TEMTEM SEMSEM
TissueStandard PreparationStandard Preparation
Chem.Fixation
CryoFixation
Chem.Fixation
CryoFixation
Rinse/store
Rinse/storeEn blocstaining
Substitution
Cryo-sectioningDehydration
Dehydration Dehydration
Resin infiltration
SectioningPost staining
Drying
Mounting
Coating
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.
Dehydration
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.
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.
Embedding and Sectioning
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.
Embedding and Sectioning
Paraffin Sectioning for Light Microscopy
Embedding and SectioningEmbedding 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.
Embedding and SectioningEmbedding and SectioningTypes 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
Embedding and SectioningEmbedding 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 - 50-70 C, depending on resin mix
•U.V. - usually done to avoid heat •of polymerization. Often done at low temp.
Embedding and SectioningEmbedding and SectioningUltramicrotomy
•Mechanical Advance•Thermal Advance
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)
Embedding and SectioningEmbedding and Sectioning
Making a glass knife: •Use of a glass knifemaker to score a 1" glass square
Embedding and SectioningEmbedding and Sectioning
A scored 1" glass square (top) and the resultant glass knife:
a) Cutting edgeb) Knife angle (45o)c) Cornerd) Shelf
Making the water troughTape or plastic
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.
Setting up the Microtome
Glass Knife
SampleBlock
Knifeedge
Blockface
Tools Needed:Tools Needed:Tools Needed:Tools Needed:
Syringe - adjusting water in trough
Loop - assist picking up sections
Eyelash tools - assist with section manipulations
Sectioning - TroubleshootingSectioning - 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
SectioningSectioning
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
SectioningSectioningSectioningSectioning
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.
Embedding and SectioningEmbedding and Sectioning
Section Thickness
•Ideally, sections should be in the 55 - 60 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 85 - 90 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.
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) 0 - 1000m
•The smaller the mesh size, the greater the support (section drifting, splitting), but the less open area for viewing.
Section MountingSection Mounting
Section MountingSection 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:
Picking up sectionsPicking up sections
Mesh grids
Slot grids
Eyelash tool
Sections floatingon water
Dried on bridge, thenpunched out for viewing
Collecting on slot grids
Section Mounting
An ultrathin section on a 50m support filmed grid at 200X mag.
Post-Staining
•Normally done, even if en bloc staining (ie uranyl acetate) has been done.
•Uranyl acetate - 0.5 - 2% 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.
Staining with UA
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.
Contrast
Light Microscopy:
•Contrast achieved by:
•Selective staining•Chromatic stains selectively bind to specific components in the specimen. E.g. - Hematoxylin/Eosin
Contrast
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”).
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.
