Goals of Specimen Preparation Observe specimen near natural state as possible. Preservation of as many features as possi

1. 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)

2. Preparation of Biological Samples • Fixation and washing/rinsing • Clearing of tissue (light microscopy-optional) • Dehydration • Embedding • Sectioning • Mounting • Staining

3. Fixation A process which is used to preserve (fix) the structure of freshly killed material in a state that most closely resembles the structure and/or composition of the original living state. • Chemical crosslinking - coagulative/noncoagulative • Coagulative: • original killing agents (alcohols, Farmer’s, FAA, Bouins) • Low pH Unbuffered • Coagulates cellular components - like frying an egg. • Non Coagulative: Formaldehyde, Glutaraldehyde, Osmium Tetroxide

4. Formaldehyde Usually in form of paraformaldeyde powder or 37% to 16% aqueous solution • Low MW makes it one of the best penetrating of all the fixatives, thus it is widely used in fixation of resistant materials, such as seeds, spores, plant material, etc., usually in conjunction w/ another aldehyde. • Formalin contains many impurities, so formaldehyde for use in EM is normally prepared from the dissolution, heating, and alkalination of powdered paraformaldehyde. Since this solution contains no inhibitors, it has a shelf life of only a few weeks.

5. 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. GTA fixation is irreversible. • In TEM, buffered GTA has the reputation of providing the best ultrastructural preservation in the widest variety of tissue types of any known chemical fixative.

6. Osmium Tetroxide (OsO4) • A non-polar tetrahedral molecule with a • molecular weight of 254 and solubility water • and a variety of organic compounds. • Its principle utility is its ability to stabilize and stain lipids- preferentially unsaturated fatty acids • Commercially available as a coarse yellow crystalline material packaged in glass ampoules sealed under inert gas. Similarly packaged aqueous solutions are also available. • An additive, non-coagulative type of fixative, but lacks the ability to crosslink many proteins. • Very poor rate of penetration

7. Basic factors affecting chemical fixation pH (Isoelectric point) Total ionic strength of reagents Osmolarity Temperature Length of fixation Method of application of fixative

8. Buffers Solution containing a weak acid and its salt. Serves to hold pH steady during the fixation process. • Chemical fixation is a complex set of oxidative and reductive reactions, thus [H+] is constantly changing. • All fixatives have an optimal pH at which rate of crosslinking is greatest. • At a specific pH, all proteins have a point, the isoelectric point (IEP) where the numbers of + and - charges are equal. Fixation is most effective at the IEP.

9. Tonicity • Osmolality of fixatives, buffers, and tissue fluids can be measured with an OSMOMETER • Effect of tonicity: • 1.Isotonicity • Environment and • Sample similar • 2.Hypertonicity • Environment higher osmolarity • Water moves out of sample • 3.Hypotonicity • Environment lower osmolarity • Water enters sample 5 mOsm 5 3 8

10. Dehydration • Reasons for dehydration: • Water in incompatible with conditions inside an electron column. • Most of the materials used to infiltrate and embed specimens prior to ultrathin sectioning are hydrophobic. • Methods of Dehydration: • Organic solvent Series • Tissue is transferred through a series of organic solvents in increasing concentration. • Ethanol and acetone are the most commonly used. • Water content is slowly reduced to the point that the tissue is in 100% solvent. and is thus completely dehydrated.

11. Embedding and Sectioning • Requirements for cutting any material into thin slices: • Support - biologicals tend to be soft. Inducing hardness in them gives them the mechanical support needed for sectioning. • Accomplished by lowering temperature (freezing) or infiltration with some material that can be hardened. • Plasticity - resiliency as opposed to brittleness.

12. Embedding and Sectioning • Cryosectioning • Commonly done for light microscopy. • ie hospital operating room biopsies. • Rapid. • Preservation is usually sufficient for a rapid diagnosis. • Overall resolution is low. • Ultrathin cryosectioning • Technically demanding • Requires expensive specialized equipment • Ultrastructural preservation often poor due to freezing artifact. • Usually done only when tissue cannot be exposed to chemical fixatives...as in some immunolabeling, analytical work.

14. Embedding and Sectioning • Embedment • Light microscopy • Tissue infiltrated with molten paraffin wax - which is allowed to cool and harden. • Requires dehydration and infiltration with a paraffin solvent - aromatic hydrocarbon (xylene, toluene, benzene). • Provides sufficient support to section to about 3 micrometers minimum with a steel knife. • Paraffin can infiltrate deeply into tissue, allowing large blocks and ultimately large sections to be obtained.

15. Embedding and Sectioning Paraffin Sectioning for Light Microscopy

17. TEM Embedment • Tissue infiltrated with a resin which is polymerized by heat, chemicals, or U.V. • Provides support to section infiltrated tissue to about 40 nm minimum. • Infiltration is limited...specimens can be no more than a few mm thick. • The required thinness of the sample and the friction during cutting limits the section size to about 1 mm2 maximum.

18. Types of Resins • Acrylics - ie methyl, butyl methacrylates (plexiglass) - "Open-structured" - allows for better stain penetration and Antibody rxn • Epoxies - epon, araldite, Quetol, Spurr - for most general work • Polycarbonates - vestopal - fiberglass resin • Infiltration • In resin/solvent mixture in increasing concentration • Ethanol/resin or acetone resin often used • Propylene oxide/resin is most effective • Polymerization • Thermal - 50-70 C, depending on resin mix • U.V. - usually done to avoid heat • of polymerization. Often done at low temp.

19. Ultramicrotomy • Ultramicrotome Knives: • Diamond - 1.5 - 6mm cutting edge • Latta-Hartmann (glass) - 6mm cutting edge (~1mm useable) • Both use water to support and lubricate the section as it is cut (decreases friction)

20. Making a glass knife: • Use of a glass knifemaker to score a 1" glass square

21. A scored 1" glass square (top) and the resultant glass knife: Making the water trough Tape or plastic a) Cutting edge b) Knife angle (45o) c) Corner d) Shelf

22. Evaluating a glass knife - factors to consider: • Age - degrade rapidly due to edge flaking • Quality of cutting edge - flat, concave, convex • Amount of cutting edge - judged by the stress line. A "spur" is normal. • Contamination - on edge or sides.

23. Setting up the Microtome Block face Sample Block Knife edge Glass Knife

24. Syringe - adjusting water in trough Loop - assist picking up sections Eyelash tools - assist with section manipulations

25. Standard Preparation Tissue TEM SEM Chem. Fixation Cryo Fixation Chem. Fixation Cryo Fixation Rinse/store Substitution Rinse/store En bloc staining Cryo- sectioning Dehydration Dehydration Dehydration Drying Resin infiltration Mounting Sectioning Coating Post staining

26. Support Films Formvar, Carbon, Collodion -Used when sections or samples are smaller than support of grid. -100 mesh or less, slot grids -Fragile or very thin sections Avoid when possible because: Usually has holes or uneven thickness Added thickness affects clarity and contrast

27. Formvar Coating

28. Formvar coated grids Holey formvar Formvar and carbon

29. Negative Staining Positive staining - forms a complex with specimen Negative - stain and specimen do not interact and specimen remains electron transparent Advantages: 1) Improved resolution 2) Speed 3) Unique information 4) Simplicity

30. Disadvantages: 1) Repeatability 2) Limited surface topography 3) Toxicity

31. Choice of stain: 1) High density to provide high contrast 2) High solubility and minimal reaction to sample 3) High melting and boiling point (beam stable) 4) Precipitant formed is extremely fined grained Stains commonly used: Phosphotungstate, sodium tungstate, uranyl acetate and uranyl nitrate

32. Brief procedure: Small grid and support film (formvar, paraloidin. Sometimes carbon added. Thin suspension of sample and excess removed. Dry then add negative stain and remove Factors affecting staining: concentration of stain pH of stain time - Dry and view.

34. Negatively stained Ad2 (K. Boucke)

35. Bacteria with flagella

36. SARS inducing virus (coronavirus)

37. Negative stain of purified RhMV virus labelled with anti-RhMV and detected with anti- rabbit conjugated to 10 nm gold. Bar = 100 nm. Photograph provided by Fred Gildow Lab, Department of Plant Pathology, Penn State.