Rapid freezing, cryosectioning, and x‐ray microanalysis on cardiac muscle preparations in defined functional states

Abstract

Electrically stimulated heart muscle preparations can be quickly frozen in undercooled propane at defined times of the mechanically controlled contraction cycle. The apparatus for triggered freezing of the muscle strips in undercooled propane is described in detail. Freeze substitution of some strips after freezing shows the degree of ice crystal formation without the potential interference of artifacts introduced later by cryosectioning and freeze drying. Ultrathin longitudinal and transversal cryosedions are cut with a LKB cryoultramicrotome at temperatures of -130 to -140°C, freeze-dried at lop6 Torr vacuum and carbon-coated before analysis. The freeze-dried cryosections are analyzed in a Siemens Elmiskop 102 electron microscope equipped with a Kevex energy dispersive system, and the elemental concentrations (in mMoVkg d.w.1 of Na, Mg, P,S,Cl,K, and Ca are determined in subcellular compartments of muscle frozen in different functional states. The methodology of quantitation, i.e, determination of elemental net peak and continuum, correction of continuum, preparation of standards, and deconvolution of overlapping peaks are described. The minimum detectable elemental concentration using the reported methods is in the range of a few mMol/kg d.w. This also applies to Ca, which can be accumulated in heart muscle in readily detectable amounts in intracellularly located stores as well as structures connected with the cell membrane. The present report shows that cryotechniques and x-ray microanalysis can be successfully applied to heart physiology.

X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections

“…The concentration of elements in freezedried cryosections of standards was found to be lower in the case of large ice crystal damage (ice segregation zones with a hole diameter above 100 nm) than in sections with small ice crystal damage (segregation zones less than 50 nm) (Zierold, 1984a(Zierold, , 1986a. Similar observations were reported by Wendt-Gallitelli and Wolburg (1984). Hall (1986b) ascribes this effect to inappropriate correction for extraneous background.…”

“…It is not by chance that many X-ray microanalytical studies of tissues have concentrated on muscle. These cells were prepared as a few micrometer-thick single fibers or fiber bundles that can be excited and tested for viability immediately before cryofixation (Somlyo et al, 1977(Somlyo et al, , 1985Wendt-Gallitelli and Wolburg, 1984). Except for some specialized cases, for example silk moth antennae (Steinbrecht and Zierold, 19841, the proper sampling of complex tissues for cryofixation remains an important challenge for the future.…”

Presystolic calcium-loading of the sarcoplasmic reticulum influences time to peak force of contraction. X-ray microanalysis on rapidly frozen guinea-pig ventricular muscle preparations

“…During contraction these regions no longer revealed any Ca accumulation (14). This finding suggested that SR could function as the store providing Ca for the activation of "early" contraction, subsequent to this positive inotropic intervention.…”

Cryofixation during live‐imaging enables millisecond time‐correlated light and electron microscopy

“…Current approaches carefully time cryofixation to occur at a known time interval following user‐initiation of a certain dynamic process or perturbation. Such methods have allowed electron microscopy studies to provide snapshots of dynamic processes with a time resolution of ∼10 ms (Heuser et al ., ; Wendt‐Gallitelli & Wolburg, ; Knoll et al ., ; Ménétret et al ., ). However, these techniques do not allow direct correlation between live imaging and electron microscopy.…”