Cryo-electron tomography on vitrified sections: a critical analysis of benefits and limitations for structural cell biology.
The technology to produce cryo-electron tomography on vitrified sections is now a few years old and some specialised labs worldwide have gathered sufficient experience so that it is justified at this point to critically analyse its usefulness for cellular and molecular biology, and make predictions on how the method might develop from here. Remarkably, the production of vitrified sections has been introduced some 40 years ago (the very origin dates back to Christensen, 1971, and McDowall et al., 1983). However, the real breakthrough came between 2002 and 2004 when the groups of Jacques Dubochet and Carmen Manella independently resurrected the vitrified sectioning technology from its sleeping beauty state. And despite its hooks and hurdles a beauty indeed it is! When aiming at the right subjects the results obtained by vitrified sectioning and soon after by cryo-electron tomography exceeded all expectations. Molecular details of intracellular structures were imaged with never before seen clarity in a comparable setting, and the structural preservation of macromolecular assemblies within cells was stunning. However, as with every progress, the great results we now have with vitrified sectioning come at a price. The sectioning procedure and handling of vitrified sections is tricky and requires substantial training and experience. Once frozen, the specimens cannot be manipulated anymore (e.g., by staining or immuno-labelling). The contrast, as with all true cryo-EM approaches, is produced solely by small density differences between cytosol and macromolecular assemblies, membranes, or nucleic acid structures (e.g., ribosomes, nucleosomes, inner nuclear structures, etc.). Vitrified sectioning should not be seen as a competition to the more established plastic-section tomography, but constitutes an excellent complement, filling in high-resolution detail in the overview of cellular architecture. Here we critically compare the benefits and limitations of vitrified sectioning for its application to modern structural cell biology.
Bouchet-Marquis
Cédric
C
The Boulder Laboratory for 3-D Microscopy of Cells, Univ. of Colorado at Boulder, MCD-Biology, Boulder, CO 80309-0347, USA. bouchetm@colorado.edu
Hoenger
Andreas
A
D000818:Animals; D016477:Artifacts; D003585:Cell Biology / Q000295:instrumentation*; D020285:Cryoelectron Microscopy; D055032:Electron Microscope Tomography; D005629:Frozen Sections; D007091:Image Processing, Computer-Assisted