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.
Problem
Considerations for TEM- High vacuum
Support of sample
Intense heat from beam
Depth of electron penetration
TEM not widely used by biologists until 1950’s
Considerations for SEM- Size of specimen Vacuum
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
Total ionic strength of reagents Osmolarity
Temperature
Length of fixation
Method of application of fixative
• Def. - a solution containing a weak acid and its salt.
• Serve to hold pH steady during the fixation process.
Buffers
• 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.
• 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.
CONSIDERATIONS IN THE SELECTION OF A BUFFER
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.
• 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
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 2.Hypertonicity 3.Hypotonicity
• SEM specimens should be fixed under near isotonic conditions
• TEM specimens should be fixed under slightly hypertonic conditions.
5
3
8
5 mOsm
Common Buffers used in Fixation Sodium Cacodylate
Effective range is 6.4 - 7.4.
Lacks phosphates which may 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 OsO
4May 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
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 in toto (ie 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.
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.
• Mode of action - Rxn w/ proteins is not fully understood.
• Known to involve lysine side chains of proteins and pyridine derivatives are major rxn products
• 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 between 3 and 6, 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
Glutaraldehyde, cont.
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 (OsO
4)
• 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
• 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.
• 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 and respiratory tract.
• 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.
