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P Ingram

Publications and source records attributed to P Ingram.

At least 19 recordsLinked to original sources
PubMed1992

Real-time quantitative elemental analysis and mapping: microchemical imaging in cell physiology.

Recent advances in widely available microcomputers have made the acquisition and processing of digital quantitative X-ray maps of one to several cells readily feasible. Here we describe a system which uses a graphics-based microcomputer to acquire spectrally filtered X-ray elemental image maps that are fitted to standards, to display the image in real time, and to correct the post-acquisition image map with regard to specimen drift. Both high-resolution quantitative energy-dispersive X-ray images of freeze-dried cyrosections and low-dose quantitative bright-field images of frozen-hydrated sections can be acquired to obtain element and water content from the same intracellular regions. The software programs developed, together with the associated hardware, also allow static probe acquisition of data from selected cell regions with spectral processing and quantification performed on-line in real time. In addition, the unified design of the software program provides for off-line processing and analysing by several investigators at microcomputers remote from the microscope. The overall experimental strategy employs computer-aided imaging, combined with static probes, as an essential interactive tool of investigation for biological analysis. This type of microchemical microscopy facilitates studies in cell physiology and pathophysiology which focus on mechanisms of ionic (elemental) compartmentation, i.e. structure-function correlation at cellular and subcellular levels; it allows investigation of intracellular concentration gradients, of the heterogeneity of cell responses to stimuli, of certain fast physiological events in vivo at ultrastructural resolution, and of events occurring with low incidence or involving cell-to-cell interactions.

Animals↗
PubMed1991

Structural and elemental characterization of heart cells grown in a collagen matrix.

A novel preparation of spontaneously contracting heart cells embedded in a collagen strand provides an ideal experimental system for correlative structure-function experiments that utilize the techniques of electron microscopy, quantitative electron probe x-ray microanalysis (EPXMA) and imaging. Heart cells grown within the strand for 1 day possess the subcellular content and distribution of physiologically relevant elements--Na, Mg, P, S, Cl, K, and Ca--found in intact heart cell preparations. The presence of junctional specializations between, and organized myofibrils within, the majority of cells after 1 day in culture also establishes that the collagen matrix promotes vigorous cell development as well as maintains physiological integrity. EPXMA, combined with ultrastructural analyses, provides elemental content data on a cell-by-cell basis. In studies presented here, viable cells, comprising over 80% of the strand cell population, could be distinguished easily from those which had been functionally compromised, not only by aberrant structure but also by altered subcellular compartmentation of Na, K, Cl, and Ca. Within individual viable cells, compartmental differences in element content were notable especially between mitochondria and cytoplasm. However, nuclear euchromatin, but not heterochromatin, appeared approximately identical to cytoplasm in elemental and water content. In such cells, the cytoplasmic K:Na ratio was maintained at a high level (approximately 15:1). The results with respect to K, Na, and other elements demonstrated the integrity of membrane transport mechanisms regulating the movement and distribution of ions and the maintenance of ionic homeostasis in cells of the strand preparation.

Animals↗
PubMed1991

Elemental microanalysis of organelles in proximal tubules. I. Alterations in transport and metabolism.

Oxygen deprivation to the kidney causes a multifactorial series of morphological, physiological, and biochemical alterations that occur as a function of time. One of the earliest events involves significant changes in the cellular contents of the physiologically important elements (ions) Na and K. Controversy exists as to the nature of changes in the content of the regulatory ion Ca, in either its free or bound form, and much less is known regarding in situ distribution and amounts of other elements such as Mg, P, S, and Cl during physiological or pathophysiological states. The objective of these studies was to evaluate element compartmentation in proximal renal tubules by using quantitative electron probe x-ray microanalysis, during specific conditions which are at least partially manifested during oxygen deprivation. Cells from control proximal tubule suspensions were compared with those exposed to (1) ouabain, to inhibit (Na+, K+)-ATPase; (2) mitochondrial uncouplers, to rapidly deplete ATP content; or (3) calcium ionophores, to cause a rapid elevation in cytoplasmic free calcium. In parallel with electron probe x-ray microanalysis imaging of subcellular elemental content, total cell potassium and ATP contents, enzyme release, oxygen consumption, cytoplasmic free calcium levels, and ultrastructural alterations were assessed. Results indicated that ATP depletion was, in the short term, more deleterious to renal proximal tubules than any of the tested ionic alterations. Intracellular organelles including mitochondria and nuclei appeared to be readily permeable to Na, K, and Cl, altering their concentrations of these ions in parallel with cytoplasmic concentrations. Lysosomes exhibited evidence of Cl accumulation, consistent with an inwardly directed proton ATPase with accompanying Cl transport. Whereas in the cytoplasm Na, K and Cl appeared to be mostly free, a large fraction of these ions within intracellular organelles seemed bound.

Animals↗
PubMed1991

Elemental microanalysis of organelles in proximal tubules. II. Effects of oxygen deprivation.

This communication describes the effects of anoxia on rabbit proximal renal tubule element (ion) content by using high-resolution electron probe x-ray microanalytical imaging to obtain quantitative elemental data from subcellular compartments not previously resolvable with low-resolution imaging. These organelles and regions include the heterochromatin and euchromatin of the nucleus and the microvilli of the apical brush border, in addition to mitochondria, lysosomes, and cytoplasm. Anoxia of 40-min duration caused the expected decrease in K and increase in Na and Cl concentrations in the tubules with the cytoplasmic K:Na ratio declining to 0.13:1. These changes were accompanied by decreases in ATP and total K contents, and an increase in lactate dehydrogenase release. Swelling occurred in some cells as evidenced by ultrastructural changes. No alterations were evident after oxygen deprivation in Ca content of cytoplasm (control, 6.7 +/- 0.6 versus anoxia, 7.6 +/- 0.7 nmol/mg dry wt) or mitochondria (control, 4.0 +/- 0.4 versus anoxia, 4.9 +/- 0.6 nmol/mg dry wt) or in S content of recognizable lysosomes (control, 314 +/- 11 versus anoxia, 325 +/- 12 nmol/mg dry wt). Brush border (microvillus) Ca content was higher than cytoplasmic Ca content during normoxia (10.7 +/- 0.9 nmol/mg dry wt) and increased further during anoxia (17.0 +/- 1.0 nmol of Ca/mg dry wt). The finding of higher Ca content within the brush border region during normoxia is unexpected and novel, because such results suggest that Ca homeostasis in the apical elaboration of the proximal cell may be different from that in the cytoplasm. The results also raise the possibility that an increase in Ca content in the brush border membrane region may be involved in the pathogenesis of renal cell injury.

Animals↗
PubMed1990

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Animals↗
PubMed1990

Calcium measurements with electron probe X-ray and electron energy loss analysis.

This paper presents a broad survey of the rationale for electron probe X-ray microanalysis (EPXMA) and the various methods for obtaining qualitative and quantitative information on the distribution and amount of elements, particularly calcium, in cryopreserved cells and tissues. Essential in an introductory consideration of microanalysis in biological cryosections is the physical basis for the instrumentation, fundamentals of X-ray spectrometry, and various analytical modes such as static probing and X-ray imaging. Some common artifacts are beam damage and contamination. Inherent pitfalls of energy dispersive X-ray systems include Si escape peaks, doublets, background, and detector calibration shifts. Quantitative calcium analysis of thin cryosections is carried out in real time using a multiple least squares fitting program on filtered X-ray spectra and normalizing the calcium peak to a portion of the continuum. Recent work includes the development of an X-ray imaging system where quantitative data can be retrieved off-line. The minimum detectable concentration of calcium in biological cryosections is approximately 300 mumole kg dry weight with a spatial resolution of approximately 100 A. The application of electron energy loss (EELS) techniques to the detection of calcium offers the potential for greater sensitivity and spatial resolution in measurement and imaging. Determination of mass thickness with EELS can facilitate accurate calculation of wet weight concentrations from frozen hydrated and freeze-dried specimens. Calcium has multiple effects on cell metabolism, membrane transport and permeability and, thus, on overall cell physiology or pathophysiology. Cells can be rapidly frozen for EPXMA during basal or altered functional conditions to delineate the location and amount of calcium within cells and the changes in location and concentration of cations or anions accompanying calcium redistribution. Recent experiments in our laboratory document that EPXMA in combination with other biochemical and electrophysiological techniques can be used to study, for example, sodium and calcium compartmentation in cultured cardiac cells. Such analyses can also be used to clarify the role of calcium in anoxic renal cell injury and to evaluate proposed ionic defects in cells of individuals with cystic fibrosis.

Animals↗
PubMed1989

Correction for specimen movement after acquisition of element-specific electron microprobe images.

Because a long time is generally required to generate X-ray maps of specific elements by electron beam methods, images are subject to a loss of resolution due to stage movement. Methods have been previously described for correcting stage drift during exposure by sensing the drift and deflecting the beam to follow the stage; but these methods require modifications of the equipment. When the drift is not excessive, it is possible to correct a series of images after the exposure series is finished. Here we demonstrate two methods for correcting the drift, one based on manual assignment of specimen position and one on the use of cross-correlation functions to determine objectively the misalignment of images in the series. The success of the methods is illustrated in calcium-specific images of a bone section that show the collagen periodicity after drift correction.

Bone and Bones↗
PubMed1989

Crystalloids in salivary gland pleomorphic adenomas.

Two types of crystalloids in salivary gland pleomorphic adenomas were studied by light microscopy and electron microscopy. The first type of crystalloid, the previously described tyrosine-rich crystalloid, was identified in three (1.5%) of 205 cases. The crystalloids by light microscopy assumed a radial configuration, resulting in the characteristic petal-shaped morphology. Transmission electron microscopy revealed them to be electron-dense, lobular projections without internal structure. Scanning electron microscopy demonstrated a range of morphology from rounded and intact doughnutlike structures to aggregates of irregular, loosely cohesive plates. The crystalloids were backscatter positive by backscattered electron imaging, and by x-ray microanalysis exhibited prominent calcium, phosphorus, and magnesium peaks that were not present in the adjacent tumor tissue; these three elements may be important in the formation and structure of tyrosine-rich crystalloids. The second type of crystalloid was intraductal and birefringent and was identified in 26 (12.7%) of 205 cases. In 21 of these 26 cases the crystalloids were lost on 10% formaldehyde fixation and paraffin embedding. Histochemical stains and x-ray microanalysis did not reveal a definite chemical composition, but did suggest a predominantly organic nature.

Adenoma, Pleomorphic↗
PubMed1988

Quantitative microchemical imaging of calcium in Na-K pump inhibited heart cells.

Quantitative electron probe X-ray imaging techniques have been utilized to determine simultaneously the element content within a single cultured embryonic chick heart cell and its intracellular compartments as well as the average elemental content of several heart cells within a population. These features of microchemical imaging have permitted establishment of data regarding: (1) the heterogeneity of calcium accumulation in mitochondrial, cytoplasmic and nuclear compartments under conditions which elevate total cell calcium without producing irreversible cell injury; and (2) the variability of calcium accumulation from cell to cell within the population sampled. The results indicate that during Na-K pump inhibition (K-free HT-BSS, 10(-4) M ouabain, 60 min) elevation of mitochondrial calcium, measured in situ by electron probe X-ray microanalysis, to levels more than 100 times greater than in the basal state, may not cause irreversible mitochondrial uncoupling and cell death.

Calcium↗
PubMed1988

Frontiers in electron probe microanalysis: application to cell physiology.

The application of electron probe microanalysis techniques, using X-ray and electron energy loss instruments, to problems in cell physiology is reviewed. The details of the special methodological requirements for the analysis of cryosections at high spatial resolution in an analytical electron microscope are discussed together with a comprehensive review of data obtained on major organ systems and cell types.

Animals↗
PubMed1988

Microprobe analysis of chlorpromazine pigmentation.

We describe the histochemical, ultrastructural, and microanalytical features of a skin biopsy specimen obtained from a patient with chlorpromazine pigmentation. Golden-brown pigment granules were present in the dermis, predominantly in a perivascular arrangement. The granules stained positively with the Fontana-Masson stain for silver-reducing substances and negatively with Perl's stain for iron. Electron microscopy revealed dense inclusion bodies in dermal histiocytes, pericytes, endothelial cells, and Schwann cells, as well as lying free in the extracellular matrix. These "chlorpromazine bodies" were quite dense even in unosmicated, unstained ultrathin sections, indicating that the pigmentation is related, at least in part, to the inclusions. Microprobe analysis of the chlorpromazine bodies revealed a striking peak for sulfur, which strongly suggests the presence of the drug or its metabolite within these inclusions.

Adult↗
PubMed1986

Heterogeneity of calcium compartmentation: electron probe analysis of renal tubules.

The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) "Viable" cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of approximately 4:1. Their measured Ca content is 4.1 +/- 1.4 (SEM) mmol/kg dry wt in the cytoplasm and 3.1 +/- 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of approximately 3.8 mmol/kg dry wt. 2) "Nonviable" cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of approximately 1. The Ca content is 15 +/- 2 mmol/kg dry wt in the cytoplasm and 685 +/- 139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is approximately 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions (approximately equal to 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in total Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusions in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that the in situ mitochondrial Ca content of viable cells from the kidney proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.

Animals↗
PubMed1985

Microprobe analysis in human pathology.

This tutorial paper reviews the literature on the application of microprobe analysis to practical problems in diagnostic human pathology. The goal is to allow the reader ready access to the literature on specific clinical problems. Specimen preparation and commonly encountered artifacts are also considered. It is concluded that energy dispersive x-ray microanalysis and back-scattered electron imaging are at present the most generally useful microprobe techniques for clinical work, and are no longer solely research tools. The findings often have diagnostic, therapeutic, and/or legal implications.

Body Fluids↗
PubMed1979

Preparative techniques for freezing and freeze-sectioning macrophages for energy dispersive x-ray microanalysis.

In order to study the subcellular distribution of normal intracellular electrolytes and of metal pollutants, rabbit alveolar macrophages and mouse peritoneal macrophages were maintained in standard tissue culture medium with or without various concentrations of cadmium chloride or ammonium vanadate. A variety of preparative techniques were employed to study both monolayers and cell pellets by light microscopy, transmission electron microscopy, scanning electron microscopy and energy-dispersive x-ray microanalysis. Pellets of macrophages centrifuged in narrow bore centrifuge tubes were successfully snap-frozen in liquid-nitrogen-cooled liquid propane and either sectioned on a cryoultramicrotome or freeze-substituted with 1% osmium tetroxide in acetone and embedded in Epon. Spot probes of freeze-dried, frozen thin sections for normal intracellular electrolytes such as potassium, phosphorus and sulfur showed good localization to the cells and differences between organelles. Monolayers were freeze-dried and directly embedded in Epon. When Epon thin sections of these cells and of the freeze-substituted, Epon embedded pellets were obtained with a dry knife, intracellular electrolytes such as potassium, phosphorus and cadmium could still be detected by energy-dispersive x-ray microanalysis. It is concluded that in studies using snap-freezing for element localization, maximum information is obtained with the simultaneous application of a combination of preparatory techniques.

Animals↗