Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 161, Issue 3, Pages 359-371Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2007.09.002
Keywords
ultrastructural preservation; macromolecular structure; electron tomography; neuroanatomy; cryofixation; Synapses; virus entry
Funding
- NCRR NIH HHS [P41 RR004050-178230, RR004050, P41 RR004050, P41 RR004050-187694, S10 RR016699-01] Funding Source: Medline
- NIDA NIH HHS [DA016602, R01 DA016602] Funding Source: Medline
- NIGMS NIH HHS [GM065937, R01 GM034220, R37 GM034220, GM072881, R01 GM072881, R01 GM065937, GM34220, R01 GM072881-03] Funding Source: Medline
- NINDS NIH HHS [R01 NS014718, NS14718] Funding Source: Medline
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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. (C) 2007 Elsevier Inc. All rights reserved.
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