect of
increasing false negatives;
adding salt and pepper noise to an inverted ground truth image, multiplying the resultant image
with an inverted ground truth image, inverting this image and then applying it as a mask to the
uorescence image to examine the eect of increasing false positives;
adding salt and pepper noise to the ground truth image and applying this as a mask to the uorescence
image to examine the eect of increasing false negatives and false positives.
The eect of adding this noise to simulate increasing numbers of false negatives and/or false positives
caused by the image processing algorithm is shown in Figure 6. For each noise density value the simulation
was run 50 times and the mean and standard deviation of the IOI and the OI Mean (c.f. Section 3.4.2).
Unsurprisingly an increase in false negatives leads to a drop in the IOI as fewer pixel values are summed,
however the OI Mean hardly changes as the drop in number of pixels to divide by seemingly keeps pace
with the drop in IOI. However this is not the case for the OI Mean for increasing false positives, as the
OI Mean drops, and the eect is more pronounced if there are both increasing false negatives and false
positives. When applying cell mask to a uorescent image to determine the average uorescence per cell it
is therefore important to consider the type of error in the mask determined by image processing as compared
with a ground truth version. False positives will eect the OI Mean of a masked image which could lead to
erroneous results if the amount of false positives changes with time.
12
0 5 10 15 20 25 30 35 40 45 50
1.2
1.4
1.6
1.8
2
2.2
2.4
x 10
6
Noise density (%)
IO
I
(a) IOI for increasing false negatives
0 5 10 15 20 25 30 35 40 45 50
1.05
1.1
1.15
1.2
Noise density (%)
O
I M
e
a
n
(b) OI Mean for increasing false negatives
0 5 10 15 20 25 30 35 40 45 50
1.2
1.4
1.6
1.8
2
2.2
2.4
x 10
6
Noise density (%)
IO
I
(c) IOI for increasing false positives
0 5 10 15 20 25 30 35 40 45 50
1.05
1.1
1.15
1.2
Noise density (%)
O
I M
e
a
n
(d) OI Mean for increasing false positives
0 5 10 15 20 25 30 35 40 45 50
1.2
1.4
1.6
1.8
2
2.2
2.4
x 10
6
Noise density (%)
IO
I
(e) IOI for increasing false negatives and false positives
0 5 10 15 20 25 30 35 40 45 50
1.05
1.1
1.15
1.2
Noise density (%)
O
I M
e
a
n
(f) OI Mean for increasing false negatives and false positives
Figure 6: Plots of the integrated optical intensity (IOI) and mean optical intensity (OI Mean) for a cell
mask applied to uorescent image of cells with increasing noise density. For (a) and (b) the noise is applied
only to the cell area of the mask; in (c) and (d) it is only applied to the non cell area of the mask; and in (e)
and (f) it is applied to the whole mask. The mean and standard deviation of 50 runs for each noise density
value are shown.
13
Finally it is worth noting that the uorescent images that the simulation was run had a high background
uorescence. In images with a lower background uorescence it is likely that the eect of false positives on
the OI Mean would be far more pronounced.
4.2 Standard stability
To be of use in experiments looking at uorescence over time, the standard itself has to have high temporal
stability. Given that the main purpose of the standard is to adjust for lamp uctuations it is extremely
dicult to determine the true stability of the standard alone using a light source which is not highly stable.
0 50 100 150 200 250 300
0
0.1
0.2
0.3
0.4
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0.9
1
Time (sec)
O
I M
e
a
n
(a) Constant exposure, full dynamic range
0 50 100 150 200 250 300
0.066
0.0665
0.067
0.0675
0.068
0.0685
0.069
0.0695
0.07
0.0705
Time (sec)
O
I M
e
a
n
(b) Constant exposure, zoomed to t
0 100 200 300 400 500 600
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (sec)
O
I M
e
a
n
(c) Break in exposure, full dynamic range
0 100 200 300 400 500 600
0.0379
0.038
0.0381
0.0382
0.0383
0.0384
0.0385
0.0386
Time (sec)
O
I M
e
a
n
(d) Break in exposure, zoomed to t
Figure 7: OI Mean measurements for uorescein slides, scaled to show either the full dynamic range of
intensities (0-1) or zoomed to show detail. In (a)/(b) an image was taken every 10 seconds and the camera
exposure was 6 seconds. In (c)/(d) an image was taken every 17 seconds and the camera exposure was 10
seconds, except for a 2 minute 30 second break where no images were taken. Lamp exposure was constant
except in the break in image capture.
In the rst experiments of stability, timescales of a few minutes were measured, with the uorescein being
constantly exposed to the light source. In the rst measurements taken of uorescein, a clear although very
small drop in intensity of around 0.0035
4
was noted after constant exposure to the lamp for 5 minutes as can
be seen in Figure 7b. To determine whether this drop in intensity was due to the uorescein photobleaching
or the lamp uctuating a further experiment was carried out where the exposure of the uorescein to the
exciting light source was halted midway through an experiment for a few minutes and then resumed. If the
change was due to the bleaching of uorescein then any decrease in intensity should continue on at the same
4
The intensity scale runs from 0-1 and so this change is eectively a change of 0.35 percentage points.
14
rate when the uorescein was re-exposed to the light. In Figure 7d it can be seen that this is not the case.
This indicates that any decrease in intensity from photobleaching of the uorescein is much smaller than
lamp uctuations on these small timescales.
When the stability of the uorescein was examined over a longer periods of time there was still no evi-
dent decrease in the intensity due to photobleaching when the uorescein was constantly exposed to the
light, as can be seen in Figure 8. Looking at the overall uctuation across the entire period, and dening
this to be the dierence between the maximum and minimum intensity across the time period, it was found
to lie in the region of 0.0132 to 0.0185. A separate experiment where the uorescein was only exposed while
the camera was capturing an image was found to have a uctuation of 0.0210, adding more credence to the
assumption that all the changes in intensity were due to lamp uctuations and not to any change in the
uorescein.
0 10 20 30 40 50 60 70 80 90 100
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (mins)
O
I M
e
a
n
(a) Constant exposure to light
0 10 20 30 40 50 60 70 80 90 100
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (mins)
O
I M
e
a
n
(b) Constant exposure to light
0 10 20 30 40 50 60 70 80 90 100
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Time (mins)
O
I M
e
a
n
(c) Only exposed to light when image taken
Figure 8: OI Mean measurements for uorescein slides for: (a) 30 second camera exposure constantly
exposed to light with image taken every 10 minutes; (b) the same experiment as (a) on a separate day; and
(c) 6 second camera exposure with uorescein only exposed to light while image was taken, with an image
taken every 3 minutes.
As it is the high concentration of the uorescein that makes it uoresce steadily over extended periods,
four dierent concentrations of uorescein were exposed to the light source for 30 minutes each to test their
stability. The results of this experiment can be seen in Figure 9. Two important features can be noted from
this: that the stability of the uorescein does decrease with decreasing concentration and that the initial
intensity of the uorescein increases with decreasing concentration. The uctuation of the intensity, as
15
dened above, increased from 0.0056 at 9.2% w/v, to 0.0109 at 4.6% w/v, to 0.0402 at 2.3% w/v and nally
to 0.1943 at 1.15% w/v. It would appear from these results that the relationship between the concentration
and stability is not linear. On the timescale of half an hour, 9.2% w/v and 4.6% w/v concentrations are
relatively stable, whereas at the lower concentrations it is clearly much less stable.
0 5 10 15 20 25 30
0
0.1
0.2
0.3
0.4
0.5
0.6
Time (mins)
O
I

M
e
a
n


1.15% fluorescein and 1s exposure
2.3% fluorescein and 2s exposure
4.6% fluorescein and 4s exposure
9.2% fluorescein and 20s exposure
Figure 9: Plot showing the measured intensity of four dierent concentrations of uorescein over 30 minutes
of continuous lamp exposure. Camera exposure times were adjusted to avoid over or under exposure.
4.3 Spatial correction
As stated in Section 3.4.1, the standard can be used to correct spatial as well as temporal inhomogeneities.
The OI CV is a good measure of the spatial inhomogeneity, as it measures the variance in intensity across
an image compared with the mean intensity of the image. Using Equation 5 to correct a raw image should
lead to a drop in OI CV as the variance due to an inhomogeneous light eld is removed. When a standard
image was applied to a raw cell image, a drop in the OI CV from 15.80% to 14.74% was achieved. When
a standard image was used to correct another standard image taken straight after the rst, the change in
OI CV was from 10.20% to 2.93%. Ideally two standard images taken one after the other should be exactly
the same and on division should have a OI CV of 0%, however due to random uctuations in the light eld
and the response of the camera this is not the case. With increasing time the dierence between standard
images due to these random uctuations grows, as can be seen in Figure 10 where the OI CV of standard
images, corrected by a standard image taken just before the rst time point, were measured.
4.4 Intensity of the standard
The camera exposure time used by Model and Burkhardt to obtain images of their standard was only 0.2
seconds. It was found that to obtain images in a suitable intensity range an exposure time of at least 6
seconds was required, although 30 seconds was usually used. The cause of this was that the lamp used in
experiments was of a lower intensity than that used by Model and Burkhardt, which will be discussed in
Section 5.2.
When imaging cells it is important that they are exposed to as little of the excitatory light as possible
without compromising image quality to avoid photobleaching of the biosensor or phototoxicity to the cell.
This can be done by keeping camera and light source exposure low but may necessitate the inclusion of
16
0 10 20 30 40 50 60 70 80 90 100
2.95
3
3.05
3.1
3.15
3.2
3.25
3.3
Time (mins)
O
I

C
V

(
%
)
Figure 10: OI CV of standard images corrected by a separate standard image taken just before the standard
image at time zero.
neutral density lters. The standard also needs to be exposed for long enough to capture an image of good
quality but not too much longer than the cells are exposed. If it is exposed for much longer then it will
average the lamp uctuations over that time rather than creating a snapshot of the lamp intensity at the
time of imaging the cells. I therefore examined the factors aecting the intensity in the standard.
The choice of objective aects the intensity of light incident on the slide and therefore the uorescence
intensity. Figure 11 shows that as the magnication is increased, so does the OI Mean of an image, except
at 40 magnication. The reason why this does not follow the trend is not entirely clear although their was
a noticeable shadow across the images at 40 that probably caused this unusual result. As it is necessary to
use the same optical setup in images taken of the standard and of cells the magnication cannot be changed
to reduce the exposure time.
0 5 10 15 20 25 30 35 40 45
0
0.1
0.2
0.3
0.4
0.5
Magnification
O
I

M
e
a
n
Figure 11: Mean and standard deviation of the OI Mean for four images at 4, 10, 20 and 40 magni-
cation, with a camera exposure time of 30 seconds.
According to theory, the depth of the uorescein solution should not aect the intensity, as it would at
17
lower concentrations. As the uorescein solution covers the whole of the coverslip only, the area covered by
uorescein does not really change with changing volume, only the depth of the uorescein does. To test the
theory, slides made with dierent volumes (5, 10, 15, 20 and 25 L) of uorescein were imaged and their OI
Mean was calculated. An image at each volume was taken one after the other and the processed repeated
four times to try and remove the eect of lamp variations. To test the hypothesis that the intensity does not
change with increasing depth a one way ANOVA was used to compare the ve distributions to determine
if they were all from the same distribution. The p value for this test was 0.0354, which is enough to accept
that there is no change in intensity at the 99% condence level, and therefore conclude that the depth of
solution is not a factor aecting intensity.
Some factors that did have a small but noticeable aect on some of the image of the standard were de-
bris on the slide and camera noise. The debris included small particulate matter and hairs and could cause
areas of the standard slide to look darker than they should be as the debris absorbed the uorescent light.
Getting the slide clean enough to avoid this debris was challenging and was not achieved here. The cam-
era noise that was a concern here was not dark current noise, as we found this to be very low, but from
dead pixels in the image. While these had no noticeable eect on the mean intensity it did lead to a few
high intensity pixels, making the maximum much higher than it should be. This causes a problem when
attempting to rescale intensity values after correction by the standard to view the image.
To increase the intensity of the standard without changing the exposure time a lower concentration of
uorescein can be used. The non-linear increase in initial intensity with decreasing concentration can be
seen in Figure 12. This increase in intensity though comes at a cost of decreased stability. The intensity of
the slide could also be increased by increasing the temperature of the standard as mentioned in Section 2
although this was not tested.
1 2 3 4 5 6 7 8 9 10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Concentration (% w/v)
N
o
r
m
a
l
i
s
e
d

i
n
i
t
i
a
l

i
n
t
e
n
s
i
t
y
Figure 12: Initial intensity for dierent concentrations of uorescein, normalised to 1 second camera exposure
levels.
18
4.5 Fluorescence, cell and image segmentation
Figure 13 shows T-ask cultures of mESC expressing GFP-tagged Oct-4. The contrast in the image is poor
because of a high background level of background uorescence not associated with the GFP. This background
comes from the autouorescence
5
of proteins in the cell media. There will also be autouorescence from
cellular proteins but this will be much smaller than that from the cell media. This background uorescence
is a problem as those cells which arent expressing GFP will appear to be uorescent when they are not and
the mean uorescence per cell will therefore be raised.
Figure 13: Fluorescence image of mESC expressing GFP-tagged Oct-4 after correction by the standard.
To remove the background uorescence the global threshold technique described in Section 3.4.5 was applied
to the image in Figure 13 and a binary image of areas associated with cell uorescence and those that were
not was produced. The results of this were compared with a human determined ground truth binary image
of the cell uorescence and an F score calculated to be 0.8026. An F score for the processed cell mask
produced from a phase contrast image of the cells was also calculated and found to be 0.8234. This would
seem to indicate that thresholding does not improve the results until one considers that what we are most
interested in is the average uorescence per unit cell area. The processed cell mask was applied to the
thresholded image and the F score was then calculated to be 0.8895. When the OI Mean was calculated for
pixels corresponding to cells when the background uorescence had not been removed it was found to be
1.1666; however with thresholding to reduce background uorescence intensity values to zero it was calcu-
lated as 0.9114, much closer to the value of 0.8791 calculated by using the ground truth image to reduce all
background uorescence to zero.
By using the Jaccard method it is possible to segment the image in to cell and none cell areas, and by
using the threshold method in Section 3.4.5 the image can also be segmented in to areas of biosensor u-
orescence and background uorescence and by combining the two it is possible to get a better measure of
biosensor uorescence per unit cell area. As discussed in Section 3.4.4 it may also be useful to segment the
image in to dierent sections so that the uorescent properties of dierent areas of the eld of view can be
compared. In Figure 14 the image has been segmented in to four equal sized sections and the OI Mean of
pixels corresponding to cells, calculated with and without uorescence segmentation. It shows that while
the thresholding brings the OI Mean to around the same level as the ground truth GFP uorescence the
5
The natural uorescence of a substance.
19
relationship between dierent segments is not the same in thresholded image compared with the ground
truth GFP uorescence. It should be noted that the ground truth GFP is determined by human subjectivity
and as such dierent people would create a slightly dierent ground truth image. This or the ecacy of the
thresholding methodmay account for the dierence observed.
1 2 3 4
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Segment
O
I

M
e
a
n


Ground truth and cell mask
Thresholding and cell mask
Cell mask only
Figure 14: OI Mean for four segments of a uorescence image for pixels corresponding to cells as determined
by applying a cell mask made using the Jaccard method.
20
5 Discussion
5.1 Biosensor and standard
The choice of uorescent dye to use for a uorescent standard is determined ultimately by the biosensor
being imaged. To ensure that the standard is imaged using the same optical setup as the biosensor it must
be imaged using the same lter block as the biosensor. There should therefore be a close match in the
excitation/emission spectral peaks of both uorescent dye and biosensor and the transmittance peak of the
lter block. For the wild type GFP that are expressed in the cells imaged here the excitation spectral
peak is at 395-397 nm or 470-475 nm
6
and the emission spectral peak is at 504nm [38]. For uorescein the
excitation/emission spectral peaks 460 nm/515 nm [39]. The FITC lter block that is used transmits at
465-495 nm and 515-555nm, with the dichroic mirror reect wavelengths shorter than 505 nm[40]. There
is some margin for error in the matching of excitation, emission and transmission peaks that is determined
by the FWHM of the peak, however too large an oset of the excitation/emission spectral peaks from the
transmission/reection of the lter block will lead to a decrease in the signal to noise ratio as uorescence
signal drops. The agreement here between all three was reasonably good.
There are many other biosensors that could be used instead of GFP, including a whole range of GFP
mutants. Reasons for choosing dierent biosensors may include the specic biological activity they are
reporting, their quantum eciency or photostability. They can also be chosen to improve the signal to
noise ratio in images. In Section 4.5 the impact of autouorescence on the images was discussed. Aut-
ouorescence in animal tissue is usually emitted in the blue/green region, the notable exceptions being the
porphyrins which emit in the red [41]. To reduce the amount of autouorescence in images, a biosensor
which emits in the red part of the spectrum, where there are fewer autouorescent species, could be used.
A widely used red uorescent protein that can be used as a marker for gene expression is mCherry. The
excitation/emission spectral peaks reported by Seefeldt et al. [42] for mCherry was 586 nm/607 nm. The
only uorescent dye other than uorescein to be tested by Model and Burkhardt was rhodamine 6G, which
has excitation/emission spectral peaks at 525 nm/550 nm [41] making it unsuitable for use with mCherry.
Other possible uorescent dyes are sulphorhodamine 101 or Texas Red with excitation/emission spectral
peaks at 596 nm/615 nm; Alexa Fluor

568 with excitation/emission spectral peaks at 578 nm/603 nm[43];

or TRITC with excitation/emission spectral peaks at 555 nm/580 nm[41]. The solubility of these dierent
compounds is not well documented in the literature although the AlexFlour

dyes are said to be more water

soluble than their uorescein or rhodamine counterparts, to have a high extinction coecient and be less
susceptible to self-quenching[44]. Ideally the dye used should not just match the spectra of the biosensor
being used but be soluble in an easy to handle solvent such as water
7
, be of a good brightness, photostable,
inexpensive and in a suitable form. Finally the matching of the biosensor and dye with the lter block set
needs to be considered. The microscope used in these experiments was equipped with a TRITC lter which
transmits at 515-565 nm and 550-660nm, with the dichroic mirror reect wavelengths shorter than 565
nm[40]. This lter set would probably not be ideal with mCherry, sulphorhodamine 101 or Alexa Fluor

568 and so a new lter set would need to be considered.

5.2 Illumination
As was mentioned in Section 4.4, the lamp that was used in these experiments was probably illuminating at
a much lower intensity than that used by Model and Burkhardt. Although their bulb was 100 W and the one
6
There are two excitation peaks for wt GFP.
7
Model and Burkhardt found that methanol was a dicult solvent to work with because of how quickly it evaporated.
21
used in these experiments was 130 W, the intensity of mercury lamps is known to change throughout their
lifetime. The drop in intensity over the lifetime of both mercury and metal-halide lamps can be signicant
as can be seen in Figure 15. Nikon gives a guideline lifetime for their C-LHGFI HG lamp
8
of 2000 hours
[45] and the runtime of the lamp used was over 2500 hours. As well as being liable to failure its intensity
could well have been less than 50% of its original intensity during its use in these experiments.
!
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'!!
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4
5
6
,
5
2
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6
7

-
8
3

9:;
<,6=>?.=>*@,
<,1A017
Figure 15: Comparison of intensity change during lifetime of three dierent uorescence microscopy light
sources. Reproduced from Beacher [46].
It is worth considering whether a better illumination light source for quantitative uorescence might be
LEDs rather than mercury arc-lamps, given its improved stability and lifetime as evident in Figure 15.
LEDs have other advantages besides an improved stability and lifetime. These include a narrow waveband
so that the light-source can be chosen to match the biosensor being used. In wide waveband light-sources
such as mercury arc-lamps, wavelengths not associated with the excitation of the biosensor are blocked by
lter sets. This means that a lot of power is wasted on wavelengths which arent needed, but also the ltering
is not a 100% eective and this leads to noise in the uorescence images from photons not associated with
the biosensor. They could also be phototoxic to cells. There is also no need to allow for warm up time when
using an LED light-source. With LEDs now capable of an output intensity similar to mercury arc-lamps
and available in a range of dierent wavelengths they are a viable alternative.
5.3 Fluorescent standard design
As described in Section 3.1, the uorescent standard used in this project was prepared on a glass slide and
sealed with a glass coverslip. This approach would not be suitable for integration with the microuidic
bioreactor platform developed by Reichen et al [9, 10]. Instead the standard must either be included with
the bioreactor chip or miniaturised and included elsewhere on the platform in a position where it can still
be imaged with the same optical set up as the cells in the bioreactor.
5.3.1 Integrated standard
The microuidic bioreactor described by Reichen et al. [9] consists of a polydimethylsiloxane (PDMS) chip
which is clamped by top and bottom plates made of polycarbonate (PC) on to a tissue culture polystyrene
(TC-PS) slide. The main issue with including the standard on the same chip as the bioreactor is the per-
meability of PDMS. A well lled with uorescein solution included on-chip would dry up, as water is known
8
An ultrahigh pressure mercury lamp.
22
to diuse in to PDMS [47], leading to bubble formation whose optical eects would destroy the corrective
ability of the standard. To avoid drying uorescein solution could instead be pumped through the chip in a
separate channel to the cell culture medium and imaged in the channel. This would also require a separate
inlet, outlet and bottle for the uorescent dye which would increase the size of the chip considerably. The
diusion of water in to the PDMS is not the only problem with using uorescein solution with PDMS. Other
uorescent molecules of a similar molecular weight as uorescein have been shown to be absorbed by PDMS
[47]. There would need to be a suitable separation between the standard channel and the bioreactor to avoid
leakage of uorescein. As the chip is imaged using an inverted microscope the bottom of the channel would
need to be the TC-PS slide; a PDMS channel bottom would absorb uorescein that would be exposed to
the exciting light source. The PDMS would absorb a nite amount of the uorescein which would then be
photobleached leading to a drop in signal not related to changes in the exciting light source. Also, with new
solution being pumped through the imaging area constantly, this standard would have more in common
with the microcapillaries mentioned in Section 1.3 than the Model-Burkhardt standard.
While there are diculties in including a uorescein standard on the same chip as the bioreactor it would
still be possible. However the permeability issues of PDMS become much more severe when uorescent dyes
such as Rhodamine 6G, which require methanol as a solvent, are used instead of uorescein. Methanol is
highly volatile and will evaporate much faster than water, leading to drying occur much more quickly than
with water. It is also highly likely that methanol could permeate in to the bioreactors, causing damage to
the cells. Methanol also causes a swelling of around 2% to occur in PDMS [48], which would cause damage
to the chip.
5.3.2 Separate standard
To save on space and complexity a separate or o-chip standard could simply be a well lled with
uorescent solution instead of a channel with uorescent solution pumped through it. To avoid the problems
of drying and absorption of uorescent molecules as described in Section 5.3.1 it would need to be made
of a highly impermeable material while still being optically transparent. Glass would be eective but due
to its brittle nature it is dicult to machine. A more versatile, easily machinable and cheaper material is
!"#$%&'(")&*+
-.(-*'&*+
!/01 *&2+
0#."'+-%+)*
-"#.3") 4+##
5#&627)8 -%'+4
9 66
Figure 16: Top down view of a possible design for a separate uorescent standard with approximate scale.
polycarbonate. A well of an easily machinable but arbitrary depth and with a diameter large enough to
easily include the entire objective focused light eld (a diameter greater than 1 mm should achieve this for
a 10 objective) could be cut in the polycarbonate. The well would then be lled with enough uorescent
solution that a convex meniscus forms above the plane of the polycarbonate substrate. To ensure that a
23
tight seal is form and no bubbles are included in the well, a polycarbonate cover could be screwed on to the
polycarbonate substrate with PTFE tape between the two to ensure a tight bond. The excess uorescent
solution in the meniscus should be forced out when the two pieces of polycarbonate are sealed together
forcing any air out of the vicinity of the well with it. Figure 16 shows a what such a standard chip might
look like.
6 Conclusion
Of all the options investigated the use of a Model-Burkhardt as a standard to correct for spatial and tempo-
ral inhomogeneities in the excitatory light source and the microscopes optical components response appears
to be the best for the integration in to a microuidic bioreactor system for the on-line analysis of live cells.
It has been show to be stable for up 100 minutes of constant exposure with intensity uctuations of only 1
to 2 percentage points, which can be explained by uctuations of the mercury arc-lamp that was used in the
experiment. When images of cells were corrected they showed a decrease in their CV of around 1 percentage
point and the background uorescence was homogeneous enough to successfully use a global threshold to
remove most of it. The threshold method employed here could be improved by further post processing of
the thresholded image.
The system that is proposed for integration in to the microuidic bioreactor of Reichen et al. [9, 10] is
a well of uorescent dye on a polycarbonate chip no larger than about 2 1 cm that can be easily placed
somewhere in the incubator housing near to the bioreactor chips and with the uorescing layer in the same
imaging plane as the cells in the bioreactors. The well would need to be sealed well enough to avoid the
loss of solvent and inclusion of bubbles. In terms of the biosensor/uorescent dye that would be used in
the system, GFP with uorescein was found to work quite well, with uorescein being very easy to prepare
and handle. It was found that the background uorescence in this part of the spectrum was high due to
autouorescence. While this could be removed with a global threshold method a much more precise way
of increasing the signal to noise ratio would be to use a biosensor/uorescent dye in the red part of the
spectrum. A selection of possible dyes to use with mCherry, a red uorescent protein, were suggested. Fi-
nally a switch from mercury arc-lamp excitation to LEDs was proposed because of their narrower waveband,
increased stability and longer lifetimes.
Acknowledgements
I would like to thank my supervisors Dr Lewis Grin and Dr Nicolas Szita for the opportunity to work on
this project and for the help and guidance they provided. I would also like to thank those working in the
microuidics lab, especially Dr Matthew Davies for his practical advice in the lab on slide cleaning and for
his invaluable help on the issues of integrating the standard in a microuidic setting. Finally I would like
to thank Nicolas Jaccard for his helpful advice, listening ear, the pictures supplied for the project and also
for his guidance in the lab.
24
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