Fluorescence and Confocal

Microscopy

Yvona Ward
Cell and Cancer Biology Branch
 OUTLINE

1. Immunofluorescent Staining
2. Conventional Fluorescent Microscopy
3. Confocal Microscopy
4. Applications
 Immunofluorescent Staining
Immunofluorescent staining makes use of antibodies to locate and
identify patterns of protein expression in cells.

Primary antibody binds to antigen.

Antibody-antigen complex is bound by a secondary antibody

conjugated to a fluorochrome.

Upon absorption of high energy light, the fluorochrome emits light at

its own characteristic wavelength (fluorescence) and thus allows
detection of antigen-antibody complexes.

Suitable for: 1. frozen, non-fixed tissues and ethanol fixed tissues

2. paraformaldehyde-fixed or methanol/acetone-fixed
cells
 Basic Staining Technique
Cell Preparation
1. Culture cells on a glass coverslip in a 24-well plate. Cells may be transfected directly on the
coverslip.
2. Fix cells using paraformaldehyde or methanol/acetone and then wash them 3 times in PBS

Cell Permeabilization
1. Incubate fixed cells in 1% Triton X-100 in PBS+0.02%BSA for 2 minutes at room temperature.
2. Wash the cells 3 times with PBS.

Immunofluorescent Cell Staining

1. Incubate cells with a blocking solution to minimize non-specific staining
2. Incubate cells with a polyconal or monoclonal antibody specific for the protein of interest.
3. Incubate cells with a secondary antibody directed against the primary antibody.
The secondary antibody must be conjugated to a fluorochrome

ANTIGEN PRIMARY SECONDARY FLUOROCHROME

ANTIBODY ANTIBODY
Giannakakou et al., Nature Cell Biology,2000

PRIMARY ANTIBODY PRIMARY ANTIBODY

sheep anti-p53 polyconal mouse anti-a tubulin monoclonal

SECONDARY ANTIBODY SECONDARY ANTIBODY

Texas Red conjugated anti-sheep FITC conjugated anti-mouse
 Direct Staining of Cell Structures
Organelle Probes
Mitochondria MitoTracker mitochondrial membrane potential

Lysosomes LysoTracker hydrolytic activity of enzymes

ER and Golgi Lectin conjugates lipid composition

Other Probes
Stress fibers Phalloidin-conjugaes bind F-actin

Nuclei DAPI binds to minor groove of ds-DNA

MitoTracker-Orange CMTMRos 4’,6-diamidino-2-phenylindole (DAPI)

TRAP1 Mouse Monoclonal

+ Goat anti-Mouse-FITC

Felts et al., JBC, 2000

 microtubules centrosomes

Anti-tubulin MoAb Anti-pericentrin PoAb

Goat anti-mouse-Rhodamine Goat anti-mouse-FITC

DAPI
nucleus MERGE
 Stress Fibers Focal Adhesions

Anti-vinculin MoAb
Rhodamine-Phalloidin
Goat anti-mouse-FITC
 Translocation of mutated protein to the mitochondria

deep red-mitotracker GFP-fusion protein MERGE

 Use of Biotinylated Antibodies
Streptavidin is a bacterial protein that specifically
binds biotin. This interaction may be used to label
cellular components.

PRIMARY SECONDARY
ANTIGEN FLUOROCHROME
ANTIBODY ANTIBODY

BIOTIN STREPTAVIDIN
 Rabbit anti-Factor H
Guinea Pig anti-Insulin PoAb Biotinylated Goat anti-Rabbit
Donkey anti-Guinea Pig-Cy5 Streptavidin-FITC

Mouse anti-Glucagon MoAb

Goat anti-Mouse-Rhodamine Martinez et al., J. Endocrinol., 2001
Conventional
Fluorescent
Microscopy
 Confocal Microscopy Core

Inverted Scope Upright Scope

 Preparation of Stained Specimens
For Microscopy
Specimen Mounting
In order for the stained specimen to be visualized on a fluorescent
microscope, it needs to be mounted onto a slide using an appropriate
mounting medium.

Mounting medium is usually a PBS/Glycerol mix and is commercially

available.

•Biomeda Corporation Aqueous Mounting Medium

•Molecular Probes SlowFade
Specimen Photobleaching
 One of the major problems in microscopic examination of
fluorescent specimens is the tendency of fluorochromes to lose
fluorescence upon excitation by a high energy light source.

 Free radicals generated during fluorochrome excitation are

responsible for this quenching or photobleaching.

 Various chemical agents that scavenge free radicals may be

added to the mounting medium to preserve specimen brightness.

Sigma trans-pyridine-2-azo-p-dimethylaniline (PADA)

Fluorescence
Molecules absorbing the energy of electromagnetic radiation
will jump to a higher energy level. When certain excited molecules
return to the ground state they emit radiation. This phenomenon
is known as fluorescence. Fluorescent molecules are known as
fluorochromes or fluorophores.
Absorption Spectra of Fluors Commonly
Conjugated to Secondary Antibodies

Fluorochrome Absorption Emission

Cascade Blue 400 420

Fluorescein 494 518
Rhodamine 570 590
Texas Red 595 615
Cy5 650 670
Fluorescence Microscopy
Since the molecules used for immunofluorescence emit light in the
visible range, it is possible to detect them with a microscope.
 A mercury lamp is used to illuminate the sample with UV light through the
objective lens. A dichroic mirror reflects short l and transmits longer l.

 The fluorescence emitted from the sample passes back through

this mirror, but the UV light does not.

 An excitation filter in front of the mirror will control the excitation wavelength.

 An emission filter in front of the eyepiece will control the wavelength

of the emitted light. eyepiece

excitation
emission filter
filter

Hg
* dichroic mirror

specimen
 Numerical Aperature (NA)
A solid cone of light that hits the specimen

Lenses with a high NA have a short working distance but,

allow more light to be captured from the specimen.

Example:
Phase contrast lens low NA long working distance
High resolution 100x high NA short working distance
Confocal Microscopy
CCBB Confocal Core Facility (1999-2006) UV 351,364nM
Argon 488nM
Zeiss LSM510 with 4 color capability HeNe I 543nM
HeNe 2 633nM

Building 37 Room 1035

 What is Confocal Microscopy?
Laser Scanning Confocal Microscopy
Confocal Scanning Laser Microscopy

Confocal microscopy is a powerful tool for generating high-resolution images

and 3-D reconstructions of a specimen.

In confocal microscopy a laser light beam is focused onto a fluorescent specimen

through the objective lens. The mixture of reflected and emitted light is captured
by the same objective and is sent to the dichroic mirror. The reflected light is
deviated by the mirror while the emitted fluorescent light passes through a
confocal aperature (pinhole) to reduce the “out of focus” light. The focused light
then passes through the emission filter and proceeds to the photomultiplier.

In order to generate an entire image, the single point is scanned in an X-Y manner
as the laser focus is moved over the specimen.
Simplified Optics of a Confocal Microscope
 The LSM 510

To the Specimen From the Specimen

(1) optical fibers (1) optical fibers
(4) main dichroic beam-splitter (4) main dichroic beam-splitter
(5) scanner mirrors (7,8,9) secondary dichroics
(6) scanning lens (10) pinhole diaphragm
(11) emission filters
(12) photomultipliers
 Why is Confocal Microscopy Better?

1. More Color Possibilities

Because the images are detected
by a computer rather than by eye,
it is possible to detect more color
differences.
 Insulin-Cy5 CRLR-FITC

Glucagon-Rhodamine Overlay
 Why is Confocal Microscopy Better?
2. Less Cross Talk
In most applications, fluorochromes have overlapping emission spectra. Hence, the
emission signals cannot be separated completely into different detection channels
resulting in “bleed through” or cross talk.

However, if the fluorochromes have distinct excitation spectra, the fluorochromes

can be excited sequentially using one excitation wavelength at a time. This is only
possible with confocal systems that offer the multitracking feature.
 Standard
Microscopy Multitracking

Brain Slice nerve fibers (FITC)

cell nuclei (propidium iodide)

Courtesy Dr. Schild, University of Gottingen

 Why is Confocal Microscopy Better?
3. Optical Sectioning of Objects Without Physical Contact

Zebra fish embryo wholemount

Neurons (green)
Cell adhesion molecule (red)

Monika Marks, Martin Bastmeyer

University of Konstanz
Cultured Cells
Formation of Acini in a 3-D Matrigel Matrix
Three-dimensional culture
of MCF10A mammary
epithelial cells on a
reconstituted basement
membrane leads to the
formation of polarized, growth
arrested acini-like spheroids
that recapitulate several
aspects of glandular
architecture in vivo.

Introduction of oncogenes into

MCF10A cells results in
distinct morphological
phenotypes
Ras V12 Empty vector

b-catenin
DAPI
MERGE
 Why is Confocal Microscopy Better?
4. Three-Dimensional Reconstruction of Specimen

3D shadow projection
Tight junctions (red)
Cytoskeletal structures (green)

Prof. Wunderli-Allenpach
ETH, Zurich
 Animated 3-Dimensional Reconstruction

Laser Scanning Microscopy

LSM510
3D for LSM
www.Zeiss.com
 Animated 3-Dimensional Reconstruction

Mitosis
www.Zeiss.com
 Why is Confocal Microscopy Better?
5. Improved Resolution

Rat Cerebellum
Astrocytes (green)
Mn dismutase (red)

Jorg Lindeman
University of Magdeburg
 Applications
1. Colocalization

2. Live Cell Imaging

FRAP/FLIP
GFP-Fusion

3. FRET
 Colocalization of Proteins

Colocalization of up to 4
different proteins

Colocalization does not

mean interaction

Decreased cross talk with

multitracking feature
Colocalization of insulin and calcitonin receptor-like receptor

Insulin-Cy5 CRLR-FITC

Glucagon-Rhodamine
Colocalization
p53 a-tubulin

Proteins may colocalize but not necessarily interact

Fluorescence Resonance Energy Transfer

The high resolution of

a confocal microscope
allows us to study the
physical interaction of
protein partners.
What is FRET?
FRET is the non-radioactive transfer of photon energy from an excited fluorophore (the donor)
to another fluorophore (the acceptor) when both are located within close proximity (1-10nm).
Using FRET one can resolve the realtive proximity of molecules beyond the optical limit of a
light microscope to reveal
(1) molecular interactions between two protein partners,
(2) structural changes within one molecule (eg. enzymatic activity or DNA/RNA conformation),
(3) ion concentrations using special FRET-tools like the CFP-YFP cameleon

CFP is excited by light and emits light

No FRET CFP is more than 10nm from YFP
Signal YFP is not excited and does not emit light

CFP is excited by light and emits light

FRET CFP is in close proximity to YFP
Signal YFP emits light
 The Principle of FRET
An excited fluorophore (donor) transfers its excited state energy to a light
absorbing molecule (acceptor). This transfer of energy is non-radioactive due
primarily to a dipole-dipole interaction between donor and acceptor.

There are only certain pairs of fluorophores suitable for FRET experiments
since, besides other prereqisites (eg. dipole orientation or sufficient fluorescence
lifetime), the donor emission spectrum has to overlap the excitation spectrum of
the acceptor. Known FRET pairs are CFP/YFP, BFP/GFP, GFP/Rhodamine,
FITC/Cy3.

Energy Diagram of CFP/YFP FRET:

CFP donor is excited but most of its energy
does not result in cyan emission. Instead,
It is transferred to the YFP acceptor. Thus
Resulting emission is mostly yellow.
FRET
Region of
interest

Two channel (CFP,YFP) time series

Two channel (CFP,YFP) time series

Confocal Microscopy is a
powerful tool for studying
signaling mechanisms

Yvona Ward
Building 37 Room 1066
301-594-2645
yward@helix.nih.gov