Sub-second cellular dynamics: time-resolved electron microscopy and functional correlation.
Subcellular processes, from molecular events to organellar responses and cell movement, cover a broad scale in time and space. Clearly the extremes, such as ion channel activation are accessible only by electrophysiology, whereas numerous routine methods exist for relatively slow processes. However, many other processes, from a millisecond time scale on, can be "caught" only by methods providing appropriate time resolution. Fast freezing (cryofixation) is the method of choice in that case. In combination with follow-up methodologies appropriate for electron microscopic (EM) analysis, with all its variations, such technologies can also provide high spatial resolution. Such analyses may include, for example, freeze-fracturing for analyzing restructuring of membrane components, scanning EM and other standard EM techniques, as well as analytical EM analyses. The latter encompass energy-dispersive x-ray microanalysis and electron spectroscopic imaging, all applicable, for instance, to the second messenger, calcium. Most importantly, when conducted in parallel, such analyses can provide a structural background to the functional analyses, such as cyclic nucleotide formation or protein de- or rephosphorylation during cell stimulation. In sum, we discuss many examples of how it is practically possible to achieve strict function-structure correlations in the sub-second time range. We complement this review by discussing alternative methods currently available to analyze fast cellular phenomena occurring in the sub-second time range.
Plattner
Helmut
H
Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Hentschel
Joachim
J
D000818:Animals; D020013:Calcium Signaling; D002468:Cell Physiological Phenomena; D002477:Cells / Q000648:ultrastructure*; D002923:Cilia / Q000502:physiology; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology; D008854:Microscopy, Electron / Q000379:methods; D015398:Signal Transduction; D013997:Time Factors; D014021:Tissue Preservation / Q000379:methods