26/06/2011. Electron Microscopy: TEM, Immunogold Labeling, SEM, Correlative Microscopy

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1 26/06/2011 Electron Microscopy: TEM, Immunogold Labeling, SEM, Correlative Microscopy Prof. Dr. Rainer Duden 1

2 Resolution Comparison Light vs Electron Microscopy

3 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 3

4 Electron Microscopy beams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution 4

5 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)

6 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

7 Comparison of LM and TEM

8 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

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10 Transmission Electron Microscopy (TEM) Zeiss 10/A conventional TEM Excellent for training Film only

11 Negative Staining Viruses, small particles, proteins, molecules No sectioning Same day results

12 negative staining particles Electron dense negative stain

13 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)

14 Negative Stain Ebola

15 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

16 metal shadowing - rotary

17 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

18 s e n Clathrin: a major and evolutionarily conserved coat protein a r b s V C em C m d e e d fi i u r Cr Pu clathrin heavy chain ~100kD proteins 66 ~50kD proteins 45 clathrin light chains ~20kD proteins

19 Rotary shadowing EM images of purified Clathrin triskelia

20 Rotary shadowing EM images of purified Clathrin triskelia Kirchhausen and Harrison (1981). Cell 23, : see EM image above Ungewickell and Branton (1981). Nature 289, : - reversible dissassembly of Clathrin triskelions into clathrin - coats in vitro

21 Clathrin triskelions 3 heavy chains 3 light chains

22 s e n Adaptors: a r b s V C em C m d e e d fi i u r Cr Pu essential for cargo sorting clathrin heavy chain ~100kD proteins 66 ~50kD proteins 45 clathrin light chains ~20kD proteins

23 Protein pattern of Adaptor Complexes extracted from purified brain CCVs after SDS-PAGE β1 γ AP-1 AP-2 αa β2 αc µ1 µ2 σ1 σ2

24 Rotary shadowing EM images of purified AP-2 complex

25 Adaptor proteins mediate sorting of specific cargo from different compartments α µ2 σ2 β2 γ µ1 σ1 β1 δ µ3 σ3 β3 ε µ4 σ4 β4 AP-1 AP-2 AP-3 AP-4 Margaret Robinson, Univ. Cambridge

26 Overview of Biological Specimen Preparation Killing & Fixation - Death; Molecular stabilization Dehydration - Chemical removal of H 2 O Infiltration - Replace liquid phase with resin Embedding & Polymerization - Make solid, sectionable block Sectioning - Ultramicrotome, mount, stain

27 Preparing for cutting sections for TEM 27

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30 Estimating Section Thickness Interference reflection angle from Sjöstrand (1967)

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32 Serial section 3-D reconstruction

33 The Freeze Fracture Technique

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36 Gap Junctions in negative stain, freeze fracture & TEM

37 Tight Junction structure in TEM, freeze fracture, and live fluorescence microscopy

38 Cryotechniques Ultrarapid cryofixation Metal mirror impact Liquid propane plunge Freeze fracture with Balzers 400T Cryosubstitution Cryo-ultramicrotomy Ultrathin frozen sections (primarily for antibody labeling)

39 Clathrin - coated vesicles - the minimal machinery Clathrin triskelions 3 heavy chains 3 light chains Adaptor (AP2) α - four adaptins β2 σ2 µ2 Tom Kirchhausen, Harvard Medical School

40 John Heuser s Quick Freeze Deep Etch Technique

41 Quick freeze - deep etch technique John Heuser Washington University School of Medicine, USA

42 Clathrin - coated Vesicles J. Heuser Inner layer : membrane containing cargo Middle layer : adaptors and accessory proteins Outer layer : mechanical scaffold

43 Now please put on the 3-D glasses

44 John Heuser, Washington Univ. School of Medicine

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52 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

53 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 LM Cryofixation for LM or TEM

54 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

55 Specimen Preparation for TEM Chemical fixation with buffered glutaraldehyde Or 4% paraformaldehyde with >1% glutaraldehyde Postfixation with osmium tetroxide Or not, or with subsequent removal from sections Dehydration and infiltration with liquid epoxy or acrylic resin Polymerization of hard blocks by heat or UV Ultramicrotomy 60-80nm sections Labeling and/or staining View with TEM

56 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

57 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

58 Immunolocalization LM Fluor/confocal TEM SEM with backscatter detector

59 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

60 Steps in Labeling of Sections Chemical fixation Dehydration, infiltration, embedding and sectioning Optional etching of embedment, permeabilization Blocking Incubation with primary antibody Washing Incubation with secondary antibody congugated with reporter (fluorescent probe, colloidal gold) Washing, optional counterstaining Mount and view

61 Controls! Controls! Controls! Omit primary antibody Irrelevant primary antibody Pre-immune serum Perform positive control Check for autofluorescence Check for non-specific labeling Dilution series

62 Desmosomes and IFs in primary mouse keratinocytes Duden & Franke, 988 (J. Cell Biol.)

63 Pre-embedding labelling of desmosomal vesicles in primary mouse keratinocytes Duden and Franke, 1988 (J. Cell Biol.)

64 Visualization of desmosomal vesicles in A431 cells grown on glass coverslips Duden and Franke, 1988 (J. Cell Biol.)

65 Pre-embedding labelling of desmosomal vesicles in A431 cells Duden & Franke, 1988 (J. Cell Biol.)

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67 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

68 George Palade 1974 Nobel Prize for Physiology or Medicine Analysis of the secretory pathway by a combination of EM and autoradiography ER --> Golgi --> Vesicles --> PM

69 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

70 TEM vs SEM

71 Scanning Electron Microscopy

72 SEM

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

76 Correlative Light/EM microscopy & electron tomography Diaminobenzidine (DAB) photooxidation by GFP (GalT-GFP) Grabenbaur et al., Nat. Meth

77 Correlative Light/EM microscopy & electron tomography Grabenbaur et al., Nat. Meth

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