Quantitative energy dispersive X-ray microanalysis of eight elements in pancreatic endocrine and exocrine cells after cryo-fixation

1987 ◽  
Vol 7 (11) ◽  
pp. 859-869 ◽  
Author(s):  
Robert Norlund ◽  
Norbert Roos ◽  
Inge-Bert Täljedal
Keyword(s):  
Exocrine Pancreas ◽  
Secretory Granules ◽  
Cell Nuclei ◽  
Β Cells ◽  
Tissue Samples ◽  
Copper Surfaces ◽  
X Ray ◽  
Insulin Granules ◽  
Freeze Dried ◽  
Cellular Compartments

Quantitative X-ray microanalysis of 8 elements was performed on ultrathin, freeze-dried sections of islets and pancreas pieces from non-inbred ob/ob-mice. Diffusion of elements was reduced to a minimum by rapidly freezing the tissue samples between nitrogen-cooled polished copper surfaces and avoiding the use of chemical fixatives and stains. The ultrastructural morphology was adequately maintained to allow measurements on secretory granules, mitochondria, cell nuclei, and cytoplasm free of these organelles. The distribution of the various elements between cellular compartments was similar in islet β-cells and exocrine pancreas cells. However, the insulin secretory granules were outstanding in exhibiting the highest concentrations of zinc and calcium. In comparison with cytoplasm in the β-cells, the insulin granules accumulated calcium 2-fold and zinc as much as 40-fold. As no correlation could be made for endoplasmic reticulum in the cytoplasmic measurements areas, the true accumulations above cytosol are likely to be even higher.

The Distribution of Light Elements in Biological Cells Measured by Electron Probe X-Ray Microanalysis of Cryosections

2005 ◽  
Vol 11 (2) ◽  
pp. 138-145 ◽  
Author(s):  
Karl Zierold ◽  
Jean Michel ◽  
Christine Terryn ◽  
Gérard Balossier
Keyword(s):  
Electron Irradiation ◽  
Electron Probe ◽  
Inorganic Mercury ◽  
Rat Hepatocytes ◽  
Light Elements ◽  
Cell Nuclei ◽  
X Ray ◽  
Freeze Dried ◽  
Subcellular Compartmentation ◽  
Cellular Compartments

The intracellular distribution of the elements carbon, nitrogen, and oxygen was measured in cultured rat hepatocytes by energy dispersive electron probe X-ray microanalysis of 100-nm-thick freeze-dried cryosections. Electron irradiation with a dose up to 106e/nm2caused no or merely negligible mass loss in mitochondria and in cytoplasm. Cell nuclei lost carbon, nitrogen, and—to a clearly higher extent—oxygen with increasing electron irradiation. Therefore, electron doses less than 3 × 105e/nm2were used to measure the subcellular compartmentation of carbon, nitrogen, and oxygen in cytoplasm, mitochondria, and nuclei of the cells. The subcellular distribution of carbon, nitrogen, and oxygen reflects the intracellular compartmentation of various biomolecules. Cells exposed to inorganic mercury before cryofixation showed an increase of oxygen in nuclei and cytoplasm. Concomitantly the phosphorus/nitrogen ratio decreased in mitochondria. The data suggest mercury-induced production of ribonucleic acid (RNA) and decrease of adenosine triphosphate (ATP). Although biomolecules cannot be identified by X-ray microanalysis, measurements of the whole element spectrum including the light elements carbon, nitrogen, and oxygen can be useful to study specific biomolecular activity in cellular compartments depending on the functional state of the cell.

scholarly journals Vapor fixation for immunocytochemistry and X-ray microanalysis on cryoultramicrotome sections.

1984 ◽  
Vol 32 (6) ◽  
pp. 636-642 ◽  
Author(s):  
P M Frederik ◽  
P H Bomans ◽  
W M Busing ◽  
R Odselius ◽  
W M Hax
Keyword(s):  
Exocrine Pancreas ◽  
Osmium Tetroxide ◽  
Protein A ◽  
Alpha Amylase ◽  
X Ray ◽  
Freeze Dried ◽  
Frozen Tissue ◽  
Formaldehyde Vapor ◽  
Fresh Frozen ◽  
Subcellular Level

Several organic and inorganic vapor fixatives have been tested for their ability to stabilize the ultrastructure of freeze-dried thin cryosections. The vapors from osmium tetroxide and dry formaldehyde gave a good preservation of the ultrastructure. Fixation in formaldehyde vapor preserved the immunoreactivity of alpha-amylase in exocrine pancreas, as was demonstrated with an indirect labeling technique using anti-alpha-amylase and protein A-gold. A major advantage of the use of vapor fixation is that cryosections from a specimen of fresh-frozen tissue can be used for immunocytochemistry as well as for X-ray microanalysis, as was demonstrated for the exocrine pancreas. This opens the possibility of localizing atomic species (X-ray microanalysis) and molecular species (immunocytochemistry) at the subcellular level on thin cryosections from the same tissue block.

scholarly journals The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane

2013 ◽  
Vol 24 (3) ◽  
pp. 319-330 ◽  
Author(s):  
Hao Wang ◽  
Ray Ishizaki ◽  
Jun Xu ◽  
Kazuo Kasai ◽  
Eri Kobayashi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Knockout Mice ◽  
Secretory Granules ◽  
Small Gtpase ◽  
Membrane Phospholipids ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Insulin Exocytosis

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

scholarly journals TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells: different behaviour of granule motion between normal and Goto–Kakizaki diabetic rat β-cells

2004 ◽  
Vol 381 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Mica OHARA-IMAIZUMI ◽  
Chiyono NISHIWAKI ◽  
Toshiteru KIKUTA ◽  
Shintaro NAGAI ◽  
Yoko NAKAMICHI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Release ◽  
Secretory Granules ◽  
Dynamic Change ◽  
Second Phase ◽  
Β Cells ◽  
Gk Rat ◽  
Pancreatic Β Cells ◽  
Insulin Granules ◽  
Granule Motion

We imaged and analysed the motion of single insulin secretory granules near the plasma membrane in live pancreatic β-cells, from normal and diabetic Goto–Kakizaki (GK) rats, using total internal reflection fluorescence microscopy (TIRFM). In normal rat primary β-cells, the granules that were fusing during the first phase originate from previously docked granules, and those during the second phase originate from ‘newcomers’. In diabetic GK rat β-cells, the number of fusion events from previously docked granules were markedly reduced, and, in contrast, the fusion from newcomers was still preserved. The dynamic change in the number of docked insulin granules showed that, in GK rat β-cells, the total number of docked insulin granules was markedly decreased to 35% of the initial number after glucose stimulation. Immunohistochemistry with anti-insulin antibody observed by TIRFM showed that GK rat β-cells had a marked decline of endogenous insulin granules docked to the plasma membrane. Thus our results indicate that the decreased number of docked insulin granules accounts for the impaired insulin release during the first phase of insulin release in diabetic GK rat β-cells.

scholarly journals Soft X-ray induced radiation damage in thin freeze-dried brain samples studied by FTIR microscopy

2020 ◽  
Vol 27 (5) ◽  
pp. 1218-1226
Author(s):  
Artur D. Surowka ◽  
A. Gianoncelli ◽  
G. Birarda ◽  
S. Sala ◽  
N. Cefarin ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Biological Sample ◽  
Radiation Doses ◽  
Complex Interplay ◽  
Ftir Microscopy ◽  
Tissue Samples ◽  
X Ray ◽  
Freeze Dried ◽  
Resolution Limits ◽  
First Time

In order to push the spatial resolution limits to the nanoscale, synchrotron-based soft X-ray microscopy (XRM) experiments require higher radiation doses to be delivered to materials. Nevertheless, the associated radiation damage impacts on the integrity of delicate biological samples. Herein, the extent of soft X-ray radiation damage in popular thin freeze-dried brain tissue samples mounted onto Si3N4 membranes, as highlighted by Fourier transform infrared microscopy (FTIR), is reported. The freeze-dried tissue samples were found to be affected by general degradation of the vibrational architecture, though these effects were weaker than those observed in paraffin-embedded and hydrated systems reported in the literature. In addition, weak, reversible and specific features of the tissue–Si3N4 interaction could be identified for the first time upon routine soft X-ray exposures, further highlighting the complex interplay between the biological sample, its preparation protocol and X-ray probe.

scholarly journals Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

1981 ◽  
Vol 90 (3) ◽  
pp. 769-777 ◽  
Author(s):  
I Z Nagy ◽  
G Lustyik ◽  
V Z Nagy ◽  
B Zarándi ◽  
C Bertoni-Freddari
Keyword(s):  
Cancer Cells ◽  
Human Cancer ◽  
Rat Kidney ◽  
Transitional Cell ◽  
Energy Dispersive ◽  
Cell Nuclei ◽  
Healthy Human ◽  
Cytostatic Treatment ◽  
X Ray ◽  
Freeze Dried

Intranuclear sodium, potassium, and chloride contents were measured by energy-dispersive x-ray microanalysis in freeze-fractured, freeze-dried, bulk-tumor samples taken from 10 patients suffering from invasive urogenital cancers. Human biopsies were carried out during the first diagnostic interventions before any cytostatic treatment had been applied. Pathohistological diagnosis established the malignancy in each case. The cancers were classified in three types: keratinizing, transitional cell, and hypernephroid carcinoma. More than 250 cell nuclei were measured from each type of cancer. The results were compared with those obtained in intact human urothelium taken from patients having no malignant processes. Proximal and distal tubular epithelial cell nuclei representing the origin of human hypernephroid cancer were also measured in rat kidney because corresponding healthy human material cannot be obtained. The analyses revealed, in all three types of cancer cells, that the average intranuclear sodium content increased more than three-fold, the potassium content decreased 32, 16, and 13%, respectively; meanwhile the chloride content increased, but to a lesser extent than did the sodium. The intranuclear Na+:K+ ratios were more than five-fold higher in the cancer cells on the average, and their distribution histograms were much broader than in the normal human urothelium and in the tubular cell nuclei of the rat kidney. The results obtained fit well with the theory of Cone, C. D., Jr. 1971. J. Theor. Biol. 30: 151-181 according to which the sustained depolarization of the cell membrane may be of mitogenic effect.

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.

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.

Electron Beam-Induced Mass Loss in Freeze-Dried Tissue and Protein Samples

1985 ◽  
Vol 43 ◽  
pp. 470-471
Author(s):  
M.E. Cantino ◽  
M.K. Goddard ◽  
L.E. Wilkinson ◽  
D.E. Johnson
Keyword(s):  
Electron Beam ◽  
Salivary Gland ◽  
Mass Loss ◽  
Counting Rate ◽  
Dose Dependence ◽  
X Ray ◽  
Large Samples ◽  
Freeze Dried ◽  
Muscle Homogenate ◽  
Large Muscle

Quantification in biological x-ray microanalysis depends on accurate evaluation of mass loss. Although several studies have addressed the problem of electron beam induced mass loss from organic samples (eg., 1,2). uncertainty persists as to the dose dependence, the extent of loss, the elemental constituents affected, and the variation in loss for different materials and tissues. in the work described here, we used x-ray counting rate changes to measure mass loss in albumin (used as a quantification standard), salivary gland, and muscle.In order to measure mass loss at low doses (10-4 coul/cm2 ) large samples were needed. While freeze-dried salivary gland sections of the required dimensions were available, muscle sections of this size were difficult to obtain. To simulate large muscle sections, frog or rat muscle homogenate was injected between formvar films which were then stretched over slot grids and freeze-dried. Albumin samples were prepared by a similar procedure. using a solution of bovine serum albumin in water. Samples were irradiated in the STEM mode of a JEOL 100C.

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