X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions

1989 ◽  
Vol 256 (2) ◽  
pp. H574-H583 ◽  
Author(s):  
M. F. Wendt-Gallitelli ◽  
G. Isenberg
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Microprobe Analysis ◽  
Ventricular Myocyte ◽  
Pulse Electron ◽  
Ca Current ◽  
X Ray ◽  
Freeze Dried ◽  
Patch Pipette ◽  
Intracellular Compartments ◽  
Junctional Sarcoplasmic Reticulum

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

Tension-voltage relations of single myocytes reflect Ca release triggered by Na/Ca exchange at 35 degrees C but not 23 degrees C

1994 ◽  
Vol 267 (2) ◽  
pp. C623-C632 ◽  
Author(s):  
M. Vornanen ◽  
N. Shepherd ◽  
G. Isenberg
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Guinea Pig ◽  
Voltage Clamp ◽  
Temperature Sensitive ◽  
Ca Current ◽  
Test Pulse ◽  
Ventricular Cells ◽  
Patch Pipette ◽  
Tension Transients

Contractile tension in response to 200-ms voltage-clamp pulses was measured in isolated guinea pig ventricular cells conditioned to constant Ca load. At 23 degrees C, the tension-voltage relation was bell shaped, decaying from a maximum at +20 mV to zero at +100 mV, but at 35 degrees C it was sigmoidal, with similar twitch tensions at +20 and +100 mV. Tension at 35 degrees C and +100 mV was reduced by ryanodine or caffeine and abolished by removal of Ca just before the test pulse. At 35 degrees C and +100 mV, twitch tension increased markedly as the Na concentration in the patch pipette ([Na]p) was varied between 0 and 20 mM. Cd (300 microM) blocked tension at all potentials at 23 degrees C, but tension remained in the presence of Cd at 35 degrees C (29% of control at +2 mV and 100% of control at +100 mV). Cd-resistant tension began to relax during the clamp pulse at all potentials (80 +/- 10 ms at +2 mV and 140 +/- 12 ms at +100 mV). Ni (3.6 mM) both reduced and slowed tension transients at all potentials. The results suggest that fast contractions due to sarcoplasmic reticulum Ca release can be triggered by Ca influx through either Ca current (ICa) or Na/Ca exchange and that those triggered through exchange are much more temperature sensitive than those triggered by ICa.

Electron-probe x-ray microanalysis studies on the intracellular calcium translocation during the contraction-relaxation cycle in the fish swimbladder muscle

1990 ◽  
Vol 48 (2) ◽  
pp. 168-169
Author(s):  
Suechika Suzuki ◽  
Naoki Hino ◽  
Haruo Sugi
Keyword(s):  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Teleost Fish ◽  
Electron Probe ◽  
Middle Portion ◽  
Relaxation Cycle ◽  
X Ray ◽  
Thin Carbon ◽  
Freeze Dried ◽  
Intracellular Ca

The swimbladder muscle of a teleost fish, Sebasticus marmoratus, contracts rapidly to produce sounds for communication and contains extremely well developed sarcoplasmic reticulum (SR), providing a material suitable for studying the intracellular Ca translocation during the contraction-relaxation cycle. We examined the change of Ca distribution along the sarcomere at various states of the fibers during the contractionrelaxation cycle in the swimbladder muscle by means of quantitative electron probe X-ray microanalysis of cryosections.The muscle fibers were rapidly frozen at rest, during sustained contraction and at 0.1 and 1.0 sec after the onset of relaxation with a dual-jet liquid propane freezing device. Cryosections (200 nm thick) were cut from the middle portion of the frozen fibers at -110°C on a LKB NOVA cryoultramicrotome, and placed on thin carbon supporting films on Nigrids. Then the cryosections were freeze-dried at 10-6 torr and below -80°C. Electron probe X-ray microanalysis was performed on a liquid N2-cooled Be-stage at -130°C in a JEOL 2000FX electron microscope operated at 80 kV and equipped with a Tracor Northern TN5500 energy dispersive X-ray microanalyzer.

Time Course of Total Myofibrillar Calcium During the Cardiac Cycle: X-RAY Microprobe Analysis of Voltage-Clamped Guinea-Pig Ventricular Myocytes

1990 ◽  
Vol 48 (2) ◽  
pp. 138-139
Author(s):  
Maria Fiora Wendt-Gallitelli ◽  
Gerrit Isenberg
Keyword(s):  
Voltage Clamp ◽  
Time Course ◽  
Microprobe Analysis ◽  
Electron Microprobe Analysis ◽  
Cardiac Cycle ◽  
Ventricular Myocytes ◽  
Cell Voltage ◽  
X Ray ◽  
Patch Pipette

We report methodological efforts and results in measuring the changes in total myofibrillar Ca-concentration during systole and diastole. The measurements were made by means of electron microprobe analysis (EPMA) from ultrathin cryosections. We believe that EPMA is the only method that can adequately measure total [Ca] at the place of interest, in this case at the overlapping myofilaments of the A-band. In combination with cryotechniques, it it can be used to assess total [Ca] without disturbing the original in vivo Ca-gradients.EPMA was performed on isolated ventricular myocytes. The cells were studied at 36 °C and 2 mM extracellular Ca using a whole-cell voltage clamp. Together with the membrane currents, contractions were recorded as unloaded shortening. In a different population of myocytes, Ca ionized in the cytosol ([Ca2+]c) was estimated by microspectrofluometry (50 μM Indo-1, sodium-salt, applied through the patch pipette). The voltage-clamp gave the opportunity of controlling the voltage-operated Ca-influx, and in turn Ca-induced release of Ca from the sarcoplasmic reticulum (SR), and thus the amount of contraction.

Subcellular localization of diffusible ions in the yeast Saccharomyces cerevisiae: quantitative microprobe analysis of thin freeze-dried sections

1976 ◽  
Vol 21 (1) ◽  
pp. 119-127
Author(s):  
G.M. Roomans ◽  
L.A. Seveus
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Microprobe Analysis ◽  
Total Concentration ◽  
Yeast Cells ◽  
Fresh Weight ◽  
Ion Distribution ◽  
Yeast Saccharomyces Cerevisiae ◽  
Thin Sections ◽  
X Ray ◽  
Freeze Dried

In yeast cells, of which the intracellular potassium had been partly replaced by rubidium or caesium, the intracellular ion distribution was studied by means of energy-dispersive X-ray microanalysis. The cells were rapidly frozen and thin sections were cut at low temperature on a cryo-ultramicrotome without the use of a trough liquid. By this dry cryosectioning procedure, complete retention of the diffusbile ions in the cells was obtained. Unless the sections had been exposed to moisture, no signs of redistribution were apparent. For quantitative determinations a gelatin standard, containing known amounts of the elements of interest, was prepared in the same way as the cells. The concentrations of potassium, rubidium, caesium and chloride in the nucleus, the cytoplasm and the vacuole could be measured. The intracellular distributions of potassium, rubidium and caesium were very similar. The concentrations of these ions in the cytoplasm were about equal to those in the nucleus and twice those in the vacuole. The total concentration in the cytoplasm was 180–190 mmol/kg fresh weight, in the nucleus 190–200 mmol/kg fresh weight and in the vacuole 75–90 mmol/kg fresh weight. The permeability of the yeast cell for chloride is markedly lower than for the cations.

Anchorfibers and the Topography of Junctional SR

1977 ◽  
Vol 35 ◽  
pp. 582-583
Author(s):  
James Junker ◽  
Joachim R. Sommer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cardiac Muscle ◽  
Single Filament ◽  
Relaxation Cycle ◽  
10 Nm Filaments ◽  
Junctional Sarcoplasmic Reticulum

Junctional sarcoplasmic reticulum (JSR) in all its forms (extended JSR, JSR of couplings, corbular SR) in both skeletal and cardiac muscle is always located at the Z - I regions of the sarcomeres. The Z tubule is a tubule of the free SR (non-specialized SR) which is consistently located at the Z lines in cardiac muscle (1). Short connections between JSR and Z lines have been described (2), and bundles of filaments at Z lines have been seen in skeletal (3) and cardiac (4) muscle. In opossum cardiac muscle, we have seen bundles of 10 nm filaments stretching across interfibrillary spaces and adjacent myofibrils with extensions to the plasma- lemma in longitudinal (Fig. 1) and transverse (Fig. 2) sections. Only an occasional single filament is seen elsewhere along a sarcomere. We propose that these filaments represent anchor fibers that maintain the observed invariant topography of the free SR and JSR throughout the contraction-relaxation cycle.

Artefacts in the x-ray microanalysis of frozen-hydrated biological samples

1987 ◽  
Vol 45 ◽  
pp. 658-659
Author(s):  
Patrick Echlin
Keyword(s):  
Electron Scattering ◽  
Biological Samples ◽  
Fracture Face ◽  
Detailed Structure ◽  
X Ray ◽  
Morphological Appearance ◽  
The Poor ◽  
Freeze Dried

A number of papers have appeared recently which purport to have carried out x-ray microanalysis on fully frozen hydrated samples. It is important to establish reliable criteria to be certain that a sample is in a fully hydrated state. The morphological appearance of the sample is an obvious parameter because fully hydrated samples lack the detailed structure seen in their freeze dried counterparts. The electron scattering by ice within a frozen-hydrated section and from the surface of a frozen-hydrated fracture face obscures cellular detail. (Fig. 1G and 1H.) However, the morphological appearance alone can be quite deceptive for as Figures 1E and 1F show, parts of frozen-dried samples may also have the poor morphology normally associated with fully hydrated samples. It is only when one examines the x-ray spectra that an assurance can be given that the sample is fully hydrated.

Revealing gross three-dimensional structure in the SEM

1987 ◽  
Vol 45 ◽  
pp. 656-657
Author(s):  
Sterling P. Newberry
Keyword(s):  
Freeze Drying ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Small Object ◽  
Final Solution ◽  
Dimensional Representation ◽  
X Ray ◽  
Three Dimensional Representation ◽  
Freeze Dried ◽  
Critical Point Drying

The beautiful three dimensional representation of small object surfaces by the SEM leads one to search for ways to open up the sample and look inside. Could this be the answer to a better microscopy for gross biological 3-D structure? We know from X-Ray microscope images that Freeze Drying and Critical Point Drying give promise of adequately preserving gross structure. Can we slice such preparations open for SEM inspection? In general these preparations crush more readily than they slice. Russell and Dagihlian got around the problem by “deembedding” a section before imaging. This some what defeats the advantages of direct dry preparation, thus we are reluctant to accept it as the final solution to our problem. Alternatively, consider fig 1 wherein a freeze dried onion root has a window cut in its surface by a micromanipulator during observation in the SEM.

Fluorescence effects in quantitative microprobe analysis

1990 ◽  
Vol 48 (2) ◽  
pp. 188-189
Author(s):  
S.J.B. Reed
Keyword(s):  
Microprobe Analysis ◽  
Depth Distribution ◽  
Exciting Radiation ◽  
Primary Radiation ◽  
List Type ◽  
Type Number ◽  
Computing Power ◽  
X Ray ◽  
Fluorescent Radiation

Characteristic fluorescenceThe theory of characteristic fluorescence corrections was first developed by Castaing. The same approach, with an improved expression for the relative primary x-ray intensities of the exciting and excited elements, was used by Reed, who also introduced some simplifications, which may be summarized as follows (with reference to K-K fluorescence, i.e. K radiation of element ‘B’ exciting K radiation of ‘A’):1.The exciting radiation is assumed to be monochromatic, consisting of the Kα line only (neglecting the Kβ line).2.Various parameters are lumped together in a single tabulated function J(A), which is assumed to be independent of B.3.For calculating the absorption of the emerging fluorescent radiation, the depth distribution of the primary radiation B is represented by a simple exponential.These approximations may no longer be justifiable given the much greater computing power now available. For example, the contribution of the Kβ line can easily be calculated separately.

Reduction of Potassium in Kidney Proximal Tubules to Aid in Electron Probe X-Ray Microanalysis of Calcium

1985 ◽  
Vol 43 ◽  
pp. 474-475
Author(s):  
A. LeFurgey ◽  
P. Ingram ◽  
L.J. Mandel
Keyword(s):  
Smooth Muscle ◽  
Proximal Tubule ◽  
Electron Probe ◽  
Clinical Applications ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Target Cells ◽  
X Ray ◽  
Freeze Dried

For quantitative determination of subcellular Ca distribution by electron probe x-ray microanalysis, decreasing (and/or eliminating) the K content of the cell maximizes the ability to accurately separate the overlapping K Kß and Ca Kα peaks in the x-ray spectra. For example, rubidium has been effectively substituted for potassium in smooth muscle cells, thus giving an improvement in calcium measurements. Ouabain, a cardiac glycoside widely used in experimental and clinical applications, inhibits Na-K ATPase at the cell membrane and thus alters the cytoplasmic ion (Na,K) content of target cells. In epithelial cells primarily involved in active transport, such as the proximal tubule of the rabbit kidney, ouabain rapidly (t1/2= 2 mins) causes a decrease2 in intracellular K, but does not change intracellular total or free Ca for up to 30 mins. In the present study we have taken advantage of this effect of ouabain to determine the mitochondrial and cytoplasmic Ca content in freeze-dried cryosections of kidney proximal tubule by electron probe x-ray microanalysis.

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