Preparation and imaging of biological samples in the transmission electron microscope - an introduction Graham Knott, BioEM Facility, CIME, EPFL Objectives To understand the principles for preparing cells and tissues for observation in the TEM To understand the ways that biological material can be imaged in the TEM Why do we need electron microscopes for biology? 2 Dimensions of biological structures metres (1) human millimetres (10-3 ) insects-fleas flea micrometres (10-6 ) cells - bacteria nanometres (10-9 ) viruses - proteins picometres (10-12 ) small molecules-atoms red blood cells bacteria virus particles bacteriophage The resolving power of a microscope is proportional to λ/na Introduction Silver stained neurons in the rat sensory cortex - optical image TEM image of a single synaptic connection 3 Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possible. Avoid artifacts (changes, loss or additional information) Render specimen stable for examination in environment of EM reconstruction of a single clathrin -coated vesicle Introduction
4 Considerations for EM of biological structures Samples contain atoms of low molecular weight Most biological samples contain water Intense heat of the e beam High vacuum Size of Specimen -only thin or very small samples can be imaged without sectioning jelly fish - aequoria victoria Introduction 5 General schematic for preparing biological samples Standard Preparation Tissue TEM SEM chemical fixation cryo fixation chemical fixation cryo fixation wash / store substitution wash / store en bloc staining dehydration dehydration resin infiltration mounting sectioning cryo-sectioning coating post staining Imaging Introduction 6 Fixation A process used to preserve the structure of freshly killed material in a state that most closely resembles the structure and/or composition of the original living state. Chemical - coagulative/noncoagulative Coagulative: original killing agents (alcohols, Bouin s), Low ph, unbuffered, coagulates cellular components. Non-coagulative: formaldehyde, glutaraldehyde, osmium tetroxide Egyptian mummy - few thousand years old Freeze Fixation Baby mammoth, 37,000 years old Fixation
7 Chemical fixation of biological samples - considerations ph (Isoelectric point) Total ionic strength of reagents Osmolarity Temperature Length of fixation Method of application of fixative 8 Osmolarity 0.2% glutaraldehyde 2% glutaraldehyde 8% glutaraldehyde in 0.1M phosphate buffer 9 Chemically fixing biological samples Pieces should be small to allow rapid and complete penetration of the fixative. The volume of fixative should exceed the volume of tissue by factor of 10 eg. - plants - small pieces may be excised and dropped directly in fixative. Very small plants and algae may be fixed whole Insects, other invertebrates can be dropped into the fix Larger tissues should be perfused with the fixative
10 Interpreting EM images of fixed tissue perfused unperfused TEM images of kidney tubules 11 Interpreting EM images of fixed tissue choroid plexus - perfused tissue Typical fixatives - aldehydes 12 Formalin Low MW - makes it one of the best penetrating of all the fixatives, widely used in fixation of resistant materials, such as seeds, spores, plant material, Formalin contains many impurities, so formaldehyde for use in EM, prepared from paraformaldehyde Glutaraldehyde Glutaric acid dialdehyde, a 5 carbon dialdehyde, is the most widely applied fixative in both scanning and transmission electron microscopy. Most highly cross-linking of all the aldehydes. Glutaraldehyde fixation is irreversible.
Action of aldehydes on proteins causes the cross-linking of proteins 13 Effects of aldehydes fixatives 14 glutaraldehyde formaldehyde TEM images of rat liver 15 Over-fixing - artefacts Muscle tissue fixed for 3 days at room temperature in 2.5% glutaraldehyde
Other fixative (and membrane marker) - Osmium Tetroxide 16 Non-polar tetrahedral molecule MW 254, solubility in water and a variety of organic compounds Ability to stabilize and stain lipids - preferentially unsaturated fatty acids Yellow crystalline material packaged in glass ampoules sealed under inert gas. Very poor rate of penetration Vapors rapidly fix exposed mucous membranes such as those in the eyes (eventually causing blindness) and respiratory tract (lung edema). Mode of action - reacts primarily w/ double bonds and sulfhydryl groups of proteins, causing major conformational changes in the structure of proteins* dendritic spine in mouse cerebral cortex / Staining Other heavy metal stains 17 Lead citrate (Reynolds stain; stains poysaccharides, eg. glycogen) Uranyl acetate (basic stain - eg DNA) uranyl acetate - staining of the basic structures (DNA and RNA) lead citrate (polysaccharides-eg glycogen) sample: adult rat liver - aldehyde fixed and resin embedded Staining 18 Standard Preparation Tissue TEM SEM chemical fixation cryo fixation chemical fixation cryo fixation wash / store substitution wash / store en bloc staining dehydration dehydration resin infiltration mounting sectioning cryo-sectioning coating post staining Imaging
19 Freeze fixation Reasons for freeze fixation halt rapid events structures are fixative sensitive. removal of water changes topography/morphology Disadvantages Specialized equipment required Freeze Damage Limited view of specimen or difficulty manipulating frozen material Hazards of using some cryogens 20 Cryogens Melting pt Boiling pt Freon 13-181 - 81 Isopentane -160 28 Propane -189-42 Nitrogen -209-196 Ethane -183-88 Helium -272 (1 o K) -269 Freeze Fixation 21 Freezing Equipment Device Freezing depth (microns) cost Plunge freezer 10-20.50-50 Spray freezer 10-20 10-50 Slam freezer 20-40 2K Propane Jet 40 10K High Pressure 50-100 150K plunge freezing bath slam freezing device high pressure freezer Freeze Fixation
22 Standard Preparation Tissue TEM SEM chemical fixation cryo fixation chemical fixation cryo fixation wash / store substitution wash / store en bloc staining dehydration dehydration resin infiltration mounting sectioning cryo-sectioning coating post staining Imaging Freeze Fixation 23 Embedding in resin Epoxy resin - araladite Acrylic resin - methyl methycrylate Resin Embedding 24 Block trimming with a glass knife Thin sectioning
Tissue blocks are sectioned with diamond or glass knives using ultramicrotomes 25 Serial thin sectioning 50 nm thick sections floated on water - 800 microns wide Thin sectioning Typical EM grids for holding sections 26 300 trous 2 mm single slot grid multi hole grid cryo sections placed on grid Thin sectioning TEM of adult mouse S1 layer IV 27 TEM imaging - thin sections
serial TEM images of adult neuropil 50nm thick sections 28 TEM imaging - thin sections Immunocytochemistry - is a means of localizing particular molecular complexes (antigens) antigens within tissue and cells using antibodies 29 pre-embedding (before resin embedding this tissue) post-embedding (after embedding in the tissue) Example of pre-embedding immunocytochemistry TEM imaging - thin sections 30 TEM image of a single synapse in the adult brain Schematic diagram of vesicle trafficking in a single bouton
31 Plunge freezing a sample of protein complex in solution Cryo-electron microscopy image of clathrin-coated vesicles embedded in vitreous ice 32 Cheng et al. Journal of Molecular Biology (2007) vol. 365 (3) pp. 892-899 33
34 Cheng et al. Journal of Molecular Biology (2007) vol. 365 (3) pp. 892-899 35 Cheng et al. Journal of Molecular Biology (2007) vol. 365 (3) pp. 892-899 36 Cheng et al. Journal of Molecular Biology (2007) vol. 365 (3) pp. 892-899
37 Cheng et al. Journal of Molecular Biology (2007) vol. 365 (3) pp. 892-899 38 Schematic drawing of a single mitochondria based on results from Palade (Palade, G. The fine structure of mitochondria. Anat. Rec. 114 (1952), pp. 427 451) 39
Single particle staining at ambient temperature - negative staining 40 Principles of negative staining Papillomavirus Hepatitis b virus 41 Virus phage image recorded with ORIUS SC1000 at TEM magnification of 43,000x and 120kV. Sample courtesy of Dr. Hans-W. Ackermann, MD, Department of Medical Biology, Laval University, Quebec Canada 42 Virus phage imaged with TEM, negative staining