Introduction to Electron Microscopy Andres Kaech

Transcription

1 Center for Microscopy and Image Analysis Introduction to Electron Microscopy Andres Kaech The types of electron microscopes Transmission electron microscope (TEM) Scanning electron microscope (SEM) 1

2 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 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 2

3 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 Electron microscope Transmission electron microscope 3

4 Electromagnetic lenses Electromagnetic lens of a transmission electron microscope The focal length can be changed by changing the current: No movement or exchange of the lens is required for focusing or changing magnification! Specimen holders and stages - TEM Specimen holder Specimen on a TEM grid 3 mm 4

5 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 - SEM 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) 5

6 Electron microscopes are high vacuum systems Example: Transmission electron microscope Cathode IGP mbar Ion getter pump Specimen holder IGP mbar Turbo molecular pump Oil diffusion pump TMP mbar Viewing screen Rotary pump RP Atmosphere: 1000 mbar 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) 6

7 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 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 7

8 Physical demands of electron microscopy What is (was) this? 2 cm Main preparation pathways for TEM 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 8

9 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 Room temperature processing for TEM 9

10 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 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 10

11 Cryo preparation for TEM Fixation Well frozen mouse cerebellum Dehydration Embedding Thin sectioning Not well frozen mouse cerebellum Staining TEM 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 11

12 Cryo preparation for TEM Fixation Plunge/slam freezer Propane jet freezer Dehydration Embedding High-pressure freezer Thin sectioning Staining TEM Relative sizes Cryo preparation for TEM Fixation Dehydration Freeze-substitution Substitution of water/ice with solvent (ethanol, acetone) Usually combined with simultaneous fixation with chemicals (OsO 4, Uranyl-acetate ) 0 Embedding Thin sectioning Temperature ( C) C acetone -80 Staining Time (h) TEM 12

13 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 Ultrastructural details, interaction of organelles Are these objects related to each other? From: J.C.Russ, D 2D 13

14 3D reconstruction of 2D projections of a 3-dimensional object 2D projections 3D rendered object Reconstruction 3D reconstruction based on: I) Imaging of serial sections Thickness of section for EM: nm Z resolution limited to section thickness Imaging in TEM Imaging in SEM (newer application) 14

15 3D reconstruction based on: II) Serial imaging of block faces Focused ion beam SEM Ablation of material with Ga-ions Ultra-microtome in SEM High-pressure frozen and embedded mouse brain (hippocampal slice culture) 1 µm 15

16 High-pressure frozen and embedded C. elegans Specimen courtesy of K. Stutz, Institute of Molecular Life Sciences, UZH 3D reconstruction based on: III) Projections of different tilts 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) 16

17 Aligned projections of a Pancreatic beta cell line: Double-tilt series, ±60, 1.5 increment, section 400 nm thick Marsh 2005 Calculation of tomograms based on projections virtual sections Marsh

18 Segmentation of objects of interest Software available for automated segmentation Marsh 2005 Partly manual: Very time consuming Rendering of segmented structures Marsh

19 3D reconstruction based on: IV) Projection of a random particle orientation (cryo-tem) Single particle imaging Imaging in a Cryo TEM 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 19

20 3D reconstruction of the cyclic nucleotide gated potassium channel MloK1 H. Stahlberg, Center for Cellular Imaging and Nanoanalytics (C-CINA), Biozentrum, University of Basel Main preparation pathways for SEM 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 20

21 Room temperature processing for SEM Fixation Dehydration Critical point drying Coating Mouse kidney (glomerulus) SEM Room temperature processing for SEM Fixation Critical point of H2O: 374 C and 221 bar Critical point of CO2: 31 C, 74 bar Dehydration CO2 74 bar Critical point drying Coating 31 C SEM 21

22 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) Cryo processing for SEM Fixation Freeze-fractured mouse intestine Freeze-fracturing < 100 C Sublimation (partial freezedrying) Coating Cryo-SEM Electron microscopy ETH Zurich 22

23 Immuno electron microscopy Correlative light and electron microscopy Immunolabelling on electron microscopy level Immunolabelling: Localization of proteins H/K -ATPase in cimetidine-treated resting gastric parietal cells (rabbit). 100 nm Sawaguchi et al. 2004, Journal of Histochemistry & Cytochemistry 23

24 Immuno electron microscopy Immunolabelling on sections Correlative light and electron microscopy (TEM) Light microscopy Protein localization -> function Electron microscopy Morphology -> structure 24

25 Correlative light and electron microscopy (TEM) Correlative light and electron microscopy (SEM) Cryo scanning electron microscopy green...calcein red...autofluorescence of symbiont Khalifa et al. 2016, Biomineralization of Foraminifers... 25