Scanning Transmission Electron microscope Studies of Deep-Frozen Unfixed Muscle Correlated with Spatial Localization of Intracellular Elements by Fluorescent X-Ray Analysis

Abstract

Thin sections of deep-frozen unfixed muscle were studied in a scanning electron microscope modified for transmission imaging and equipped with a "cryostage" for vacuum compatibility of hydrated tissue.With an energy-dispersive x-ray analysis system, intracellular atomic species in the scan beam path were identified by their fluorescent x-rays and spatially localized in correlation with the electron optical image of the microstructure. Marked differences are noted between the ultrastructure of deep-frozen hydrated muscle and that of fixed dehydrated muscle. In frozen muscle, myofibrils appear to be composed of previously undescribed longitudinal structures between 400-1000 A wide ("macromyofilaments"). The usual myofilaments, mitochondria, and sarcoplasmic reticulum were not seen unless the tissue was "fixed" before examination. Fluorescent x-ray analysis of the spatial location of constituent elements clearly identified all elements heavier than Na. Intracellular Cl was relatively higher than expected.The present body of knowledge relating the microstructure and physiologic function of muscle is based primarily on (1) electron microscope studies of fixed tissue, dehydrated so as to be compatible with the vacuum required for electron optical imaging and (2) (Fig. 1).To obtain sufficient contrast in the electron optical images of a thin section of tissue, the normal reflected electron imaging operation of the SEM was converted to transmission imaging-causing it to function as a transmission scanning electron microscope. The contrast obtained in unstained unfixed tissue was enhanced by partial dark-field techniques that were developed for this purpose. A detailed description of the cryostage and transfer system and transmission imaging technique will be reported separately (16).X-Ray Analysis. In the SEM, a 20 keV electron beam focused to a small spot, about 75 A in diameter, is scanned 3423

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl−

“…During deoxygenation, [Cl − ] i increases through exchange with HCO 3 − (chloride shift or Hamburger shift), which may amount to 10% of the total Cl − concentration of erythrocytes [22]. However, intracellular Cl − and other ions are compartmentalized [29,30]. Therefore, the Cl − concentration at the Cl − binding sites of the Na + /Mg 2+ antiporter and the alterations of [Cl − ] i at these sites during deoxygenation are not exactly known.…”

The preparation, examination and analysis of frozen hydrated tissue sections by scanning transmission electron microscopy and X‐ray microanalysis

“…Thinner sections or higher accelerating voltages may be needed. Thinner sections present a separate set of problems since sectioning is more difficult, the elemental quantities are smaller (Bacaner et al, 1973), and since the volume of water is smaller in thinner sections, a very small amount of sublimation or contamination might greatly alter the elemental mass fractions. However, the use of higher accelerating voltages results in a reduction in intensity of low voltage continuum radiation which in turn improves the peak to background ratios for lighter elements.…”

Electron‐probe X‐ray Microanalysis: Techniques and Recent Applications in Biology

“…Perhaps the most serious attempt to analyse diffusible ions in skeletal muscle was that of Bacaner, Broadhurst, Hutchinson & Lilley (1973) using rabbit psoas muscle frozen by contact with a liquid-nitrogen cooled copper surface, sectioned (ultrathin) at -85 O C and examined without freeze-drying. In the scanning transmission mode of imaging, myofibrils and ' macromyofilaments ' varying from 40-60 nm thick in the I-bands to IOO nm in the A-zones could be resolved, as well as dense areas corresponding to the Z-bands.…”