The structure of the cytoplasmic matrix preserved by freeze-drying and freeze-substitution

This paper reports a study of the cytoplasmic matrix in whole cultured cells examined by high voltage electron microscopy. In order to acquaint ourselves with the influence of preparation procedures on the morphology depicted, PtK2 cells were prepared for examination by first freezing in propane at -185 degrees C and then drying while maintained at -95 degrees C. Other cells of the same type were first fixed with glutaraldehyde and OsO4 and then frozen dried. Others were prepared by freeze-substitution and eventually dried by the critical-point method from CO2. And finally, some were preserved by conventional techniques of glutaraldehyde and OsO4 followed by dehydration in alcohol and critical-point drying. The observations are presented in stereo images. The morphologies after these various procedures are quite similar. All show the characteristic three-dimensional lattice or meshwork of slender filaments called microtrabeculae. In cells rapidly frozen and then dried from the frozen state, there is less evidence of shrinkage and probable change in the trabecular structure than in cells first fixed with glutaraldehyde and then frozen dried. The differences we relate to the demonstrable failure of glutaraldehyde to penetrate the cell quickly and fix instantly any component that is in active motion. Other differences that can be observed between all four types of preparation are not recognizably striking and are thought to reflect as much as anything morphological diversity in the original cells. In some instances, the amorphous ice of the initial freezing at -185 degrees C was allowed to crystallize at -80 degrees C. The small crystals that form push aside the microtrabeculae and leave obvious imprints on the structure. The essential message from these experiments is that the cytomatrix is structured.