Soft Tissue Preparation

In order to examine soft tissue samples in “as near to life-like condition as possible”, chemical fixation is required. Without chemical fixation, post-mortem autolysis and decomposition would distort even the surface of the specimen of interest. Even with the surface “stabilized” through conductive coating, internal degradation would lead to glaring artifacts of surface morphology. In addition, chemical fixation will permit observation of internal structures using cryo (cold) techniques and allow for the correlative preparation of SEM samples for the TEM via epoxy resin embedment and ultra-thin sectioning. The preparation of soft tissue samples for examination in the SEM begins in an identical manner to the preparation of samples for the TEM (see Unit 1 - Preparation of Biological Samples for TEM). A generalized protocol follows:

•Primary Aldehyde Fixation •Buffer Wash

•Secondary Osmium Tetroxide Postfixation •Optional Buffer/DH2O Wash

•Dehydration Series (Ethanol/Acetone) •Intermediate Fluid Series (Freon TF/113)

•Critical Point Drying (in Transitional Fluid - liquid CO2 or Freon 13) •Mounting (adhesive on 15mm dia. aluminum stub)

•Conductive Coating (Sputter Coater or Vacuum Evaporator)

Firstly, the tissues of interest must be excised and placed into the primary fixative. As for TEM preparation, we typically use a combination method of in situ and immersion

fixation. The tissue blocks for SEM preparation can be somewhat larger since the

aldehydes (primary fixative) can penetrate through at least 3mm of sample. Tissue blocks

are therefore best minced to the dimensions of 2-3mm3_{. If strips of tissue are cut, they}

should be no thicker than approximately 2-3mm on one of the sides.

Minced tissue blocks can be transferred to vials containing the primary aldehyde

fixative and remain for one hour. Once again the best single aldehyde choice is the doubly

reactive, cross-linking, glutaraldehyde. As for TEM sample primary fixation, we use a 3% concentration of glutaraldehyde carried in the proper isotonic buffer vehicle (for most soft mammalian tissues, 0.2M phosphate buffer, pH 7.2-7.4 is ideal). The chemical action of aldehydes (stabilization of the cellular protein matrix) and other fixatives along with the purpose of buffer solutions and their preparation are covered in the chapters on TEM specimen preparation.

Once the primary fixation is complete, three buffer washes of ten minutes duration each are conducted to remove unbound aldehyde and prevent the undesirable precipitate

Following the buffer washes, the samples are placed into the secondary fixative, osmium

tetroxide. The OsO₄ will react mainly with unsaturated lipids and impart an increase in

conductivity to the biological sample. The OsO₄ is also carried in the same buffer vehicle

as for the glutaraldehyde. You will recall that the benefit to osmium tetroxide fixation for TEM was the introduction of an electron dense stain (for phospholipid membranes and other osmophilic structures) for added contrast. In SEM, the generation of a signal from the surface is of paramount concern. Incorporation of the heavy metal osmium to the sample allows for increased surface signal emission when contacted by the primary

electron beam of the SEM. Later in this unit, I will discuss how osmium incorporation can be enhanced by the addition of agents such as tannic acid and thiocarbohydrazide (TCH), so much so, that conductive surface coating may be avoided.

An optional buffer or distilled water wash of two changes for ten minutes duration each may be performed, although, I have not observed any artifacts arise due to the omission of this step.

Next, the tissue must undergo complete dehydration, but not for the same reason as TEM sample dehydration. Residual water in the tissue would cause surface tension collapse as it dried. For this reason, the common process of critical point drying - CPD (or some alternative) is employed in the preparation of samples for the SEM. The CPD process, which is carried out under liquid carbon dioxide, would be ineffective if water remained in the tissue block. A complete description of CPD theory and procedure is found later in this unit. By contrast, you will recall that samples are dehydrated for TEM in order that the epoxy resins infiltrate the tissue blocks (epoxies are not miscible with water). Both acetone and ethanol (EtOH) are common dehydrating agents used. Since shrinkage of tissue blocks and the resultant surface distortion is undesirable for SEM specimens, the dehydration schedule is more gradual for SEM, starting at 30% EtOH. The usual ascending series is 30%, 50%, 70%, 95% EtOH for ten minutes each, followed by 100% EtOH for two changes of ten minutes each with the vials being filled to capacity as usual. The preparation of such a dehydration series using 95% (not 100%) ethanol has been described in the unit on TEM specimen preparation.

The final steps in the soft tissue protocol will involve the preparation of tissue blocks for

critical point drying which has traditionally been performed in either liquid carbon

dioxide or liquid freon (Freon 13). Usually, before the critical point drying process, tissue blocks are passed through an ascending intermediate fluid series of Freon TF/113 since it is miscible with both ethanol and the transitional fluid used in critical point drying. The freon is diluted with 100% ethanol, not water, in order to produce the concentration series. The usual ascending series is 30%, 50%, 70%, 95% Freon TF/113 for ten minutes each, followed by 100% Freon TF/113 for two changes of ten minutes each.

As the scientific community has become more aware of the environmental impact of freons on the degradation of the protective ozone layer (which partially shields the earth from harmful UV radiation), alternatives to its use have been discovered and will be considered following the discussion of critical point drying. A common past alternative to Freon TF

was amyl acetate with its characteristic strong, banana-like odor. It was typically used

as an intermediate fluid between ethanol and liquid CO₂. Due to its toxicity, it should

always be purged out of a critical point dryer into a fume hood. A benefit to its use was that one always knew it had been completely purged from the critical point dryer be the marked absence of its trademark odor.

The process of chemical fixation of biological soft tissues have been carried out in a liquid environment. If in the final step, these tissues are simply allowed to air dry, tremendous surface tension distortion will occur leading to obvious surface artifacts. It should be noted that water can exert a surface tension force of over 2,000 psi. In order to prevent this surface tension damage, the technique of critical point drying (CPD) has been utilized since 1968.

The principle of CPD involves an understanding of phase (solid-liquid-gas) boundaries, especially, for our purposes, the boundary between liquid and vapor. Every fluid possesses

what is known as a critical density (D_c) at which the boundary or interface (the actual

liquid surface) between the liquid phase and the vapor phase becomes indistinguishable. At first, when a fluid is introduced to a sealed container, an equilibrium exists between the liquid and vapor phases. In order to attain critical density, this equilibrium must be shifted to the vapor phase through heating and the related increase in pressure. Critical

density (D_c) is attained at the critical temperature (T_c) and critical pressure (P_c) of the

given fluid. Therefore, to eliminate the interfacial boundary between liquid and vapor and avoid the associated surface tension force, the fluid must be elevated to its critical

temperature and pressure.

Since the critical temperature and pressure of water is excessive, at 374˚C and 3,184 psi respectively, and would damage delicate biological soft tissues, other liquids typically serve as transitional fluids (so named because they make the transition between liquid

and vapor phases). The most common of these is liquid carbon dioxide (LCO₂) and liquid

freon 13. Due to its higher price and negative impact on the environment, liquid freon has

become the less popular of the two transitional fluids. Liquid CO₂ has a critical

temperature and pressure of 31˚C and 1,073 psi, respectively, compared with liquid freon

13 at 28.9˚C and 561 psi. The liquid CO₂ is introduced to the critical point dryer at the

high pressure (600 -800 psi) of its storage tank. The storage tank is specially ordered with

a siphon tube which takes the liquid CO₂ from the bottom of the tank since as the tank is

emptied, CO₂ gas rises and collects at the top of the tank.

CAUTION: Liquid carbon dioxide is under high pressure and is very cold. Use care in handling the tanks and opening the tank valve. The use of a regulator is not required, however, the tank pressure is between 600-800 psi. Make sure the hose between the CPD and the tank is threaded and tightened properly, and is a hose rated for high pressure applications (a minimum burst

pressure rating should be printed on the hose - the hose used in this lab is rated at 17,000 psi). In addition, the storage tank

should be chained or strapped to a wall or a tabletop to prevent it from accidentally falling and rupturing. Under its high pressure, the tank could act as a missile causing serious injury. Finally, CO2 is a colorless, odorless gas and should be vented from the CPD into a fume hood to prevent asphyxiation as it displaces the normal atmosphere.

✥ Critical Point Dryer Operation

The CPD is a thick walled metal chamber which can withstand pressure in excess of 2,000 psi. The chamber is constructed of a metal of excellent heat conductive properties such as brass, copper or bronze. The chamber is usually machined to accommodate some type(s) of specimen holder(s) which will retain the tissue blocks throughout the procedure. The chamber also includes three high pressure needle valves, the inlet valve (for the

introduction of the LCO₂), the drain valve (to drain off the intermediate fluid - freon TF/

113) and the vent valve (to vent the CO₂ gas). Additionally, the CPD chamber must have

some provision for heating the transitional fluid. In the more expensive models, there is an electrical heater. In the least expensive models, the entire CPD chamber is simply lowered into a container or bucket of hot water. The main drawback to this method is that residual external water on the unit might contact the tissues as they are removed, rehydrating them. Our Pelco Jumbo Dryer (same as the Polaron Jumbo) is designed with a water jacket just external to the drying chamber. Hot and/or cold water can be routed through the jacket by means of plastic tubing attached to a faucet. A temperature gauge is added in order to monitor the water temperature, although, the only required gauge is a chamber pressure gauge. The Pelco CPD is equipped with a safety valve which is designed to

rupture at 2,000 psi.

CAUTION: The CPD is a high pressure device which if used improperly can lead to serious injury or death. Follow all manufacturer directions carefully. If equipped with a thick quartz view window (as in the case of the Pelco Jumbo Model), it MUST BE EXAMINED FOR CRACKS BEFORE EACH USE!!! NEVER

REMOVE the protective Lexan shield, if so equipped, just outside the view glass - it is there for your safety!! NEVER LOOK DIRECTLY INTO THE VIEW GLASS (use a mirror if you are curious) - if it were to break, the high pressure would

produce many high velocity, very sharp projectiles! Ideally, the CPD should be located in a fume hood. For obvious reasons stated above, the CPD is alternatively known as “the BOMB”.

In the routine operation of the Pelco CPD, the unit is placed into the fume hood and the

high pressure hose is connected tightly to the liquid CO₂ tank. The inlet water hose is

attached to the faucet and the outlet water hose is run into the hood sink. The drain hose (optional) is also run into the hood sink. The heavy rear door is removed using the steel rod tool. If this is the first CPD run of the day and the bomb is at room temperature, you

will not have to cool the unit by running cold water through the water jacket. If the bomb

has been heated, it will have to be cooled to approximately 20˚C before use. It is often a good idea to perform a “dry run” of the unit to check it for any leaks (valves, window and rear door dowdy seals may require tightening) - it is better to test the unit than to sacrifice good tissue blocks which will ultimately dry down with surface tension collapse in a bomb which cannot maintain pressure! All three needle valves should be closed clockwise. The valve on

the liquid CO₂ tank can now be opened.

Tissue blocks in their second change of 100% freon TF/113 can now be loaded into the CPD boat. Most CPD units come with a device to contain any number of specimen holders. The Pelco unit comes with a three reservoir/channel boat into which some nine specimen baskets, which resemble sewing thimbles, can be loaded. The boat has a large rear

stainless steel pin which interfaces with a hole in the rear door of the CPD. At the front of the boat is a spring loaded valve which when inserted into the CPD, opens to allow the intermediate fluid to drain out. Firstly, the CPD boat is filled with freon TF/113. At this point, the tissues are transferred from their vials to the tissue baskets. Sine most tissues will float in 100% freon TF/113, they are easily transferred by pouring the vials contents into the baskets over a sink.

CAUTION & REMINDER: All potentially toxic chemicals used in the

fixation process should be handled using disposable gloves and wearing goggles, ideally within a fume hood!

Once transferred to the baskets, the baskets are quickly placed into one of the three CPD boat channels which are full of the intermediate fluid, freon TF/113, to prevent premature drying down through a liquid interface. Sheets are available (fig. 9) to identify the specific tissue which occupies a specific basket in the boat. Once a channel contains three baskets, a wire mesh cover is placed on top of the baskets to prevent tissue loss in the bomb. The CPD boat can hold nine baskets at a maximum.

Once the boat is loaded with tissue baskets, the boat is carefully carried over to the bomb and gently inserted. A slot on the rear underside of the boat must align with a

corresponding slug in the rear floor of the CPD chamber. The central hole of the rear door is lined up with the stainless steel pin of the boat and the rear door is threaded clockwise until tight. Use the steel rod tool to tighten the rear door and prevent leaks. Your tissues are now ready to undergo critical point drying.

With the tissues properly loaded into the CPD and with all valves closed, the three

valves will be used (or as I like to call it, juggled) in order to introduce the liquid CO₂ and

flush out the freon TF/113 (fig. 10). Initially, the upper inlet and vent valves are opened. Do not be alarmed at the amount of noise coming from these high pressure needle valves. The vent valve must be opened to allow for the escape of gas and permit the required volume of transitional fluid to enter the bomb. You should be able to observe the liquid

CO₂ enter the bomb and rise to a desired level above the boat. As the liquid CO₂ is

under pressure.

Fig. 9

At first, a clear fluid will be observed flowing down and out of the drain line, followed by a

solid CO₂ “snow”. Allow the solid CO₂ to exit the drain line for a few seconds, ensuring

complete flushing out of the freon TF/113, and then close the drain valve. Using the inlet

and vent valves, allow the liquid CO₂ to reach a volume just above the top of the boat, then

close both valves. At this point, you should not hear any “hissing” which would be typical of a seal or valve leak. The pressure gauge should be holding steady at 600-800 psi (The

Fig. 10

Now, hot water can be flowed into the water jacket. The flow rate should be moderate (as viewed directly at the outlet hose) and the water temperature should be approximately 40- 45˚C. As time passes and the heat is conducted from the water jacket to the CPD chamber, the temperature gauge and pressure gauge will rise accordingly. At approximately, 29˚C

and 1,000psi, you will observe a turbulence at the liquid CO₂ interface, followed by the

PEDESTAL