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 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

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:

5 mOsm

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

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 OsO 4 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. (OsO 4 is an exception)

•

Pieces should be as small as possible to allow rapid and complete penetration of the fixative, particularly when OsO 4 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

•

Serine

•

Glycine

•

Glycylglycine

•

Arginine

•

Reactions outside a neutral range (pH 7.0 – 9.0) are reversible.

•

Never use a buffer that contains primary amines

•

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 (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.

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.