Electron Microscopy Transmission Electron Microscopy SEM = Scanning Electron Microscopy TEM = Transmission Electron Microscopy Sara Henriksson, UCEM 2017-02-16 E. coli, William E. Bentley, Maryland, USA 2 For more information, kindly refer to UCEM website: http://www.kbc.umu.se/platforms/electron-microscopy.html Our new microscopes Stereocilia on a hair cell in the inner ear SEM Light microscop SEM Figure 9-51 Molecular Biology of the Cell ( Garland Science 2008) TEM TEM Titan Krios Talos Scios Figure 9-50 Molecular Biology of the Cell ( Garland Science 2008) Figure 12-9c Molecular Biology of the Cell ( Garland Science 2008) TEM Electron Tomography Full cell volume of Fission yeast Johanna Höög Figure 9-58 Molecular Biology of the Cell ( Garland Science 2008) 1
100 nm + β β +GTP α α - Tubulin: Nogales et al. 1998 In vitro polymerized microtubules, Linda Sandblad Tubulin: Nogales et al. 1998 Tubulin: Nogales et al. 1998 Light Microscopy is limited by the diffraction limit Figure 9-3b Molecular Biology of the Cell ( Garland Science 2008) Light microscopy can only resolve structures bigger than 0.2 µm Resolution is limited by the wave length of light Electrons Electrons are used instead of light waves for illumination Electrons are easily absorbed & scattered by different forms of matter, e.g. heavy metals or proteins, this interaction forms the image Electrons have a low penetration depth->sectioning is necessary High vacuum is necessary because electrons have an extremely low mass and easily give up their energy in collisions with gas atoms and molecules 14 Resolution Resolution is determined by the wavelength of the illumination (electron source) Higher kv of electron source -> shorter wavelength ->smaller details can be resolved The amount of electrons (current density) and the size of the electron beam (spot size) determines resolution The source of electrons determines the coherence of the electron beam Resolution is normally limited by imperfections in the optics, noise and sample preparation artifacts! Light Microscope TEM JEOL TEM 1230 Cathode CCD Camera Figure 9-42 (part 1 of 2) Molecular Biology of the Cell ( Garland Science 2008) 17 Datum 18 2
FEI Talos Electron microscopy for higher resolution Microscope column and cameras nicely wrapped in a box No oculars, camera filming the fluorescent screen Electrons that reach the fluorescent screen forms a bright spot and electrons that do not reach the screen form dark spot The varying degree of intensity of electrons form the image with a varying degree of grey Datum 20 2D projection of a 3D object The organells in a eucaryotic cell Since biological materials generally have a low atomic number, the dispersion is poor Very poor dispersion means very poor contrast in the image formation How do biologists increase the image contrast??? Figure 9-45 Molecular Biology of the Cell ( Garland Science 2008) Mitochondria Golgi apparatus Chloroplast 25 26 3
Bacteria Proteins in solution Microtubule Negative staining electron microscopy 3 ways to work with biological/soft/hydrated material Drying and staining with heavy metal salts Negative Staining Dehydration and embedding in plastic Ultra microtome sectioning Cryo Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing 28 Linda Sandblad 30 Negative staining Recombinant human keratin Adenovirus Protein in solution Heavy metal salt What you see in the TEM 31 K1 + K10 100 nm K5 + K14 100 nm Carin Årdahl & Linda Sandblad From a living cell to a EM specimen 3 ways to work with biological/soft/hydrated material Drying and staining with heavy metal salts Negative Staning Dehydration and embedding in plastic Ultra microtome sectioning Cryo Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing Chemical fixation: Osmification: Dehydration: Resin infiltration: Polymerization: Sectioning: Post staining: Preserve the cell morphology Protect tissue against disruption Keep antigens at their original localization Glutaraldehyde and paraformaldehyde Fixative and provides contrast to membranes Replace water by organic solvent A plastic material The support needed for sectioning Plasticity - specimen becomes less brittle Heat or UV-light crosslink polymers Ultra microtome and diamond knife Contrasting of proteins, membranes and sugar 35 4
Figure 9-44 Molecular Biology of the Cell ( Garland Science 2008) Figure 9-44 Molecular Biology of the Cell ( Garland Science 2008) Sectioning Sections of cells on a EM grid Sections of cells on a EM grid A microtome makes physical sections of large tissue Section thickness 50-80nm: Determined by inteference colours Glass or diamond Best contrast and maximum resolution 37 Sidfot Sidfot Sidfot Linda Sandblad 40 41 42 How to interpret your image Tokuyasu Characteristic non-membrane staining with good possibilities for immuno-gold-labeling Lenore Johansson Sara Henriksson Lenore Johansson and Roland Rosqvist, YopE-immunogold on Yersinia Svitlana Vdovikova: Macrophage with Listeria 43 45 5
HPF and AFS High Pressure Freezing + Freeze Substitution 3 ways to work with biological/soft/hydrated material Drying and staining with heavy metal salts Negative Staining Dehydration and embedding in plastic Ultra microtome sectioning Cryo Vitrification - HPF + AFS, HPF + cryo ultra microtome sectioning, plunge freezing Instant fixation (6ms) under high pressure (2000 bar) gives perfect fixation of your sample Sample is dehydrated and embedded in plastic at low temperatures to avoid ice crystal formation Haemophilus influenzae Chemical fixation RT HPF and AFS Linda Sandblad 48 TEM methods @ UCEM Gives better and more native structure of your specimen More time consuming and expensive. Each type of sample might require optimization to get a nice result Facility service: Chemical fixation, plastic embedding and ultra microtome sectioning Tokuyasu frozen frozen/cryo ultramicrotome sectioning Negative staining Immunolabeling for EM High pressure freezing (HPF) Automated freeze substitution (AFS) Correlative light and electron microscopy (CLEM) together with BICU=National Microscopy Infrastructure, NMI Method development: Cryo EM for structure biology Electron tomography FIB-SEM Future facility: Cryo-CLEM Service and method development: Negative staining with improved resolution for single proteins visualization The Electron Gun Filament (20-100 KV) Bias (Wehnelt) Cylinder Anode How are electrons generated? Thermionic emission Tungsten (W) filament Lanthanum hexaboride (LaB6) filament Field emission gun Linda Sandblad and Tomas Edgren: Yersinia membrane vesicles and flagella 52 stream of electrons originating from outer shell of filament atoms 53 W hairpin LaB6 crystal FEG 6
The Electron Gun Field emissions Condenser lens system Filament (20-100 KV) Bias (Wehnelt) Cylinder V 1 V 2 Anode An extremely high field is produced at the sharp tip of the cathode. This reduces the potential barrier and permits electrons to tunnel out. The condenser aperture must be centered stream of electrons originating from outer shell of filament atoms 55 C1 controls the spot size C2 changes the convergence of the beam Electron beam Electron interaction with the specimen Image formation occurs by electron scattering Electron strike the atomic nuclei and get dispersed This disperse electrons form the image Visible light X-rays Back-scattered electrons (D) Secondary electrons (E) Electron interaction with the specimen 4. Some electron (D) get backscattered instead of getting transmitted through the specimen 1. Transmitted electrons (A) of the beam passes straight through the specimen on to the screen 2. Some electron (B) of the beam lose a bit of their energy while passing through the specimen & get deflected a little from their original axis of the beam à inelastically scattered electrons 3. Some electron (C) interact with atoms of specimen & get elastically scattered without losing energy. Electron deviate widely (C ) Elastically scattered electrons (B) Inelastically scattered (A) electrons Transmitted electron 5. In some cases the electrons get absorbed by the atoms of the specimen & instead low energy electron (E) are emitted. These electron are termed secondary electron. These are very useful for forming the image in the SEM 6. Some atom emit x-ray & light energy 58 59 Chose a suitable grid Chose a suitable grid EM sample preparation for proteins or microorganisms in vitro Which metal? What surface, open, formvar, carbon, holy carbon Mesh size, how large is your sample, how stable is your surface film? Do you need to tilt? Finder grids for correlative microscopy Make your one prefect surface - in the negative staining practical Pull a formvar film on a glass slides Floating a thin film on the water surface Carbon coating Glow discharge 62 Linda Sandblad 63 7
TEM FACILITIES @ UCEM EM sample preparation for microorganisms, cells and tissue EM sample preparation for microorganisms, cells and tissue JEOL 1230 Transmission Electron Microscope Unfixed tissue/cells/molecules Unfixed tissue/cells/molecules Cryo fixation methods Ultra microtome sectioning Carbon coater and glow discharge equipment Chemical fixation at room temperature Plastic embedding High pressure freezing Freeze substitution Plunge freezing (Sample + fiducial marker) Chemical fixation at room temperature Plastic embedding High pressure freezing Freeze substitution Plunge freezing (Sample + fiducial marker) Ultra microtome sectioning Fiducial marker application TEM of this sections Tomography Ultra microtome sectioning Fiducial marker application TEM of this sections Tomography EM sample preparation for microorganisms, cells and tissue Unfixed tissue/cells/molecules Chemical fixation at room temperature Plastic embedding High pressure freezing Freeze substitution Plunge freezing (Sample + fiducial marker) Ultra microtome sectioning Fiducial marker application TEM of these sections Tomography 8