TEM

1
Tissue
Standard Preparation
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
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 - 50-70 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 - 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)

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 lowcompression too highchatter
• 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 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.

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) 0 -
  1000m
• 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 - 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.

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.