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.

Differential mass loss in biological microanalysis determined by digital image analysis of STEM images

1988 ◽  
Vol 46 ◽  
pp. 638-639
Author(s):  
B. J. Hurley
Keyword(s):  
Salivary Gland ◽  
Mass Loss ◽  
Cardiac Muscle ◽  
Dose Dependence ◽  
X Ray ◽  
Video Images ◽  
Salivary Gland Cells ◽  
Freeze Dried ◽  
Dose Information

Quantitative x-ray microanalysis of biological specimens requires that the extent of electron beam induced mass loss in the tissue prior to and during analysis be determined. Previous studies using x-ray counting methods have determined the extent and dose dependence of mass loss in some freeze-dried tissues. This method is limited by the requirement of large scan areas to determine low dose information. The large scan areas yield mass loss values that are cell averages and do not allow determination of differential mass loss fractions between cellular organelles. Knowledge of such differential mass loss is important for example, in the determination of ionic gradients between mitochondria and adjacent myofibril space in cardiac muscle. In the work described here, digital STEM video images of cardiac muscle and salivary gland were used to quantify the extent of mass loss in specific cellular regions. Mass loss values in mitochondria and myofibril regions were compared in cardiac muscle whereas secretory granule and nuclear mass loss fractions were compared in salivary gland cells.

Quantification of losses during X-ray microanalysis of mercury

1978 ◽  
Vol 36 (2) ◽  
pp. 12-13
Author(s):  
T. Bistricki
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Mass Loss ◽  
Atomic Number ◽  
High Vacuum ◽  
Analytical Electron Microscopy ◽  
X Ray ◽  
Low Level ◽  
Analytical Electron ◽  
Concentration Levels

Diminution of the X-ray signal from the elements of low atomic number and mass loss associated with electron excited X-ray microanalysis have been demonstrated elsewhere. The elements of higher atomic number did not receive sufficient attention although under severe conditions of analytical electron microscopy, the losses of any of the specimen's elemental components exposed to the electron beam bombardment in high vacuum are to be expected and have been indicated previously. Unlike the elements of low atomic number, the losses of heavier elements, like mercury, are usually not detected unless a mass of the element within the excited volume is at a very low level (10-11 g range or less) or analytical conditions are extremely unfavourable, in which case considerable losses may occur even when concentration levels are by orders of magnitude higher (10-9 g range or higher).

Aspects of microanalysis in a transmission electron microscope

1978 ◽  
Vol 36 (1) ◽  
pp. 510-511
Author(s):  
R. Sinclair ◽  
B.E. Jacobson
Keyword(s):  
Grain Size ◽  
Electron Beam ◽  
Electron Microscope ◽  
Chemical Analysis ◽  
Transmission Electron Microscope ◽  
Vapor Deposition ◽  
Critical Currents ◽  
X Ray ◽  
Fine Grain ◽  
Transmission Electron

INTRODUCTIONThe prospect of performing chemical analysis of thin specimens at any desired level of resolution is particularly appealing to the materials scientist. Commercial TEM-based systems are now available which virtually provide this capability. The purpose of this contribution is to illustrate its application to problems which would have been intractable until recently, pointing out some current limitations.X-RAY ANALYSISIn an attempt to fabricate superconducting materials with high critical currents and temperature, thin Nb3Sn films have been prepared by electron beam vapor deposition [1]. Fine-grain size material is desirable which may be achieved by codeposition with small amounts of Al2O3 . Figure 1 shows the STEM microstructure, with large (∽ 200 Å dia) voids present at the grain boundaries. Higher quality TEM micrographs (e.g. fig. 2) reveal the presence of small voids within the grains which are absent in pure Nb3Sn prepared under identical conditions. The X-ray spectrum from large (∽ lμ dia) or small (∽100 Ǻ dia) areas within the grains indicates only small amounts of A1 (fig.3).

X-ray micro tomography in the scanning electron microscope

1978 ◽  
Vol 36 (1) ◽  
pp. 106-107 ◽  
Author(s):  
W. Brünger
Keyword(s):  
Electron Beam ◽  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Target Surface ◽  
The Body ◽  
X Rays ◽  
Cross Sectional ◽  
X Ray ◽  
Micro Tomography ◽  
Scanning Electron

Reconstructive tomography is a new technique in diagnostic radiology for imaging cross-sectional planes of the human body /1/. A collimated beam of X-rays is scanned through a thin slice of the body and the transmitted intensity is recorded by a detector giving a linear shadow graph or projection (see fig. 1). Many of these projections at different angles are used to reconstruct the body-layer, usually with the aid of a computer. The picture element size of present tomographic scanners is approximately 1.1 mm2.Micro tomography can be realized using the very fine X-ray source generated by the focused electron beam of a scanning electron microscope (see fig. 2). The translation of the X-ray source is done by a line scan of the electron beam on a polished target surface /2/. Projections at different angles are produced by rotating the object.During the registration of a single scan the electron beam is deflected in one direction only, while both deflections are operating in the display tube.

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.

Searching for the causes why some of the paintings by Mihaly Munkacsy are darkening

1990 ◽  
Vol 48 (2) ◽  
pp. 204-205
Author(s):  
Imre Pozsgai ◽  
Klara Erdöhalmi-Torok
Keyword(s):  
Electron Beam ◽  
Cross Section ◽  
Polyester Resin ◽  
National Gallery ◽  
X Ray ◽  
Art Historians ◽  
Made In ◽  
Grinding And Polishing

The paintings by the great Hungarian master Mihaly Munkacsy (1844-1900) made in an 8-9 years period of his activity are deteriorating. The most conspicuous sign of the deterioration is an intensive darkening. We have made an attempt by electron beam microanalysis to clarify the causes of the darkening. The importance of a study like this is increased by the fact that a similar darkening can be observed on the paintings by Munkacsy’s contemporaries e.g Courbet and Makart. A thick brown mass the so called bitumen used by Munkacsy for grounding and also as a paint is believed by the art historians to cause the darkening.For this study, paint specimens were taken from the following paintings: “Studio”, “Farewell” and the “Portrait of the Master’s Wife”, all of them are the property of the Hungarian National Gallery. The paint samples were embedded in a polyester resin “Poly-Pol PS-230” and after grinding and polishing their cross section was used for x-ray mapping.

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.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Elemental mass loss in silicate minerals during x-ray analysis

1990 ◽  
Vol 48 (4) ◽  
pp. 804-805
Author(s):  
P.E. Champness ◽  
R.W. Devenish
Keyword(s):  
Mass Loss ◽  
Current Density ◽  
Damage Mechanism ◽  
Beam Current ◽  
Single Chain ◽  
Plagioclase Feldspar ◽  
X Ray ◽  
Alkali Ions ◽  
Structural Order ◽  
Sheet Silicate

It has long been recognised that silicates can suffer extensive beam damage in electron-beam instruments. The predominant damage mechanism is radiolysis. For instance, damage in quartz, SiO2, results in loss of structural order without mass loss whereas feldspars (framework silicates containing Ca, Na, K) suffer loss of structural order with accompanying mass loss. In the latter case, the alkali ions, particularly Na, are found to migrate away from the area of the beam. The aim of the present study was to investigate the loss of various elements from the common silicate structures during electron irradiation at 100 kV over a range of current densities of 104 - 109 A m−2. (The current density is defined in terms of 50% of total current in the FWHM probe). The silicates so far ivestigated are:- olivine [(Mg, Fe)SiO4], a structure that has isolated Si-O tetrahedra, garnet [(Mg, Ca, Fe)3Al2Si3AO12 another silicate with isolated tetrahedra, pyroxene [-Ca(Mg, Fe)Si2O6 a single-chain silicate; mica [margarite, -Ca2Al4Si4Al4O2O(OH)4], a sheet silicate, and plagioclase feldspar [-NaCaAl3Si5O16]. Ion- thinned samples of each mineral were examined in a VG Microscopes UHV HB501 field- emission STEM. The beam current used was typically - 0.5 nA and the current density was varied by defocussing the electron probe. Energy-dispersive X-ray spectra were collected every 10 seconds for a total of 200 seconds using a Link Systems windowless detector. The thickness of the samples in the area of analysis was normally 50-150 nm.

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