Fluorescence and Confocal Microscopy

Yvona WardCell and Cancer Biology Branch

OUTLINE

1. Immunofluorescent Staining2. Conventional Fluorescent Microscopy3. Confocal Microscopy4. Applications

Immunofluorescent StainingImmunofluorescent 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 atits own characteristic wavelength (fluorescence) and thus allowsdetection 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 TechniqueCell Preparation1. 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 Permeabilization1. 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 Staining1. Incubate cells with a blocking solution to minimize non-specific staining2. 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 PRIMARYANTIBODY

SECONDARY ANTIBODY

FLUOROCHROME

Giannakakou et al., Nature Cell Biology,2000

PRIMARY ANTIBODYsheep anti-p53 polyconal

SECONDARY ANTIBODYTexas Red conjugated anti-sheep

PRIMARY ANTIBODYmouse anti- tubulin monoclonal

SECONDARY ANTIBODYFITC conjugated anti-mouse

Direct Staining of Cell StructuresOrganelle 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)

4

TRAP1 Mouse Monoclonal+ Goat anti-Mouse-FITC

Felts et al., JBC, 2000

nucleus MERGE

microtubules centrosomes

Anti-tubulin MoAbGoat anti-mouse-Rhodamine

Anti-pericentrin PoAbGoat anti-mouse-FITC

DAPI

Anti-vinculin MoAbGoat anti-mouse-FITCRhodamine-Phalloidin

Stress Fibers Focal Adhesions

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 labelcellular components.

ANTIGEN PRIMARYANTIBODY

SECONDARY ANTIBODY FLUOROCHROME

STREPTAVIDINBIOTIN

Guinea Pig anti-Insulin PoAbDonkey anti-Guinea Pig-Cy5

Rabbit anti-Factor HBiotinylated Goat anti-RabbitStreptavidin-FITC

Mouse anti-Glucagon MoAbGoat anti-Mouse-Rhodamine Martinez et al., J. Endocrinol., 2001

ConventionalFluorescentMicroscopy

Confocal Microscopy Core

Inverted Scope Upright Scope

Preparation of Stained SpecimensFor Microscopy

Specimen MountingIn order for the stained specimen to be visualized on a fluorescentmicroscope, it needs to be mounted onto a slide using an appropriatemounting medium.

Mounting medium is usually a PBS/Glycerol mix and is commerciallyavailable.

•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)

FluorescenceMolecules absorbing the energy of electromagnetic radiationwill jump to a higher energy level. When certain excited moleculesreturn to the ground state they emit radiation. This phenomenonis known as fluorescence. Fluorescent molecules are known as fluorochromes or fluorophores.

Absorption Spectra of Fluors CommonlyConjugated to Secondary Antibodies

Fluorochrome Absorption Emission

Cascade Blue 400 420Fluorescein 494 518Rhodamine 570 590Texas Red 595 615Cy5 650 670

Fluorescence MicroscopySince the molecules used for immunofluorescence emit light in thevisible 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 and transmits longer .

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.

Hg

eyepiece

specimen

* dichroic mirror

emission filterexcitation filter

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 distanceHigh resolution 100x high NA short working distance

Confocal Microscopy

CCBB Confocal Core Facility (1999-2006)Zeiss LSM510 with 4 color capability

Building 37 Room 1035

UV 351,364nMArgon 488nMHeNe I 543nMHeNe 2 633nM

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

Confocal microscopy is a powerful tool for generating high-resolution imagesand 3-D reconstructions of a specimen.

In confocal microscopy a laser light beam is focused onto a fluorescent specimenthrough the objective lens. The mixture of reflected and emitted light is capturedby the same objective and is sent to the dichroic mirror. The reflected light isdeviated 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 manneras the laser focus is moved over the specimen.

Simplified Optics of a Confocal Microscope

To the Specimen(1) optical fibers(4) main dichroic beam-splitter(5) scanner mirrors(6) scanning lens

From the Specimen(1) optical fibers(4) main dichroic beam-splitter(7,8,9) secondary dichroics(10) pinhole diaphragm(11) emission filters(12) photomultipliers

The LSM 510

Why is Confocal Microscopy Better?

1. More Color PossibilitiesBecause 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 TalkIn most applications, fluorochromes have overlapping emission spectra. Hence, theemission signals cannot be separated completely into different detection channelsresulting in “bleed through” or cross talk.

However, if the fluorochromes have distinct excitation spectra, the fluorochromescan be excited sequentially using one excitation wavelength at a time. This is onlypossible with confocal systems that offer the multitracking feature.

MultitrackingStandard Microscopy

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 BastmeyerUniversity of Konstanz

Cultured Cells

Formation of Acini in a 3-D Matrigel Matrix

Three-dimensional culture of MCF10A mammary epithelial cells on areconstituted basement membrane leads to the formation of polarized, growtharrested acini-like spheroidsthat recapitulate several aspects of glandular architecture in vivo.

Introduction of oncogenes intoMCF10A cells results in distinct morphological phenotypes

Empt

y ve

ctor

Ras

V12

-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-AllenpachETH, Zurich

Animated 3-Dimensional Reconstruction

Laser Scanning MicroscopyLSM5103D for LSMwww.Zeiss.com

Animated 3-Dimensional Reconstruction

Mitosiswww.Zeiss.com

Why is Confocal Microscopy Better?5. Improved Resolution

Rat Cerebellum Astrocytes (green) Mn dismutase (red)

Jorg LindemanUniversity of Magdeburg

Applications1. Colocalization

2. Live Cell ImagingFRAP/FLIPGFP-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

Colocalizationp53 -tubulin

Proteins may colocalize but not necessarily interact

Fluorescence Resonance Energy Transfer

The high resolution of a confocal microscopeallows us to study thephysical interaction ofprotein 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 alight 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

No FRETSignal

FRETSignal

CFP is excited by light and emits lightCFP is more than 10nm from YFPYFP is not excited and does not emit light

CFP is excited by light and emits lightCFP is in close proximity to YFPYFP 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 experimentssince, besides other prereqisites (eg. dipole orientation or sufficient fluorescencelifetime), 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

Two channel (CFP,YFP) time series

Two channel (CFP,YFP) time series

Region ofinterest

Confocal Microscopy is a powerful tool for studyingsignaling mechanisms

Yvona WardBuilding 37 Room 1066301-594-2645yward@helix.nih.gov