Basic procedures of sample preparation for standard TEM
Method 1.

(for whole cells, cell fragments, bacteria, (nanobes!), and isolated organelles)

quick-freeze—freeze-fracture—deep-etch—platinum replicate.

We developed this basic preparative procedure for EM over twenty years ago. Its main advantage over other preparative techniques is that it eliminates the need for any sort of chemical fixation or dehydration of biological samples. This, plus the rapidity of freezing accomplished by our particular method of “quick-freezing” against a block of ultrapure copper cooled to liquid helium temperature (minus 269°C), has allowed us to capture fast biological processes such as exocytosis of neurotransmitters at living synapses, myosin crossbridge cycles in living muscles, and dynein crossbridge cycles in living cilia and flagella. Additionally, this technique provides images with any “depth” of exposure that is desired - - from zero etching (which yields pure freeze-fracture views of membranes) through “shallow etching” (which yields exposures comparable to the views of thin plastic sections), to “deep-etching” (which can give as much exposure a particular biological sample’s topology will permit).

Method 2.

(for isolated organelles, (bacteria & viruses), and macromolecules)

adsorb onto mica flakes—quick-freeze—freeze-fracture—deep-etch—platinum replicate.

This is the basic procedure we originally developed for viewing purified macromolecules in the EM, based on our finding that molecules wholly suspended in ice did not yield readily interpretable images when exposed by the “deep-etching” method, above. What was lacking was some sort of surface-adsorption to provide a stable substrate during the etching process. Freshly cleaved mica was found to be the optimal substrate, in terms of its physical and chemical properties. Mica “pulverized” into tiny flakes was found to be the optimal form for achieving good freezing and controllable fracturing over the mica surface. (Note that bacteria, viruses and cell organelles can be prepared by either “Methods 1 or 2”, e.g. with or without adsorption to mica flakes before quick freezing, depending on the amount of material available (“method 2” requiring much less material)

Method 3.

(again, for isolated organelles, (bacteria & viruses), and macromolecules)
(also for Tokuyasu-type frozen thin sections)

adsorb to polylysine-treated glass—glutaraldehyde fix—wash in H2O—quick-freeze—totally freeze-dry—platinum replicate.

(Note that in contrast to “Methods 1 & 2”, samples here are adsorbed to glass not to mica, are aldehyde-fixed not unfixed, and are wholly freeze-dried not just deep-etched.)

The major advantage of this method is that it eliminates freeze-fracturing and thereby yields solely membrane surfaces - and vastly broader expanses of them. Hence, it provides much more information about the surface coats on cell membranes & their associated cytoskeletons. The disadvantage of this method is that it involves aldehyde-fixation and washing in water before a sample can be totally freeze-dried. (Otherwise, an impenetrable “scum” of salts is left behind, which wholly obscures the surface of the glass.) A further disadvantage of this method is that freeze-dried surfaces lie deeper beneath the surface of the sample (hence are less well frozen), and during freeze-drying these surfaces must be exposed to the vacuum for longer periods before platinum replication. Thus, the resolution of molecular details with this method is significantly poorer than in “Methods 1&2” - roughly 4 nm vs. ~2 nm).

Method 3/immuno.

Despite the limitations mentioned for “Method 3” above, the single greatest advantage of this technique is that it is directly amenable to immunoEM by the standard techniques of gold-labeled secondary antibodies. Only slight modifications need to be introduced to add this step to “Method 3” in general. First, the glass will be coated with carbon so that later, when the replica is floated off of it, the gold will not be damaged by exposure to hydrofluoric acid (the glass-platinum separating agent). Second, formaldehyde fixation will be substituted for glutaraldehyde fixation for the usual reasons: to preserve antigenicity & reduce background. Finally, the samples will be labeled with primary and 10nm gold-tagged secondary antibodies by standard protocols, prior to the usual quick freezing and freeze drying of the glass.

It is important to stress here is that frozen thin sections, adsorbed to glass and thawed for indirect gold immunocytochemistry exactly as is done for thin sections on EM grids in the classical Tokuyasu or Slot and Geuze techniques, are perfectly suitable samples for “Method 3/immuno” as well. After labeling, the thin sections are simply refrozen by the quick-freeze method, freeze-dried and replicated with platinum. This specifically overcomes the chronic problem of the low intrinsic membrane contrast that plagues the frozen thin section technique and makes the interpretation of its images often so difficult.

Method 2/immuno.

It should be stressed that direct immunoEM can also be done—and at significantly higher resolution and higher sensitivity—in “Methods 1 and 2” above. The improvement in resolution and sensitivity derives from the fortunate circumstance that primary antibody molecules themselves, applied directly to the sample before freezing, can be clearly resolved by deep-etch EM. As a consequence, no gold labeled secondary antibodies are needed, so antigenic sites are more closely targeted and the signal is increased (e.g., the intrinsic loss in signal involved in getting a second labeled antibody to bind to the primary antibody is obviated). In essence then, this technique simply involves direct exposure of samples to primary antibodies immediately before adsorption to mica & quick freezing.

New methodologies recently introduced into this laboratory, which are adaptations of the five above procedures, are the following:

Method 1/thin sectioning +/- immuno.

After quick-freezing of whole cells, cell fragments (bacteria) or isolated organelles, they need not be cryofractured and replicated with platinum, but instead can be freeze- substituted at –80 C and imbedded in plastics for traditional thin sectioning. This is rapidly becoming a strong contender to classical Tokuyasu-type frozen thin sectioning for immunoEM, since it often preserves a respectable degree of antigenicity while invariably improving tissue morphology. When this approach is used for gold-secondary immunoEM, it can be termed “Method 1/thin sectioning/immuno”.

Method 2/molecular dynamics.

Samples adsorbed to mica flakes have recently been found to exist in a relatively metastable state, in that they remain capable of undergoing internal conformational changes or depolymerization, and in some instances are even capable of moving about on the surface of the mica and undergoing polymerization reactions. (It will be interesting to learn whether macromolecules are actually capable of shuttling about on the surface of the mica, or instead, are reversibly desorbing/readsorbing from a narrow layer of unstirred solvent immediately overlying the mica.) In any case, such changes can be provoked simply by washing mica flakes in suitable reactivation/polymerization solutions following the adsorption of macromolecules.

Method 3/freeze fracture.

Samples adsorbed to glass are traditionally freeze-dried completely, as described above. (This requires only 10 min of vacuum sublimation at –100C, since the samples lie in but a thin film of water). However, we have recently realized that it is not very difficult to freeze-fracture this thin film of water without dislodging the glass substrate from the cryostage. This has permitted us to back off from total freeze drying of such samples and, instead, simply deep-etch them for 1-2 min exactly as is done in “Methods 1 and 2”, above. This results in a significant improvement in membrane resolution (via the shorter exposure to the vacuum). However, more importantly, it allows us to eliminate the aldehyde fixation and water wash that was formerly required for freeze-drying of samples on glass. This has opened a whole new world of image-improvements for us, totally changing our view of several surface-structures whose conformation we find was only partially and imperfectly preserved by aldehyde fixation. This is an important new wrinkle in our methodology!