Quantitative mapping of skeletal muscle fibers by x-ray microanalysis: Effect of long-time storage of freeze-dried cryosections

Abstract

Electron probe X-ray microanalysis (EPXMA) is uniquely capable of performing quantitative topochemical analyses with high spatial resolution of elemental concentrations in cryosections from quickfrozen biological material. EPXMA requires very expensive instrumentation and maintenance, and data acquisition is very time consuming. For economic reasons, it is most desirable that any EPXMA facility serve several users who, while waiting for their turn, may need to store cryosections for a time. Storage of cryosections is associated with loss of structure and, under certain circumstances, also with changes in elemental distribution after short periods of time. We have re-examined several cryosections by EPXMA mapping that had been stored under vacuum for very long periods of time.Materials and Methods:Single, intact semitendinosus muscle fibers from R. temporaria were quick-frozen as previously described. The cryosections were freeze-dried in a Balzers FDU 101 and carbon-coated before external transfer to plastic desiccators in which they were stored under vacuum. Quantitative EPXMA maps were obtained as previously described (Figs.1A-F).Results and Conclusions:In the maps [Ca] was very high in the JSRs and, occasionally, (Fig. 1D) seen in hot spots that may correspond to spotty accumulations of negatively charged electron-dense material often seen in the free SR away from JSR. Overall, only small variations in elemental distribution or concentration were observed after storage for over one year; the significance of these variations (especially with such elements as Na) is difficult to assess because of the small number (4) of muscle fibers examined. The lighter region in the center of Fig. 1B is a result of slight etching due to extended static probes that were taken from this area in addition to mapping. JSR [Ca] remained remarkably constant after one year of storage. Freeze-dried cryosections revealed some smearing of structural detail, ranging from minimal to moderate, especially in the myofibrillar compartment (cf. Figs 1A,B). Our findings show that, when handled properly, carbon-coated cryosections can be stored over extended periods of time with minimal loss of structure or changes in elemental distribution. Thus, quantitative EPXMA image maps after long storage present a rather accurate representation of the original. Proper storage of freeze-dried cryosections is a viable procedure for the effective participation of independent research groups in multiple-user EPXMA facilities, access to which may occasionally require prolonged storage periods.