Cryofixation: A Tool In Biological Ultrastructural Research

Plattner, Helmut; Bachmann, L.

doi:10.1016/s0074-7696(08)61676-9

Cited by 193 publications

(121 citation statements)

References 300 publications

Supporting

Mentioning

120

Contrasting

Order By: Relevance

“…2-4 are micrographs from control muscles that illustrate the typical degree of preservation achieved in our experiments. The uppermost superficial layer of the muscle fibers (muscle--copper block interface), where the maximum heat transfer occurs, is well preserved (1,16,19,40). The depth of this layer, marked by the dotted line in Fig.…”

Section: Control Musclesmentioning

confidence: 98%

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Torri-Tarelli¹,

Grohovaz²,

Fesce³

et al. 1985

The Journal of Cell Biology

151

View full text Add to dashboard Cite

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca 2+, and 10 -4 M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of <1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15°K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5-10/~m, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was ~3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released.Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release. The vesicle hypothesis holds that each quantum is confined within one of the synaptic vesicles that populate the nerve terminal and is released by exocytosis when the membrane of that vesicle fuses with the axolemma at one of the many specialized regions called "active zones" (8,13,51).A number of workers have shown that at frog neuromuscular junctions vesicles fuse with the axolemma of stimulated 1386 terminals and some of the remaining vesicles become labeled with extracellular tracers (9)(10)(11)25). Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent...

show abstract

Section: Control Musclesmentioning

confidence: 98%

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Torri-Tarelli¹,

Grohovaz²,

Fesce³

et al. 1985

The Journal of Cell Biology

151

View full text Add to dashboard Cite

show abstract

“…To date however, ultrarapid freezing techniques (9,15,21,31,34), when applied to tissue samples, have been limited by the need to excise and mount specimens before freezing, thereby introducing the possibility of time-and damage-related alterations to membrane structure in the samples . To reduce these variables, we have developed two techniques for freezing tissue in a condition as close as possible to that existing in the living animal (37,38), and at set intervals after animal death .…”

mentioning

confidence: 99%

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

Green¹,

Severs²

1984

The Journal of Cell Biology

View full text Add to dashboard Cite

By using two ultrarapid freezing techniques, we have captured the structure of rat and rabbit cardiac gap junctions in a condition closer to that existing in vivo than to that previously achieved . Our results, which include those from fully functional hearts frozen in situ in the living animal, show that the junctions characteristically consist of multiple small hexagonal arrays of connexons . In tissue frozen 10 min after animal death, however, unordered arrays are common. Examination of junction structure at intervals up to 40 min after death reveals a variety of configurations including dispersed and close-packed unordered arrays, and hexagonal arrays . By use of an isolated intercalated disk preparation, we show that the configuration of cardiac gap junctions in vitro cannot be altered by factors normally considered to induce functional uncoupling .These experiments demonstrate that, contrary to the conclusions of some earlier studies

show abstract

“…Commercial instruments are available for rapid flow technologies, including stopped-flow and quenched flow (Dunford, 1983;Gore, 2000). Stopped-flow operates with short activa tion times, whereas quenched-flow allows stimulation of samples, also by rapid mixing with a stimulant, for different time intervals, followed by rapid quenching (Moffat and Henderson, 1995;Plattner and Bachmann, 1982). Stopped-flow techniques are used mainly to measure very rapid processes and, thus, require vigorous mixing with intense shearing forces.…”

Section: A Appreciation Of Methods Availablementioning

confidence: 99%

“…as documented in this review mainly by work from our lab. this technical principle has recently been adapted to rapid kinetic analysis of proteins (Cherepanov and De Vries, 2004 (Plattner and Bachmann, 1982;Sitte, 1996). EM analy sis can include freeze-fracturing.…”

Section: A Appreciation Of Methods Availablementioning

confidence: 99%

Sub-Second Cellular Dynamics: Time-Resolved Electron Microscopy and Functional Correlation

Plattner¹,

Hentschel²

2006

International Review of Cytology

Self Cite

View full text Add to dashboard Cite

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 analyzino 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.

show abstract

Cryofixation: A Tool In Biological Ultrastructural Research

Cited by 193 publications

References 300 publications

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Gap junction connexon configuration in rapidly frozen myocardium and isolated intercalated disks.

Sub-Second Cellular Dynamics: Time-Resolved Electron Microscopy and Functional Correlation

Contact Info

Product

Resources

About