Goals of Specimen Preparation

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Goals of Specimen Preparation Observe specimen
near natural state as possible. Preserv
ation of as many features as possible. Avoid
artifacts (changes, loss or additional
information)
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Preparation of Biological Samples

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

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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, Farmers, FAA,
  Bouins)
• Low pH Unbuffered
• Coagulates cellular components - like frying an
  egg.
• Non Coagulative Formaldehyde, Glutaraldehyde,
  Osmium Tetroxide

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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.

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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.

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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

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

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• 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
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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.

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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.

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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.

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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.

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Embedding and Sectioning
Paraffin Sectioning for Light Microscopy
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• 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.

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• 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.

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• 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)

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

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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
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• 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.

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Setting up the Microtome
Block face
Sample Block
Knife edge
Glass Knife
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Syringe - adjusting water in trough Loop -
assist picking up sections Eyelash tools -
assist with section manipulations
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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
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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
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Formvar Coating
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Formvar coated grids
Holey formvar
Formvar and carbon
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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
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Disadvantages 1) Repeatability 2) Limited
surface topography 3) Toxicity
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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
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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.
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Negatively stained Ad2 (K. Boucke)
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Bacteria with flagella
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SARS inducing virus (coronavirus)
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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.