The emergence of electron tomography as a tool for three dimensional structure determination
of cells and tissues has brought its own challenges for the preparation of thick sections.
High pressure freezing in combination with freeze substitution provides the best method
for obtaining the largest volume of well-preserved tissue. However, for deeply embedded,
heterogeneous, labile tissues needing careful dissection, such as brain, the damage
due to anoxia and excision before cryofixation is significant. We previously demonstrated
that chemical fixation prior to high pressure freezing preserves fragile tissues and
produces superior tomographic reconstructions compared to equivalent tissue preserved
by chemical fixation alone. Here, we provide further characterization of the technique,
comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were
(1) high pressure frozen without fixation, (2) high pressure frozen following fixation,
and (3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to
freezing produces ultrastructural preservation superior to that obtained through chemical
fixation alone that is close to that obtained when cells are fast frozen without fixation.
We demonstrate using a variety of nervous system tissues, including neurons that were
injected with a fluorescent dye and then photooxidized, that this technique provides
excellent preservation compared to chemical fixation alone and can be extended to
selectively stained material where cryofixation is impractical.