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
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 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.
•Buffering capacity is highest here.
•As pH is changed on either side, the capacity to hold pH steady
is decreased.
•A buffer must be selected which is effective at the pH desired.
•Example: tissue from acidic environment (digestive)
CONSIDERATIONS IN THE SELECTION OF A BUFFER
•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.
CONSIDERATIONS IN THE SELECTION OF A BUFFER
•Effectiveness at low concentration
•Most buffers begin to lose their capacity to control pH as they are
diluted out.
•E.g. - buffers at 0.1 M/l used for most plant and animal tissue,
however some freshwater invertebrates require dilution over a
thousand times to maintain isotonicity.
•Phosphate and cacodylate are no longer buffers at this conc.
CONSIDERATIONS IN THE SELECTION OF A BUFFER
•Cytotoxicity
•If buffer is to be used to rinse or temporarily hold specimens prior
to fixation, it should support life and NOT be toxic.
•Ringer Solution - a solution for temporarily maintaining the
normal physiological conditions of an organism.
•E.g. - phosphate buffered physiological saline (0.89% for
mammals).
• Otherwise you will be fixing dead or dying and autolysing
material.
•Cacodylate and collidine are very toxic.
•Tissue compatible (Vital) buffers - PBS, TRIS, HEPES, PIPES.
•Tonicity
•Osmolality = a measure of solute concentration.
•1 osmol of a substance = its mw / the # of freely moving particles each
molecule liberates in solution.
•Osmolality (DEF) - # of osmols per kilogram of solvent. For biological
systems, this is normally expressed as mOsm/ liter of water.
•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
•Osmolality of fixatives, buffers, and tissue fluids can be measured
with an OSMOMETER
•Effect of tonicity:
1.Isotonicity
Environment and
Sample similar
5
2.Hypertonicity
Environment higher osmolarity
Water moves out of sample
3
3.Hypotonicity
Environment lower osmolarity
Water enters sample
8
5 mOsm
SEM specimens should be fixed under near isotonic conditions
TEM specimens should be fixed under slightly hypertonic conditions.
Common Buffers used in Fixation
Sodium Cacodylate
Effective range is 6.4 - 7.4.
Lacks phosphates that could interfere with cytochemical
studies.
Incompatible with uranyl salts and should be rinsed out
thoroughly if planning to do "en-bloc" staining with UA.
Can add Calcium and Magnesium without precipitation.
Used extensively with animal tissues.
Contains arsenic, which is toxic. Avoid contact with acids to
avoid production of arsenic gas.
Phosphate Buffers
(Millonigs, Sorensens, PBS)
Effective range 5.0-8.0
More physiological than other buffers
Found in living systems in the form of inorganic phosphates
and esters.
Non-toxic to cells grown in culture.
Not as sensitive to temperature changes
Cause artifacts such as electron dense particles or nuclear
shrinkage.
Phosphate buffers should not be used when calcium is to be
added to the fix solution.
Infrequently Used Buffers
Collidine
Pyridine derivative with buffering capacity is 6-8 but most
efficient around 7.4.
Primarily used with Formaldehyde in tissue storage
Does not react with OsO4
May possibly extract proteins, but is good with high
concentrations of aldehydes
Is not recommended for EM
Has a bad smell.
Veronal Acetate
Effective between 4.2 and 5.2
Should not be used with aldehydes, since it reacts with these
fixatives
Does not precipitate UA
Preserves membranes well when used with OsO4
Tris
Poor buffering capacity below 7.5 and is a biological inhibitor.
Reacts with GA
Causes excessive extraction of cellular components
Not recommended for EM
HEPES
Zwitterionic buffer effective at 7.3 at 37 C
May interfere with amine-aldehyde rxns.
Appears to stabilize membranes.
Compatible with divalent cations.
PIPES
Another zwitterionic buffer effective at 6.66 at 37 C.
Thought to be better buffer and reduce lipid loss.
Has been reported to produce multivesicular myelin figures in rat cortex.
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)
Low pH
Unbuffered
Coagulates cellular components - like frying an
egg.
- Non Coagulative: Formaldehyde,
Glutaraldehyde, Osmium Tetroxide
•The volume of fixative should exceed the volume of tissue by 10X or
greater. (OsO4 is an exception)
•Pieces should be as small as possible to allow rapid and complete
penetration of the fixative, particularly when OsO4 is used.
•Plants - small pieces may be excised and dropped directly in fixative.
•Some very small plants and algae may be fixed as a whole (e.g.
Duckweed).
•Insects, other invertebrates.
•For SEM, its usually possible to get a good fix by dropping entire
bug into the soup.
•Cuticle is very hard to penetrate if its to be embedded and sectioned.
•On some with very tough cuticles, it may be necessary to puncture
the cuticle in places. Desired parts may be dissected away after
fixation.
•Agar or gelatin enrobement - for single-celled and very small
organisms
•Microorganisms (bacteria, protozoa, fungi, microscopic plants
and animals)
•Colonies on agar plates may be flooded with fix, then pieces of
agar cut out for further processing.
•Liquid cultures may be concentrated by gentle centrifugation.
•Cells may be attached to coverslips with polylysine, etc.
Aldehydes
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.
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.
Glutaraldehyde, cont.
•Does not prevent lipid extraction.
•Nucleic acids not preserved directly, but may be retained due to the
preservation of associated proteins in eukaryotes. Bacterial and viral
DNA not preserved.
•Soluble carbohydrates not preserved, but some glycogen may be retained.
•Reagent grade GTA is a 25 or 50% aqueous solution with a pH around 3 4, and containing impurities such as ethanol, methanol, glutaric acid, and
oxidation products. These products can exert a considerable influence on
fixation, it is advisable to use distilled GTA prepared specifically for EM
sealed in glass ampoules under inert gas
•Mode of action - Rxn w/
proteins is variable
depending on the available
reactive residues of the
proteins –but no general
agreement.
•Reported to react with:
•Lysine
•Tyrosine
•Tryptophan
•Phenylalanine
•Histidine
•Cysteine
•Proline
•Reactions outside a neutral range (pH 7.0 – 9.0)
are reversible.
•Never use a buffer that contains primary amines
•Serine
•Glycine
•Glycylglycine
•Arginine
•It is particularly important to match the osmolarity
of your tissue to avoid distortion.
Acrolein
•A 3 carbon olefinic aldehyde, introduced to EM by Luft (1959) for its utility as a
rapidly penetrating primary fixative in applications where penetration tends to be
a problem.
•Highly volatile and reactive compound
•Has a reputation for being hazardous and difficult to handle.
•Smells like scorching fat.
•Volatility gives it application as a vapor fixative, especially in cases where
anhydrous fixation is necessary, such as in the
stabilization of mineralized tissues where mineral loss or translocation may be a
problem.
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 lipidspreferentially unsaturated fatty acids
•Although it is widely used in preparative schemes for SEM, this must
be due at least in part to arbitrary whole-cloth adoption of TEM
fixation schemes for SEM. Except for cases where lipid retention is
essential,
the aforementioned qualities of this compound have much less to
offer the area of SEM.
•Commercially available as a coarse yellow crystalline material
packaged in glass ampoules sealed under inert gas. Similarly
packaged aqueous solutions are also available.
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*
Osmium tetroxide
•Causes tissue hardening, imparting mechanical strength and affording
some extra resistance to shrinkage and distortion during subsequent steps.
•Imparts a small measure of conductivity, which is useful in specimens with
extremely detailed surfaces or with deep recesses.
•Also serves as a mordant for some of the chemical coating deposition
techniques which will be mentioned later.
Pre-embedding "Stains"
Also known as "en bloc" staining.
All will enhance electron contrast of specific components.
Staining can occur during fixation and/or dehydration or after
embedding.
Generally staining after embedding is better because:
Sometimes pre-embedded stain can make tissue hard and difficult
to section
The reagent may affect the tissue and introduce artifacts
The reagent may interfere with subsequent cytochemical rxns.
Uranyl Acetate
Reacts strongly with cacodylate buffer and phosphate buffers
- should be rinsed extensively prior to exposure.
Incubate either before or after osmium fix.
- 2% aqueous
- 1% methanolic UA
Tannic Acid
Improves fixation with aldehydes of proteins and reacts with OsO4
Mix the glutaraldehyde with TA
Must be prepared fresh and filtered before use.
Alcian Blue
Sufficiently electron dense
Is used during aldehyde fixation at physiological pH – causes
reactive areas to become osmiophilic
Binds extracellular glycoproteins, mucins.