Protocol for freeze drying & replicating Tokuyasu FTS's
The tiny coverslips you will receive from us were cut down to their 3mm x 3mm size and their "Nevada" shape by scoring #1 glass coverslips with a diamond pencil and then breaking them up. (The tiny size of these coverslips was to facilitate their later quick-freezing.) After breaking them, they are cleaned with chromic acid and rinsed umpty-ump times in Milli-Q water to get rid of any trace of chromate. Then they are air-dried, mounted in a vacuum-evaporator and coated with 100nm of carbon by standard techniques. (The reason for carbon coating the glass like this, which is the critical "trick" that makes this whole procedure work, is that it prevents the immunolabelled sections from coming into contact with hydrofluoric acid later, when the replica is separated from the glass. Otherwise, the HF will tend to dissolve away the gold immunolabel.) Finally, just before shipment to you, the cleaned and dried fragments of coverslip will be "glow-discharged" to make them hydrophilic. (Ideally, this should be done just a few hours before they are used to receive the Tokuyasu-type frozen thin sections, since the charge on them dissipates pretty fast.)

Upon receipt of your sections back from you (still in the glutaraldehyde postfix that you will apply after all your immunodecoration is finished), they will be carefully removed from their Eppindorf shipping tubes, by holding them carefully on the very corner with curved Dumont #7 forceps. Next, they will be washed extensively in distilled water over ~1 hour to remove all the glutaraldehyde, and to remove any salts or sugars that the glutaraldehyde may have been dissolved in. (The reason for this water-wash, which is admittedly the most problematic step in our whole procedure --simply because it exposes the labeled sections to such an unnatural environment -- is that ANY residual salt or sugar will leave an unacceptable “scum” on the coverslips after freeze drying.)

Next, without allowing any time for the coverslips to dry out, each will be mounted on a 3x3mm (and 0.8mm thick) slab of rabbit lung (itself aldehyde-fixed and stored in water for months on end). This thin slab of fixed lung will serve as a “cushion” for the next step of "impact-freezing" or "slamming," which involves an abrupt gravity-drop onto an ultrapure copper block cooled to 4 degrees above absolute zero via a spray of liquid helium. Thereafter, the frozen coverslips will be stored in liquid nitrogen until they are ready for mounting in a Balzers’ model 301 or model 400 vacuum evaporator. In one of these devices, each one will next be freeze-dried by warming it to minus 80 degrees Celsius for 15 min, and then rotary-replicated with a thin (~2nm) film of platinum evaporated over 5-10 sec from an electron beam gun mounted 15-20 degrees above the horizontal, all while the coverslip is rotated at 5Hz. This Pt ‘replica’ of the coverslip with the cryosections attached to it will then be immediately supported or “backed” by evaporating ~10nm of pure carbon onto it, using a standard carbon-arc supply mounted some 10 degrees off the vertical. (This takes approx. 4 sec. and is done while the coverslip continues to rotate, to insure the generation of a strong, uniform film of carbon.)

Next, the coverslip will be removed from the Balzers device, allowed to thaw, and the replica is floated off of it by immersing it at a ~45 degree angle into full strength (47%) hydrofluoric acid (HF). Immediately thereafter, the replica will be picked up off the surface of the HF with a glass rod and transferred via the same rod through several washes of distilled water, before being picked up on a 75 mesh formvar-coated EM grid. For electron microscopy, the grid is mounted in a eucentric side-entry goniometer stage of a JEOL 200CX electron microscope, imaged at 30-70K magnification, and photographed in stereo at +/- 10 degrees of tilt off the vertical axis.

For the production of final “anaglyph” stereo images, the two stereo micrographs representing each field are placed in proper register on a Bessler copy-stand and photographed at an additional 3-6x magnification with a Kodak EOS1Ds Mark II digital camera, producing a 4000x6000 pixel, ~18 MB B&W .jpg file for each view. The files are next sorted into left and right views by direct inspection on the computer screen, and then using Adobe Photoshop, the right view is converted to a pure red-channel RGB image and the left view to a blue+green channel RGB image. Next, either one of these colored images is copied directly onto the other, and the two are imaged simultaneously by selecting the “screen” command in the “Layers” menu of Photoshop. This creates an anaglyph stereo image of the original field, in a roughly 30MB .psd file. The anaglyph is finally brought into perfect alignment by using the “free translate” command in Photoshop on one of the two layers. (This operation in Photoshop even allows for correction of slight mismatches in magnification between the two original electron micrographs.) The final digital anaglyph stereo image is then transferred to a standard dye-sublimation printer for publication.

Refs for the original use of the technique of quick-freezing, freeze-drying, and platinum rotary replication with platinum:

Heuser, J.E. 1980. Three-dimensional visualization of coated vesicle formation in fibroblasts. J. Cell Biol. 84: 560-583.

Heuser, J.E. 1989. Effects of cytoplasmic acidification on clathrin lattice morphology. J. Cell Biol. 108: 401-411.

Ref for the original use of the technique of quick-freezing:

Heuser, J.E., T.S. Reese, L.Y. Jan, Y.N. Jan, M.J. Dennis and L. Evans. 1979. Synaptic vesicle exocytosis captured by quick-freezing and correlated with quantal transmitter release. J. Cell Biol. 81: 275-300.

Refs for the original use of the technique of "deep-etching" or freeze-drying:

Heuser, J.E. and S.R. Salpeter. 1979. Organization of acetylcholine receptors in quick-frozen, deep-etched and rotary-replicated Torpedo postsynaptic membrane. J. Cell Biol. 82: 150-175.

Heuser, J. 1981. Preparing biological samples for stereomicroscopy by the quick-freeze, deep-etch, rotary-replication technique. Meth. Cell Biol. 22: 97-122.

Heuser, J.E. and M.W. Kirschner. 1980. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J. Cell Biol. 86: 212-234.

Refs for the original use of the technique of imaging molecules on mica:

Heuser, J.E. 1983. Procedure for freeze-drying molecules adsorbed to mica flakes. J. Mol. Biol. 169: 155-195.

Heuser, J.E. 1989. Protocol for 3-D visualization of molecules on mica via the quick-freeze, deep-etch technique. J. Elect. Micro. Tech. 13: 244-263.