Comparison of the effects of critical point-drying and freeze-drying on cytoskeletons and microtubules.

We have compared the effects of critical point-drying (CPD) and freeze-drying (FD) on the morphology of Triton-resistant cytoskeletons and microtubules by scanning (SEM) and transmission electron microscopy (TEM). In general, cytoskeletons attached to Formvar films suffer less structural damage than cells or cytoskeletons attached to glass, because the Formvar film absorbs some of the stress associated with shrinkage during drying. However, as seen in stereo-pair electron micrographs, the three-dimensional structure of cytoskeletons prepared by FD is better preserved and shows fewer artefacts than those prepared by CPD. CPD specimens are flatter, often have a concave and apparently collapsed nuclear matrix and show large cracks both in the perinuclear zone and through the cytoskeleton. At least some of the damage appears to be due to residual water in the CO2 used as the substitution fluid, because cytoskeletons dried with a water filter attached to the CPD apparatus show substantially less damage than those dried without the filter. Freeze-dried cytoskeletons consist mostly of unbroken, smooth filaments and have no perinuclear open space. Comparison of the effects of drying on the diameters of in vitro polymerized microtubules showed that the diameter of microtubules is reduced after drying, but that FD causes significantly less shrinkage than CPD. Addition of 0.2% tannic acid to the glutaraldehyde fixative significantly reduces the shrinkage of CPD microtubules, but has no effect on FD microtubules. The observations on microtubules support the hypothesis that drying-induced shrinkage is the result of both pressure and solvent evaporation and they indicate that tannic acid stabilizes samples against the former but not the latter.