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26/06/2011

Electron Microscopy: TEM, Immunogold Labeling, SEM, Correlative Microscopy

Prof. Dr. Rainer Duden duden@bio.uni-luebeck.de

Resolution Comparison Light vs Electron Microscopy

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Microscope Resolution

• ability of a lens to separate or distinguish small objects that are close together

• wavelength of light used is major factor in resolution

shorter wavelength ⇒ greater resolution

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• beams of electrons are used to produce images

• wavelength of electron beam is much shorter than light, resulting in much higher resolution

Electron Microscopy

•Light microscopy• Glass lenses• Source of illumination is usually light of visible

wavelengths

•Electron microscopy• Electromagnetic lenses• Source of illumination is electrons

• Hairpin tungsten filament (thermionic emission)

• Pointed tungsten crystal (cold cathode field emission)

The Transmission Electron Microscope

• electrons scatter when they pass through thin sections of a specimen

• transmitted electrons (those that do not scatter) are used to produce image

• denser regions in specimen, scatter more electrons and appear darker

Comparison of LM and TEM

Specimen Preparation

• analogous to procedures used for light microscopy

• for transmission electron microscopy, specimens must be cut very thin

• specimens are chemically fixed and stained with electron dense material

Transmission Electron Microscopy (TEM)

Zeiss 10/A conventional TEM

Excellent for trainingFilm only

Negative Staining

Viruses, small particles, proteins, molecules

No sectioning

Same day results

negative staining

Electron dense negative stain

particles

negative staining

• requires minimal interaction between particle & ‘stain’

• to avoid binding, heavy metal ion should be of same charge +/- as the particle

• positive staining usually destructive of bio-particles

• biological material usually -ve charge at neutral pH

• widely used negative contrast media include:

anionic cationic

phosphotungstate uranyl actetate/formate

molybdate (ammonium) (@ pH ~ 4)

Ebola

Negative Stain

Double Immunogold Labeling of Negatively Stained Specimens

Bacterial pili serotypes dried onto grid and sequentially labeled with primary antibody, then Protein-A-5nm-gold and Protein-A-15-nm-gold before negative staining

metal shadowing - rotary

metal shadowing - rotary• Contrast usually inverted to give dark shadows > resolution 2 - 3 nm - single DNA strand detectable

- historic use for ‘molecular biology’ (e.g. heteroduplex mapping)

> good preservation of shape, but enlargement of apparent dimensions

> in very recent modification (MCD - microcrystallite

decoration), resolution ~1.1 nm

Clathrin:

a major and evolutionarily conserved coat protein

clathrin heavy chain

clathrin light chains

~100kD proteins

Crude mem

branes

Purified CCVs

200

11697

66

45

31

21.5

~50kD proteins

~20kD proteins

EM images of purified Clathrin triskelia

Rotary shadowing

EM images of purified Clathrin triskelia

Kirchhausen and Harrison (1981). Cell 23, 755-761:see EM image aboveUngewickell and Branton (1981). Nature 289, 420-422:- reversible dissassembly of Clathrin triskelions into clathrin - coats in vitro

Rotary shadowing

3 heavy chains

3 light chains

Clathrin triskelions

Adaptors:

essential for cargo sorting

clathrin heavy chain

clathrin light chains

~100kD proteins

Crude mem

branes

Purified CCVs

200

11697

66

45

31

21.5

~50kD proteins

~20kD proteins

AP-1 AP-2

γβ1

µ1

σ1

β2

µ2

σ2

αC

αA

Protein pattern of Adaptor Complexesextracted from purified brain CCVsafter SDS-PAGE

EM images of purified AP-2 complexRotary shadowing

µ1γ β1σ1

µ2α β2σ2

µ4ε β4σ4

µ3δ β3σ3

AP-1 AP-2 AP-3 AP-4

Adaptor proteins mediate sorting of specific cargofrom different compartments

Margaret Robinson, Univ. Cambridge

Killing & Fixation- Death; Molecular stabilization

Dehydration

Infiltration

Embedding & Polymerization

Sectioning

- Chemical removal of H2O

- Replace liquid phase with resin

- Make solid, sectionable block

- Ultramicrotome, mount, stain

Overview of Biological Specimen Preparation

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Preparing for cutting sections for TEM

Interference reflection angle from Sjöstrand (1967)

Estimating Section Thickness

Serial section 3-D reconstruction

The Freeze Fracture Technique

Gap Junctions in negative stain, freeze fracture & TEM

Tight Junction structure in

TEM, freeze fracture, and live fluorescence

microscopy

Cryotechniques

Ultrarapid cryofixation Metal mirror impactLiquid propane plunge

Freeze fracture with Balzers 400T

Cryosubstitution Cryo-ultramicrotomy –

Ultrathin frozen sections (primarily for antibody labeling)

Clathrin - coated vesicles - the minimal machinery

3 heavy chains

3 light chains

Clathrin triskelions

Adaptor (AP2) - four adaptins

α β2

σ2 µ2

Tom Kirchhausen, Harvard Medical School

John Heuser’s Quick Freeze Deep Etch Technique

Quick freeze - deep etch technique

John HeuserWashington University

School of Medicine, USA

Inner layer : membrane containing cargoMiddle layer : adaptors and accessory proteinsOuter layer : mechanical scaffold

Clathrin - coated Vesicles

J. Heuser

Now please put onthe 3-D glasses…

John Heuser, Washington Univ. School of Medicine

Immunolabeling for Transmission Electron Microscopy

Normally do Two-Step Method

Primary antibody applied followed by colloidal gold-labeled secondary antibody

May also be enhanced with silver

Can also do for LM

Preparation of Biological Specimens for Immunolabeling

The goal is to preserve tissue as closely as possible to its natural state while at the same time maintaining the ability of the antigen to react with the antibody

Chemical fixation of whole mounts prior to labeling for LM

Chemical fixation, dehydration, and embedment in paraffin or resin for sectioning for LM or TEM

Chemical fixation for cryosections for LMCryofixation for LM or TEM

Chemical Fixation

Antigenic sites are easily denatured or masked during chemical fixation

Glutaraldehyde gives good fixation but may mask antigens, plus it is fluorescent

Paraformaldehyde often better choice, but results in poor morphology , especially for electron microscopy

May use e.g., 4% paraformaldehyde with 0.5% glutaraldehyde as a good compromise

Specimen Preparation for TEM

Chemical fixation with buffered glutaraldehydeOr 4% paraformaldehyde with >1% glutaraldehyde

Postfixation with osmium tetroxideOr not, or with subsequent removal from sections

Dehydration and infiltration with liquid epoxy or acrylic resin

Polymerization of hard blocks by heat or UVUltramicrotomy – 60-80nm sectionsLabeling and/or stainingView with TEM

Approaches to Immunolabeling

Direct Method: Primary antibody contains label

Indirect Method: Primary antibody followed by labeled secondary antibody

Amplified Method: Methods to add more reporter to labeled site

Protein A Method: May be used as secondary reagent instead of antibody

• Colloidal gold of defined sizes, e.g., 5 nm, 10 nm, 20 nm, easily conjugated to antibodies

• Results in small, round, electron-dense label easily detected with EM

• Can be enhanced after labeling to enlarge size for LM or EM

Immunolocalization

LMFluor/confocalTEMSEM with

backscatter detector

Preembedding or Postembedding Labeling

May use preembedding labeling for surface antigens or for permeabilized cells

The advantage is that antigenicity is more likely preserved

Postembedding labeling is performed on sectioned tissue, on grids, allowing access to internal antigens

Antigenicity probably partially compromised by embedding

Steps in Labeling of Sections

Chemical fixationDehydration, infiltration, embedding and sectioningOptional etching of embedment, permeabilizationBlockingIncubation with primary antibodyWashingIncubation with secondary antibody congugated

with reporter (fluorescent probe, colloidal gold)Washing, optional counterstainingMount and view

Controls! Controls! Controls!

Omit primary antibodyIrrelevant primary antibodyPre-immune serumPerform positive controlCheck for autofluorescenceCheck for non-specific labelingDilution series

Desmosomes and IFs in primary mouse keratinocytes

Duden & Franke, 1988 (J. Cell Biol.)

Pre-embedding labelling of desmosomal vesiclesin primary mouse keratinocytes

Duden and Franke, 1988 (J. Cell Biol.)

Visualization of desmosomal vesicles in A431 cells grown on glass coverslips

Duden and Franke, 1988 (J. Cell Biol.)

Duden & Franke, 1988 (J. Cell Biol.)

Pre-embedding labelling of desmosomal vesiclesin A431 cells

Double-labeling Method

Use primary antibodies derived from different animals (e.g., one mouse antibody and one rabbit antibody)

Then use two secondary antibodies conjugated with reporters that can be distinguished from one another

Analysis of the secretory pathway by a combination of EM and autoradiography

George Palade1974 Nobel Prize for Physiology or Medicine

ER --> Golgi --> Vesicles --> PM

The Scanning Electron Microscope

• uses electrons reflected from the surface of a specimen to create image

• produces a 3-dimensional image of specimen’s surface features

TEMvs

SEM

Scanning Electron Microscopy

SEM

Correlative Light/EM microscopy - SEM: visualization of virus particles on a cell surface

Correlative Light/EM microscopy& electron tomography

Grabenbaur et al., 2005.

Nat. Meth. 2. 857-862

Diaminobenzidine (DAB) photooxidation by GFP (GalT-GFP)

Correlative Light/EM microscopy& electron tomography

Grabenbaur et al., 2005.

Nat. Meth. 2. 857-862

miniSOG

miniSOG