Goals of Specimen Preparation

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) Render
specimen stable for examination in environment of
TEM.
2
Problems
TEM not widely used by biologists until 1950s
Considerations for TEM- High vacuum Support of
sample Intense heat from beam Depth of electron
penetration
Considerations for SEM- High vacuum Size of
specimen Localized elevated temperatures Capable
of emitting signal Conductive
Early images of diatoms
3
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
4
Specimen preparation
Stabilization - Fixation and dehydration. Embeddin
g in resin for TEM Surface Preparation -
cleaning and/or exposure of new surface for SEM.
Cutting specimen to ultra-thin sections for
TEM Mounting - specimen on stub (SEM) or grid
(TEM) Staining with heavy metals for image
contrast (TEM)
5
Basic factors affecting chemical fixation
pH (Isoelectric point) Total ionic strength of
reagents Osmolarity Temperature Length of
fixation Method of application of fixative
6
Buffers

• Def. - a solution containing a weak acid and its
  salt.
• Serve 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 the
  rate of cross-linking 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.

7
CONSIDERATIONS IN THE SELECTION OF A BUFFER

• pH - each buffer has a point (pKa) where there is
  equal concentration of acid and base.
• Compatibility with fixatives components and
  stains
• E.g. - GTA reacts with buffers containing
  sulfhydryl groups (TRIS, HEPES) and phosphate
  buffer precipitates uranyl acetate, a commonly
  employed TEM stain.
• Introduction of artifacts - if elemental analysis
  is to be done.
• Effective at low concentrations
• Low Cytotoxicity

8
CONSIDERATIONS IN THE SELECTION OF A BUFFER
Tonicity Osmolality a measure of solute
concentration. Osmolality in groups of
organisms Mammals - 290- 700 mOsm (plasma)
Reptiles - 325 Marine organisms (250 - 375, 1000
or more) Freshwater invertebrates - as low as 30
mOsm Plants - differs with the tissue and species
(meristematic 400, mature vascular tissue
800)
9

• Tonicity
• 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
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) Coagulates
cellular components - like frying an egg. - Non
Coagulative Formaldehyde, Glutaraldehyde, Osmium
Tetroxide
11
Osmium tetroxide

• An additive, non-coagulative type of fixative,
  but lacks the ability to crosslink many proteins.
• Very poor rate of penetration
• Its use as a primary fixative is quite limited,
  although it is popular in some mixtures with
  other fixatives for unicellular organisms.
• Due to its extreme toxicity, low vapor pressure,
  and being a strong oxidizing agent, precautions
  are necessary for its handling.
• Vapors rapidly fix exposed mucous membranes such
  as those in the eyes (eventually causing
  blindness) and respiratory tract (lung edema).
• Mode of action - reacts primarily w/ double bonds
  and sulfhydryl groups of proteins, causing major
  conformational changes in the structure of
  proteins

12
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.

13
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.

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
16
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.

17
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

18
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.

19
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)

20
Embedding and Sectioning

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

21
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
22
The knife edge
23
Trimming the block
24
The proper size for the block face
25
Setting up the Microtome
Block face
Sample Block
Knife edge
Glass Knife
26
Tools Needed
Syringe - adjusting water in trough Loop -
assist picking up sections Eyelash tools -
assist with section manipulations
27
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.

28
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

29
Picking up sections
Mesh grids
Eyelash tool
Slot grids
30
Collecting on slot grids
Dried on bridge, then punched out for viewing
Sections floating on water
31
Section Mounting
An ultrathin section on a 50m support filmed grid
at 200X mag.
32
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).

33
Post-Staining

• Typically always used, even if en bloc staining
  (ie uranyl acetate) has been done.
• Uranyl acetate - 0.5 - 2 aqueous. Also can use
  saturated ethanolic or methanolic UA
• Lead citrate - several formulations (Venable and
  Coggeshell or Reynolds). Common is 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.
34
Staining with UA
Lead staining

• Typical protocol
• - 30 minutes UA
• - Wash with water
• - 5 minutes lead citrate
• - Wash once in 0.02M NaOH
• Wash well with water.
• Dry by wicking with filter paper

35
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
36
Formvar Coating
37
Formvar coated grids
Holey formvar
Formvar and carbon
38
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
39
Disadvantages 1) Repeatability 2) Limited
surface topography 3) Toxicity
40
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
41
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.
42
(No Transcript)
43
Negatively stained Ad2 (K. Boucke)
44
Bacteria with flagella
45
SARS inducing virus (coronavirus)
46
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.