Electron-probe X ray microanalysis of in situ calcium and other ion movements in muscle and liver.
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Animals↗
Biomedical subjects
H Shuman
Publications and source records attributed to H Shuman.
Electron energy-loss spectroscopy: quantitation and imaging.
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Bacteriophages↗
Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells.
The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phase (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.
Calcium↗
Calcium and sodium distribution and movements in smooth muscle.
Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K, Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the major intracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca2+, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise in total cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca2+, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.
Animals↗
The influence of objective lens aberrations in energy-loss spectrometry.
The conditions under which the energy resolution and collection efficiency in electron energy-loss spectrometry are limited by the spherical and chromatic aberrations of the objective lens have been studied. It is shown that, for the optimum settings of the pre-spectrometer optics, the energy resolution will be of the same order in diffraction mode as it is in magnification mode. The influence of the aberrations on the practice of energy-loss spectroscopy is discussed, and it is demonstrated that the chromatic aberration can act as a broadband filter for energy-loss electrons.
Electron Probe Microanalysis↗
Calcium release by noradrenaline from central sarcoplasmic reticulum in rabbit main pulmonary artery smooth muscle.
The subcellular composition of relaxed and noradrenaline-contracted rabbit main pulmonary artery smooth muscle cells was measured by electron probe X-ray microanalysis of cryosections of rapidly frozen tissue. Some of the preparations were made permeable with saponin and exposed to a known free Ca ion concentration, rapidly frozen, freeze-substituted, and also analysed by electron probe X-ray microanalysis. 98% of intracellular K could be replaced by Rb. This was done to remove the K peak that partially overlaps the Ca peak in the X-ray spectra. The final [Rb]i plus residual [K]i was not significantly different from the [K]i of normal tissue. The [Ca]i in Rb-containing tissue was not significantly different from the [Ca]i in normal, K-containing tissue. Non-mitochondrial micro-regions containing high [Ca] (up to 33 mmol/kg dry wt.) were found at sites 200 nm or more away from the plasma membrane. These micro-regions also contained high [P]. We consider the identification of these regions containing high [Ca] as sarcoplasmic reticulum (s.r.), validated by: (a) conventional electron micrographs that show no other structures in main pulmonary artery smooth muscle in sufficient quantity and location to account for the frequency of these regions, (b) the previous localization of strontium, a functional calcium analogue, in the central s.r. in these smooth muscles (Somlyo & Somlyo, 1971 a), (c) the present demonstration that the central s.r. in this tissue can accumulate large amounts of calcium oxalate. The proportion of regions containing high [Ca] (greater than 12.0 mmol/kg dry wt.) was significantly higher in relaxed (35 of 330 measurements) than in the contracted (14 of 337) tissues (P less than 0.005), or 26 of 34 vs. 6 of 31 high [Ca] measurements in regions identified as s.r. through their high phosphorus content (P less than 0.006). This difference is thought to represent Ca release from the central s.r. There was no significant difference (P greater than 0.05) between the distributions of P in relaxed and contracted smooth muscle. The total cell [Ca]i in relaxed Rb-containing tissue, measured with randomly positioned small probes (3.6 mmol/kg dry wt.), was the same as that measured with large defocused probes, indicating the validity of random sampling. A mathematical model was used to estimate the frequency of including s.r. (35 nm diameter and 5% of cell volume) by a randomly positioned electron probe (50 nm), because we could not visualize s.r. in the cryosections.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Detection of X-rays and electron energy loss events in time coincidence.
Experiments are described in which characteristic X-rays are detected in coincidence with the electron energy losses that are responsible for these X-rays. The possibility to use this technique for improving the detection limits of microanalysis is evaluated. It is concluded that, because of the occurrence of false coincidences, better than the state-of-the-art instrumentation will be required for most practical applications.
Calcium↗
Total cytoplasmic calcium in relaxed and maximally contracted rabbit portal vein smooth muscle.
The concentration of total cytoplasmic Ca in vascular smooth muscle was measured by electron probe microanalysis of strips of rabbit portal anterior mesenteric vein that were rapidly frozen either when relaxed or during a maintained (30 min) maximal contraction stimulated with high K and noradrenaline. Strips were also frozen and analysed after incubation in Ca-free, high-Mg2+ solution. Probe diameters of 0.1-0.2 micron and 1.0-1.5 micron were used to measure, respectively, cytoplasmic and cellular (including stored) Ca. There was a highly significant increase (P less than 0.0005) in cytoplasmic Ca of 1.0 +/- 0.2 (S.D.) mmol Ca/kg dry wt. from 0.8 +/- 0.2 (S.E. of mean) mmol/kg dry wt. (n = 262 spectra, six animals) to 1.8 +/- 0.2 (S.E. of mean) mmol Ca/kg dry wt. (n = 296 spectra, six animals), during maximal contraction. This increase is greater than can be accounted for by Ca binding to calmodulin and to myosin, suggesting the presence of other Ca-binding proteins in smooth muscle. A small amount (0.4-0.6 mmol/kg dry wt.) of cytoplasmic Ca remained after incubation in Ca-free, high-Mg2+ EGTA solution. This tightly bound, cytoplasmic Ca is insufficient to account for the total amount of divalent cation known to be bound to F-actin. We conclude that Mg is the major inexchangeably bound cation in F-actin in smooth as in striated muscle. In the contracted muscles, the cellular Ca concentration, measured with the large probes that include Ca stored in the sarcoplasmic reticulum (s.r.), was 3.2 +/- 0.3 (S.E. of mean) mmol Ca/kg dry wt. (n = 93), significantly higher than the cytoplasmic Ca concentration measured with small probes. This value of cellular Ca is probably an underestimate, as the large-diameter probes did not cover all of the peripheral s.r. The cellular Ca (measured with large probes) was highest in the contracted and lower in the relaxed tissue, and was significantly reduced in the muscles incubated in Ca-free solution. In contracted muscle, cytoplasmic Mg significantly decreased and mitochondrial Mg increased. In 0 Ca, high-Mg2+ solution, the cytoplasmic Mg increased significantly. Mitochondrial Ca did not significantly change during a maintained contraction, but was significantly lower (0.0 +/- 0.2 (S.E. of mean) mmol Ca/kg dry wt.) after incubation in Ca-free, high-Mg2+ solution than in the relaxed tissue (1.6 +/- 0.2 mmol Ca/kg dry wt.) in normal Ca-containing solution.
Animals↗
Ultrastructure, function and composition of smooth muscle.
Filamentous myosin is present in both relaxed (myosin light chains unphosphorylated) and contracted (light chains phosphorylated) vascular smooth muscle. The organization of myosin and actin filaments and the insertion of the latter on cytoplasmic and plasma membrane bound dense bodies is consistent with a mini sarcomere-like organization and a sliding filament mechanism of contraction in smooth muscle. Mitochondria are high capacity, low affinity Ca stores in smooth muscle. They do not play a role in the regulation of cytoplasmic Ca2+ at physiological levels. The localization and Ca content of the junctional sarcoplasmatic reticulum (SR) is consistent with this organelle being the major intracellular source of activator Ca released by excitatory transmitters. Repeated contractions in the absence of extracellular Ca2+ (thought to represent recycling of intracellular activator Ca2+) can be demonstrated if the excitatory agent is not allowed to remain in contact with the smooth muscle throughout relaxation.; the demonstration of "recycling" is facilitated if the efflux of cellular Ca2+ is blocked. The rise in total cytoplasmic calcium measured with electron probe analysis during a maintained (30 min) contracture in rabbit portal-anterior mesenteric vein smooth muscle (approximately 0.9 mol/kg dry cytoplasm) is greater than the amount of Ca that could be bound to calmodulin.
Actins↗
Applications of energy filtered imaging in biology.
We describe the use of a magnetic sector spectrometer positioned below the projection chamber of an electron microscope for energy filtered transmission imaging. The spectrometer used has circular pole face edges and is corrected for second order aberrations. A round EM lens is placed after the sector to form a real image of the virtual achromatic image produced by the spectrometer. A slit placed in the dispersion plane allows the passage of electrons in a selected energy range. The filtered image is projected onto a transmission phosphor and acquired with a silicon intensified TV camera and stored in digital form on computer disk. Filtered images are taken at two energies, one immediately preceding (pre-edge) and one on the characteristic energy loss (edge). To obtain images showing the distribution of elements, background subtraction is performed by either subtraction or division of edge and pre-edge images. The optical properties of the imaging system are described and the results are illustrated by energy filtered images of single ferritin molecules (Fe M2,3 and C k), the phosphorus distribution in ribosomes (PL2,3) and the localization of calcium in muscle (Ca L2, 3). The major advantage of the system, compared to other energy filtered imaging methods, is that it can be readily adapted to existing high vacuum microscopes without the necessity of modifying the column to insert a spectrometer.
Animals↗
Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis.
Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determine in situ the concentrations of Ca2+ and Mg2+ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2 x 10(-11) M Ca2+ and 2.7 x 10(-9) M Mg2+, the inexchangeably bound Mg2+ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca2+ bound to the I-band was not significantly above zero. The bound Mg2+ in the I-band was not exchangeable with Ca2+ even when the skinned fibres were exposed to 10 mM Ca2+ solution. These results clearly indicate that Mg2+, rather than Ca2+, is the divalent cation bound to F-actin in the thin filaments in situ. In the presence of 1 mM Mg2+, the exchangeable Ca2+ bound to the I-band was increased as a function of the free Ca2+, while that in the A-band was not significantly changed with [Ca2+] up to 2 x 10(-5) M, and increased to approximately 0.8 mol Ca2+ per mol myosin at 10(-4) M Ca2+. At a saturating free Ca2+ in Tris-Cl solution, the bound Ca2+ content (2-3 mol Ca2+ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na+ enhanced Ca2+ binding to the level equivalent to 3-4 mol Ca2+ per mol troponin. The depressant effect of Tris on Ca2+ binding was greater in the absence of Mg2+. High concentrations of Tris also reduced the maximum tension induced by 10(-4) M Ca2+ buffered with 10 mM EGTA. At 1.3 x 10(-7) M Ca2+, thought to be close to the cytoplasmic free Ca2+ in resting muscle, the I-band bound a significant amount of Ca2+: equivalent to about 1 mol Ca2+ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca2+ bound by the A-band at a free Ca2+ of 10(-4) M.
Animals↗
Electron probe and electron energy loss analysis in biology.
Methods, applications and limitations of quantitative electron probe analysis, X-ray mapping, electron energy loss analysis and energy filtered imaging are described, with emphasis on the analysis of thin (less than 200nm) cryosections. Energy dispersion electron probe analysis can measure reliably 5 to 10mM/Kg of biologically prevalent elements in 50nm diameter areas of 100 to 150 nm thick cryo sections during 100-300 sec counts. The minimal detectable mass (MDM) with a conventional thermionic electron source is approximately 10(-19)g Fe (100 sec count) and can be reduced to 10(-20)g through the use of a field emission gun (FEG). A spatial resolution of 8.7nm is demonstrated in two-dimensional Fourier transforms of Mo X-ray maps of stained catalase crystals. Significant biological results of quantitative electron probe analysis include the measurement of total Ca released from the Mg and K taken up by the sarcoplasmic reticulum during muscle contraction, and the demonstration that mitochondria do not contribute to the physiological regulation of cytoplasmic free Ca levels in cardiac, vascular smooth and striated muscle. Electron energy loss analysis (EELS) promises a significant improvement in sensitivity for the measurement of Ca; based on statistical errors of the measurement, 250 microM/Kg Ca should be measureable with EELS in 250 sec. through the Ca L-edge loss. The use of a doubly corrected magnetic sector spectrometer as a transmission electron microscope imaging filter outside the microscope vacuum is illustrated, and the resolution of the iron core (7.5nm) and surrounding organic shell of single ferritin molecules is demonstrated in, respectively, iron M and carbon K loss images.
Animals↗
Energy-filtered transmission electron microscopy of ferritin.
The focusing properties of a magnetic-sector spectrometer are shown to be suitable for forming high-spatial-resolution, energy-filtered transmission electron microscope images. Filtered images of ferritin molecules by using electrons scattered from the characteristic iron M2,3 and carbon K absorption edges clearly distinguish the 75-A iron core and 120-A protein shell. The minimum detectable mass is estimated to be 0.84 X 10(-20) g for Fe for an electron dose of 18 C/cm2 and 99% confidence.
Ferritins↗
Localization of adenylate cyclase in myocardial junctional sarcoplasmic reticulum. Cytochemical study after isoproterenol activation and propranolol inhibition of beta receptors.
A method for preservation of both morphological and enzymatic integrity was used to demonstrate localization of adenylate cyclase (AC) in rat myocardium. Short prefixation with a 2% glutaraldehyde solution and 5% DMSO followed by incubation in modified standard medium with 10 mM ouabain, 1 mM adenine deaminase, and 1 mM tetramisol with 10-20 mM NaF and 0.1-0.4 mM isoproterenol as activators and/or 1-100 propranolol as a beta blocker allowed regularly reproducible localization of fine granular precipitate of lead imidodiphosphate at the sites of adenylate cyclase activity. In addition to localization on the sarcolemma, the greatest precipitation was found inside the junctional sacroplasmic reticulum and subsarcolemma cisternae. Longitudinal sarcoplasmic reticulum was free of precipitate. Participation of Ca2+ ions in these areas, where calcium is usually present in high concentration, was excluded by electron probe X-ray microanalysis. The amount of precipitate was greatly enhanced after stimulation of AC with isoproterenol or NaF and was diminished by 30-min preincubation with 10-100 mM propranolol.
Adenylyl Cyclases↗
Calcium and monovalent ions in smooth muscle.
Electron probe analysis has shown that the high Cl and K contents of smooth muscle reflect the generalized cytoplasmic distribution of these elements and are not due to sequestration in organelles. These findings, in agreement with other studies, indicate that ECl is less and EK more negative than the membrane potential. The susceptibility of cellular Na content to preparatory procedures has been confirmed. Nuclei do not sequester Na, and transmitochondrial monovalent ion (Na, K, Cl) gradients are very small or nonexistent. The major identified intracellular store of Ca is the sarcoplasmic reticulum; mitochondrial Ca is low in resting as well as in maximally contracted normal portal vein smooth muscle. Observations on mitochondrial loading in saponin-skinned smooth muscle suggest that in intact cells maximal contraction may take place at less than 10(-5) M free cytoplasmic Ca2+ concentrations [Ca2+]. Problems of the composition of abnormal (hypertrophied, hypertensive, and atherosclerotic) smooth muscles are considered.
Animals↗
Parallel recording of electron energy loss spectra.
Two silicon photo diode array devices were tested as parallel recording detectors for electron energy loss spectrometry (EELS). The direct bombardment of a Reticon photodiode array detector with high energy electrons (80 keV) causes an irreversible increase in diode dark current. The dark current saturates the detector amplifier after a dose of 10(-6) C/diode making it unsuitable for EELS. A scintillator coupled SIT vidicon is sensitive enough to count two high energy electrons with a spatial resolution of 100 micrometers, corresponding to 5 eV energy resolution with the electron optical system described. The large pixel-to-pixel gain variation inherent in the scintillator and vidicon can be reduced by averaging the spectrum over a large area of the target perpendicular to the dispersion direction. The L-edge of calcium for a 4 X 10(-3) weight fraction concentration biological specimen is observable in a 40 s parallel recorded spectrum. The minimum detectable concentration of calcium is estimated to be ten times better for ELLS than EDS X-ray analysis.
Calcium↗
Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study.
Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.
Animals↗
Scanning electron probe x-ray microanalysis of elemental distributions in freeze-dried cryosections of Bacillus coagulans spores.
High-resolution scanning electron probe X-ray microanalysis had been employed to examine elemental distributions in freeze-dried cryosections of Bacillus coagulans spores. Calcium, manganese, and phosphorus were concentrated in the protoplast and the coat. Iron was found in the coat but not in the protoplast, whereas the silicon seen on the coat of other spore species was absent. Sulfur was present in the coat, but was distributed over a broader area than the other elements, which suggested that phosphorus and the metal ions were located in the outer coat layer.
Bacillus↗