How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

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Model of a Propagating Action Potential (AP) in a Twitch Skeletal Muscle Fiber Mounted in a Double-Vaseline-Gap Chamber - Currents Involved in Shaping the AP

Biophysical Journal ◽

10.1016/j.bpj.2014.11.1481 ◽

2015 ◽

Vol 108 (2) ◽

pp. 269a

Author(s):

Fatou Touré ◽

Gabor Gyurkovics ◽

Cedric R.H. Lamboley ◽

Paul C. Pape

Keyword(s):

Skeletal Muscle ◽

Action Potential ◽

Muscle Fiber ◽

Skeletal Muscle Fiber

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Side bridge geometry after quick-freezing of stimulated and unstimulated frog skeletal muscle fibers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076056 ◽

1983 ◽

Vol 41 ◽

pp. 464-465

Author(s):

J.R. Sommer ◽

R. Nassar ◽

S. Walker

Keyword(s):

Skeletal Muscle ◽

Time Course ◽

Muscle Fibers ◽

Freeze Fracture ◽

Freeze Substitution ◽

Frog Skeletal Muscle ◽

Specimen Holder ◽

Skeletal Muscle Fibers ◽

Quick Freezing ◽

The Impact

Quick-freezing allows the structural analysis of timed perturbations of morphology. We are presenting preliminary results concerning the feasibility of studying directly the side bridge geometry of actin-myosin interactions within the time course of a twitch in single intact frog skeletal muscle fibers, both by freeze-substitution and freeze-fracture after quick-freezing, and following various time intervals between stimulation and impact of the fibers on a liquid He-cooled copper block.Materials and Methods. The quick-freezing device was a "Slammer"(Polaron) for which the electronics had been redesigned; they are capable, in combination with a Grass S48 stimulator, of any stimulation interval between 0 and 1 sec prior to freezing, including tetanus. The actual elapsed time between stimulation and freezing is recorded with a digital clock. Single intact tendonto- tendon frog skeletal muscle fibers (semitendinosus of r. temporaria) or toe muscle bundles (r.pipiens) were isolated by sharp dissection and placed between coextensive Pt stimulation wires on blackened 2% agarose, the height of which on the specimen holder was adjusted appropriately with respect to a spacer ring both, to calibrate the impact time and to prevent smashing of the fibers.

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A Langendorf preparation for quick-freezing small hearts

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124276 ◽

1992 ◽

Vol 50 (1) ◽

pp. 774-775

Author(s):

Joachim Sommer ◽

Teresa High ◽

Peter Ingram ◽

Rashid Nassar ◽

Neal Shepherd

Keyword(s):

Membrane Damage ◽

Freeze Fracture ◽

Freeze Substitution ◽

Tissue Preparation ◽

Cell Structures ◽

Freeze Dried ◽

Quick Freezing ◽

Cardiac Ultrastructure

The validity of studies of cell structures at high spatial and temporal resolution depends on the fidelity with which tissue preparation maintains in vivo conditions. Optimal preservation of structural substrates of precisely timed physiological intracellular events is offered by cryopreservation followed by freeze-fracture and freeze-substitution; we have established criteria for gauging that quality of cryopreservation in skeletal and cardiac muscle. Ciyopreservation is indispensable for electron probe x-ray microanalysis (EPXMA) of freeze-dried cryosections. We have developed a Langendorf preparation for small hearts (Figs. 1,2) suitable for use in our quick-freeze device (“Cryopress”; Med-Vac, Inc., St. Louis, MO 63117) to a) investigate the spatial distribution of physiologically important elements (e.g. calcium) during excitation-contraction coupling (ECC), especially in intact avian hearts and, b) assess damage to cardiac ultrastructure that is caused by pathological conditions (e.g. ischemia), rather than by artifacts due to chemical fixation (e.g. membrane damage by glutaraldehyde). In our Langendorf preparation, the tips of hearts can be quick-frozen at optimal freezing conditions, and comparative studies of the hearts of different animal species performed.

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