Fluorescence and Confocal Microscopy

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Fluorescence and Confocal Microscopy

• Yvona Ward
• Cell and Cancer Biology Branch

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OUTLINE

• Immunofluorescent Staining
• Conventional Fluorescent Microscopy
• Confocal Microscopy
• Applications

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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-fi
  xed
• cells

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Basic Staining Technique

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

• Cell Permeabilization
• Incubate fixed cells in 1 Triton X-100 in
  PBS0.02BSA for 2 minutes at room temperature.
• Wash the cells 3 times with PBS.

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Giannakakou et al., Nature Cell Biology,2000
PRIMARY ANTIBODY sheep anti-p53
polyconal SECONDARY ANTIBODY Texas Red
conjugated anti-sheep
PRIMARY ANTIBODY mouse anti-a tubulin
monoclonal SECONDARY ANTIBODY FITC conjugated
anti-mouse
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Direct Staining of Cell Structures
Organelle Probes Mitochondria MitoTracker mitoc
hondrial membrane potential Lysosomes LysoTracke
r 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
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MitoTracker-Orange CMTMRos
4,6-diamidino-2-phenylindole (DAPI)
TRAP1 Mouse Monoclonal Goat anti-Mouse-FITC
Felts et al., JBC, 2000
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centrosomes
microtubules
Anti-tubulin MoAb Goat anti-mouse-Rhodamine
Anti-pericentrin PoAb Goat anti-mouse-FITC
DAPI
nucleus
MERGE
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Stress Fibers
Focal Adhesions
Anti-vinculin MoAb Goat anti-mouse-FITC
Rhodamine-Phalloidin
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Translocation of mutated protein to the
mitochondria
deep red-mitotracker
GFP-fusion protein
MERGE
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Use of Biotinylated Antibodies
Streptavidin is a bacterial protein that
specifically binds biotin. This interaction may
be used to label cellular components.
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Rabbit anti-Factor H Biotinylated Goat
anti-Rabbit Streptavidin-FITC
Guinea Pig anti-Insulin PoAb Donkey anti-Guinea
Pig-Cy5
Mouse anti-Glucagon MoAb Goat anti-Mouse-Rhodamine
Martinez et al., J. Endocrinol., 2001
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Conventional Fluorescent Microscopy
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Confocal Microscopy Core
Inverted Scope
Upright Scope
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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

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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-dimethyla
  niline (PADA)

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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.
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Absorption Spectra of Fluors Commonly Conjugated
to Secondary Antibodies
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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 filter
emission filter

Hg
dichroic mirror
specimen
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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
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Confocal Microscopy
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UV 351,364nM Argon 488nM HeNe I 543nM HeNe 2 633nM
CCBB Confocal Core Facility (1999-2006) Zeiss
LSM510 with 4 color capability
Building 37 Room 1035
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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.
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Simplified Optics of a Confocal Microscope
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The LSM 510

• To the Specimen
• optical fibers
• main dichroic beam-splitter
• scanner mirrors
• scanning lens

• From the Specimen
• optical fibers
• main dichroic beam-splitter
• (7,8,9) secondary dichroics
• (10) pinhole diaphragm
• (11) emission filters
• (12) photomultipliers

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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.
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Insulin-Cy5
CRLR-FITC
Overlay
Glucagon-Rhodamine
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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.
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Standard Microscopy
Multitracking
Brain Slice nerve fibers (FITC) cell nuclei
(propidium iodide)
Courtesy Dr. Schild, University of Gottingen
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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
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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
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Empty vector
b-catenin
DAPI
MERGE
Ras V12
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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
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Animated 3-Dimensional Reconstruction
Laser Scanning Microscopy LSM510 3D for
LSM www.Zeiss.com
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Animated 3-Dimensional Reconstruction
Mitosis www.Zeiss.com
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Why is Confocal Microscopy Better?
5. Improved Resolution
Rat Cerebellum Astrocytes (green) Mn dismutase
(red)
Jorg Lindeman University of Magdeburg
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Applications

• Colocalization
• Live Cell Imaging
• FRAP/FLIP
• GFP-Fusion
• 3. FRET

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Colocalization of Proteins
Colocalization of up to 4 different
proteins Colocalization does not mean
interaction Decreased cross talk with
multitracking feature
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Colocalization of insulin and calcitonin
receptor-like receptor
Insulin-Cy5
CRLR-FITC
Glucagon-Rhodamine
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Colocalization
a-tubulin
p53
Proteins may colocalize but not necessarily
interact
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Fluorescence Resonance Energy Transfer
The high resolution of a confocal
microscope allows us to study the physical
interaction of protein partners.
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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 CFP is
more than 10nm from YFP YFP is not excited and
does not emit light
No FRET Signal
CFP is excited by light and emits light CFP is in
close proximity to YFP YFP emits light
FRET Signal
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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.
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FRET
Region of interest
Two channel (CFP,YFP) time series
Two channel (CFP,YFP) time series
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Confocal Microscopy is a powerful tool for
studying signaling mechanisms
Yvona Ward Building 37 Room 1066 301-594-2645 ywar
d_at_helix.nih.gov