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.
Problems
TEM not widely used by biologists until 1950’s
Considerations for TEMHigh vacuum
Support of sample
Intense heat from beam
Depth of electron penetration
Considerations for SEMHigh vacuum
Size of specimen
Localized elevated temperatures
Capable of emitting signal
Conductive
Early images of
diatoms
Standard Preparation
Tissue
TEM
Chem.
Fixation
Rinse/store
SEM
Cryo
Fixation
Cryo
Fixation
Substitution
Rinse/store
En bloc
staining
Dehydration
Chem.
Fixation
CryoDehydration Dehydration
sectioning
Drying
Resin
infiltration
Sectioning
Post staining
Mounting
Coating
Specimen preparation
Stabilization - Fixation and dehydration.
Embedding 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)
Basic factors affecting chemical fixation
pH (Isoelectric point)
Total ionic strength of reagents
Osmolarity
Temperature
Length of fixation
Method of application of fixative
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 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.
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
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)
•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
3
8
5 mOsm
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, Farmer’s,
FAA, Bouins)
Coagulates cellular components - like frying an
egg.
- Non Coagulative: Formaldehyde, Glutaraldehyde,
Osmium Tetroxide
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*
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.
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.
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 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 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
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.
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)
Embedding and Sectioning
Making a glass knife:
•Use of a glass knifemaker to score a 1" glass square
Embedding and Sectioning
A scored 1" glass square (top)
and the resultant glass knife:
a) Cutting edge
b) Knife angle (45o)
c) Corner
d) Shelf
Making the water trough
Tape or plastic
The knife edge
Trimming the block
The proper size for the block face
Setting up the Microtome
Block
face
Glass
Knife
Sample
Block
Knife
edge
Tools Needed:
Syringe - adjusting water in trough
Loop - assist picking up sections
Eyelash tools - assist with section manipulations
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.
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:
Picking up sections
Mesh grids
Eyelash tool
Slot grids
Collecting on slot grids
Sections floating
on water
Dried on bridge, then
punched out for viewing
Section Mounting
An ultrathin section on a 50m support filmed grid at 200X mag.
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”).
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.
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
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
Formvar Coating
Formvar coated grids
Holey formvar
Formvar and carbon
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
Disadvantages:
1) Repeatability
2) Limited surface topography
3) Toxicity
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
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.
Negatively stained Ad2 (K. Boucke)
Bacteria with flagella
SARS inducing virus
(coronavirus)
Negative stain of purified RhMV virus labelled with antiRhMV 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.