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

2. 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.

3. 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.

4. 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.

5. 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.

6. Embedding and Sectioning
Paraffin Sectioning for Light Microscopy

7. Embedding and Sectioning
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.

8. Embedding and Sectioning
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
Epoxy Resins - most commonly used.
Components:
Resin - Epon 812, Araldite 502 or 6005
Hardener - DDSA - amount can be varied
Plasticizer - NSA
Accelerator - DMP-30

9. Embedding and Sectioning
Infiltration
In resin/solvent mixture in increasing concentration
Ethanol/resin or acetone resin often used
Propylene oxide/resin is most effective
When 100% resin is reached, it should be changed twice to insure that all solvent is removed
Polymerization
Thermal C, depending on resin mix
U.V. - usually done to avoid heat
of polymerization. Often done at low temp.

10. Embedding and Sectioning
Ultramicrotomy
Mechanical Advance
Thermal Advance
Ultramicrotome Knives:
Diamond mm 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)

11. Embedding and Sectioning
Making a glass knife:
Use of a glass knifemaker to score a 1" glass square

12. Embedding and Sectioning
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

13. 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.

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

15. Tools Needed: Syringe - adjusting water in trough
Loop - assist picking up sections
Eyelash tools - assist with section manipulations

16. Sectioning - Troubleshooting
Factors affecting ultrathin sectioning quality:
Embedment - poor infiltration, polymerization, too soft, too hard, brittle, etc.
Quality of Knife - sharpness, scratches
Dullness - alternate cutting and skipping.
Compression - lines perpendicular to the direction of cut.
Microgrooves
not resolvable by LM
in their absence, the knife edge will appear as a bright line under a dissecting scope
cause striations parallel to the direction of cut

17. Sectioning Factors affecting ultrathin sectioning quality:
Contamination (of knife, specimen block, or trough water) - oil, dust. Can cause lines or seen on sections
Knife angle
Usually 4o - 6o
too low=compression; too high=chatter
Environmental factors:
Building vibration - antivibration measures (inner tubes, tennis balls, granite slabs, etc…)
Static electricity - usually causes sections to be pulled down the back of the knife.
Try grounding the microtome, increasing humidity in room, or use a Zerostat or Staticmaster

18. Sectioning More factors affecting sectioning quality: Wind currents
Cutting speed - must be fast enough that vibration does not cause uneven cutting (chatter) and slow enough that the section is not compressed.
Water level in trough…
too high = block wetting; too low = compression
Block size and shape - the trapezoid.

19. Embedding and Sectioning
Section Thickness
Ideally, sections should be in the nm range.
This allows for enough stain uptake for contrast, and maximum resolution (limited in the TEM by specimen-induced chromatic aberration).
Determined by interference colors.
Maximum thickness should not exceed nm (light gold).
Thickness can sometimes be reduced by one color range by flattening sections - smooths out compression to a limited extent. Toluene, xylene, chloroform, heat.

20. Section Mounting Specimen Grids
3 mm support for TEM specimens. (a few are 2.6mm)
Different materials..usually copper...also nickel, gold, aluminum, platinum, stainless steel, beryllium, carbon, nylon.
Most are manufactured individually by electroplating; some are punched from screen stock; a few are woven.
Also differ by mesh size (bars per inch) m
The smaller the mesh size, the greater the support (section drifting, splitting), but the less open area for viewing.

21. Section Mounting A 200m grid has 60% open area; a 400m grid only 40%
Thin-bar grids...more fragile, more expensive.
Ultrathin sections can be supported on a bare grid of no greater than 200m.
Commonly used TEM grid types:

22. Picking up sections
Mesh grids
Eyelash tool
Slot grids

23. Collecting on slot grids
Dried on bridge, then
punched out for viewing
Sections floating
on water

24. Section Mounting
An ultrathin section on a 50m support filmed grid at 200X mag.

25. Post-Staining
Normally done, even if en bloc staining (ie uranyl acetate) has been done.
Uranyl acetate % aqueous or saturated ethanolic or methanolic
Lead citrate - several formulations (Venable and Coggeshell; Reynolds) mostly using lead nitrate chelated with sodium citrate.
Adequate rinsing between and after staining is essential to prevent post-stain contamination.
Particular care must be used to exclude CO2 to inhibit lead carbonate formation - black cannonballs.

26. Staining with UA

27. Contrast Light Microscopy: Contrast achieved by:
Use of special optics and filters which impart selective colors or brightness to areas differing in thickness or composition. E.g. - phase contrast, D.I.C. optics.

28. Contrast Light Microscopy: Contrast achieved by: Selective staining
Chromatic stains selectively bind to specific components in the specimen. E.g. - Hematoxylin/Eosin

29. Contrast Transmission Electron Microscopy:
Contrast is produced by the adsorption of heavy metals to specimen macromolecules.
The ability of an atom to absorb electrons is directly related to its mass.
Since biological specimens are composed mostly of low atomic # elements (C,O,H,N), they lack endogenous contrast....thus contrast is induced by "staining" with heavy metals.
Microscopists refer to the measure of a specimen's ability to absorb electrons as its electron density (vs electron “transparency”).

30. Contrast Transmission Electron Microscopy:
Heavy metals commonly used for contrasting in TEM: uranium, lead, osmium, ruthenium, molybdenum, gold, silver.
It is the differential adsorption of various heavy metals to tissue components that produces the electron image of biological thin-sectioned materials.
The image may be composed of areas ranging from completely black to completely white with all ranges of grey in between.
Images with mostly pure blacks and whites are "contrasty" images, while those containing mainly greys are "flat” images.