Monday: Y42 G53 Tuesday: Y42 G53 Wednesday: Y42 J11

Transcription

1 Locations: Irchel building 42, Level H and F Locations: Irchel building 42, Level H and F Self-study sessions: Monday: Y42 G53 Tuesday: Y42 G53 Wednesday: Y42 J11 1

2 Center for Microscopy and Image Analysis Introduction to Biological Electron Microscopy Andres Kaech The types of electron microscopes Transmission electron microscope (TEM) Electron beam Scanning electron microscope (SEM) Electron beam Specimen ~100 nm Projection Specimen Surface Hela Cells 2

3 Examples TEM Mouse cerebellum Microtubule Dendrite Synapse Ribosomes Golgi Mitochondrium Nucleus 500 nm Examples TEM Mouse cerebellum Mitochondrium Lipid bilayer Golgi Nucleus 100 nm 3

4 Examples SEM Mouse kidney 500 µm Examples SEM Mouse kidney (glomerulus) 10 µm 4

5 Examples SEM Pseudomonas aeruginosa 500 nm Properties of electrons e - Very similar to photons: Wave-particle duality Optical properties (Diffraction, chromatic abberation, spherical abberation, astigmatism etc.) Resolution depends on aperture and wavelength (Diffraction limited resolution) Abbe s equation d = 0.61 λ/na NA n sin 5

6 Resolution of electron microscopes The higher the energy of the electrons, the lower the wavelength, the higher the resolution TEM: kv Effective instrument resolution TEM: 0.5 nm (120 kv) SEM: kv Effective instrument resolution SEM: 1 nm Resolution of biological objects limited by specimen preparation: Practical resolution: > 1 nm Transmission electron microscope vs. Widefield light microscope Transmission electron microscope Widefield light microscope Illumination Condenser lens Specimen Objective lens Projector lens Final image 6

7 Scanning electron microscope vs. Confocal laser scanning microscope Scanning electron microscope Confocal laser scanning microscope Illumination Detector Lens system Beam scanner Lens system Specimen Specimen holders and stages - TEM Specimen holder Specimen on a TEM grid 3 mm 7

8 Specimen holders and stages - TEM Transmission electron microscope Goniometer: x, y, z, r Specimen size: 3 mm in diameter! Ca. 100 nm in thickness (electron transparent) Specimen holders and stages Scanning electron microscope Objective lens Specimen stub Stage Stub holder Specimen stage (x, y, z, r, tilt) Specimen size: 100 mm in diameter 2 cm in z-direction (not electron transparent) 8

9 Physical demands of electron microscopy Biology Aqueous/hydrated Soft Light elements (C, O, H, N, S, P etc.) Large Not suitable for EM Electron microscope High vacuum Electron beam Sensitive to vibration/motion (High magnifications) Physical demands of electron microscopy Biology Aqueous/hydrated Soft Light elements (C, O, H, N, S, P etc.) Large Not suitable for EM Electron microscope High vacuum Electron beam Sensitive to vibration/motion (High magnifications) Biological samples need to be transferred into a solid state......keeping the sample close to the native state Resistant to high vacuum Resistant in electron beam Thin permeable for electrons (for TEM) Contrast 9

10 Physical demands of electron microscopy Biology Aqueous/hydrated Soft Light elements (C, O, H, N, S, P etc.) Large Not suitable for EM Electron microscope High vacuum Electron beam Sensitive to vibration/motion (High magnifications) Any treatment changes the specimen! Resistant to high vacuum Resistant in electron beam Thin permeable for electrons (for TEM) Contrast Physical demands of electron microscopy What is (was) this? 2 cm 10

11 Preparation pathways overview RT specimen processing Low temperature processing Plunge freezing Bare grid technique WARM SPECIMEN Propane jet freezing FROZEN SPECIMEN Chemical fixation High pressure freezing Dehydration Freeze-substitution Freeze-fracturing/Freeze-drying/Coating Cryo-Ultramicrotomy Embedding Critical Point Drying Low-temperature embedding RT-embedding Ultramicrotomy thawing Cryo thin section Coating Immunolabeling Freeze-dried specimen Freeze-fractured/etched specimen Staining Replica RT-SEM RT-TEM, FIB-SEM RT-TEM RT-SEM Cryo-SEM Cryo-TEM Room temperature preparation for TEM Fixation Glutaraldehyde, OsO4, UAc Dehydration In a series of increasing concentration of acetone, ethanol Embedding Resin like Epon, Acrylic resins Thin sectioning Ultramicrotomy -> 70 nm section Staining Contrast enhancement with heavy metals TEM 11

12 Room temperature processing for TEM Room temperature preparation for TEM Fixation Glutaraldehyde, OsO4, UAc Dehydration In a series of increasing concentration of acetone, ethanol Embedding Resin like Epon, Acrylic resins Thin sectioning Ultramicrotomy -> 70 nm section Staining Contrast enhancement with heavy metals TEM 12

13 Cryo preparation for TEM Fixation Dehydration Cryo-Immobilization Stabilization of biological material by freezing Liquid water and vitrified water Embedding Thin sectioning Frozen water with ice crystals Staining TEM Cryo preparation for TEM Fixation Well frozen mouse cerebellum Dehydration Embedding Thin sectioning Not well frozen mouse cerebellum Staining TEM 13

14 Cryo preparation for TEM Fixation Dehydration Plunge freezing in liquid ethane/propane: Only suspensions (< 1 µm) or thin tissues containing anti-freeze Embedding Thin sectioning Slam freezing: Suspensions and thin tissues (few µm, only front well frozen ca. 1 µm) Propane jet freezing (JFD): Adequate freezing of suspensions not thicker than 15 µm Thicker specimen require anti-freeze Staining High pressure freezing (HPM) Freezing under high pressure (2100 bar) Adequate freezing of samples up to 200 µm thickness without anti-freeze TEM Cryo preparation for TEM Fixation Plunge/slam freezer Propane jet freezer Dehydration Embedding High-pressure freezer Thin sectioning Staining TEM Relative sizes 14

15 Room temperature vs. cryo preparation Thin sections of plastic embedded mouse cerebellum Conventionally fixed (glutaraldehyde) High pressure frozen Specimen courtesy of Bettina Sobottka, Neurologische Klinik, University of Zurich 500 nm Cryo preparation for TEM Fixation Cryo-Immobilization Dehydration Embedding Thin sectioning Staining TEM 15

16 Cryo preparation for TEM Images of frozen hydrated sections in a cryo-tem Green algae A. Al-Amoudi et al. / Journal of Structural Biology 150 (2005) Electron Tomography Ultrastructural details, interaction of organelles Are these objects related to each other? From: J.C.Russ, D 2D 16

17 Electron Tomography 3D reconstruction of 2D projections of a 3-dimensional object 2D projections 3D rendered object Reconstruction Electron Tomography (I) 3D reconstruction based on: I) Projection of serial sections Thickness of section for EM: nm Z resolution limited to section thickness Imaging in TEM Imaging in SEM (new application) 17

18 Electron Tomography (II) 3D reconstruction based on: II) Projection of a tilt series Thickness of sections for tilt series: ~ nm Z resolution depending on number of different projections: 5-20 nm NOTE: A combination of both methods is possible 3D high resolution of several successive sections Imaging in TEM (RT or CRYO) Electron Tomography (II) Aligned projections of a Pancreatic beta cell line: Double-tilt series, ±60, 1.5 increment, section 400 nm thick Marsh

19 Electron Tomography (II) Calculation of tomograms based on projections virtual sections Marsh 2005 Electron Tomography (II) Segmentation of objects of interest Marsh

20 Electron Tomography (II) Rendering of segmented structures Marsh 2005 Electron Tomography (III) 3D reconstruction based on: III) Projection of a random particle orientation (cryo-tem) Single particle imaging Imaging in a Cryo TEM 20

21 Electron Tomography (III) Plunge freezing Tweezers TEM grid or other carrier with specimen suspension Cryogen (propane: -186 C, ethane: -180 C) Metal container LN2 bath for cooling of secondary cryogen: C Electron Tomography (III) 3D reconstruction of the cyclic nucleotide gated potassium channel MloK1 H. Stahlberg, Center for Cellular Imaging and Nanoanalytics (C-CINA), Biozentrum, University of Basel 21

22 Electron Tomography (IV) 3D reconstruction based on: IV) Serial abrasion in a focused ion beam scanning electron microscope Slice and view Z resolution depending milling capability/specimen and surface imaging: best resolution 5 nm (voxel) Imaging in SEM Electron Tomography (IV) 22

23 Electron Tomography (IV) C. elegans embedded in Epon/Araldite Electron Tomography (IV) High-pressure frozen and embedded mouse brain (hippocampal slice culture) 23

24 Electron Tomography (V) 3D reconstruction based on: V) 3 view microtome in SEM Large areas possible Limited z-resolution: ca. 30 nm (limited by sectioning process) Room temperature processing for SEM Fixation Dehydration Critical point drying Coating Mouse kidney (glomerulus) SEM 24

25 Room temperature processing for SEM Fixation Critical point of H 2 O: 374 C and 221 bar Critical point of CO 2 : 31 C, 74 bar Dehydration Pressure solid Phase diagram of CO 2 Critical point drying liquid C S Coating E gas S Startingpoint E End point C Critical point SEM Temperature Cryo processing for SEM Fixation Cryo-Immobilization (same as for TEM) Freeze-fracturing Fracturing at low temperature and under high vacuum < 100 C Sublimation (partial freezedrying) Sublime water from frozen sample Coating Enhance contrast Cryo-SEM Image in the cold state (e.g. 120 C) 25

26 Cryo processing for SEM Fixation Freeze-fractured mouse intestine Freeze-fracturing < 100 C Sublimation (partial freezedrying) Coating Cryo-SEM Electron microscopy ETH Zurich Negative staining for TEM Contrast based on heavy metals surrounding the topography of sample: Uranyl-acetate or Phosphotungstic acid 26

27 Negative staining T4 phages on bacterium Bacterium with flagella 500 nm 500 nm Human rotaviruses 100 nm 100 nm 100 nm Immuno electron microscopy Immunolabelling on sections H/K -ATPase in cimetidine-treated resting gastric parietal cells (rabbit). 100 nm Sawaguchi et al. 2004, Journal of Histochemistry & Cytochemistry 27