Biomedical subjects

R D Leapman

Publications and source records attributed to R D Leapman.

At least 37 records · Page 2Linked to original sources

PubMed1995

Quantitative water mapping of cryosectioned cells by electron energy-loss spectroscopy.

A direct technique based on electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has been developed to map subcellular distributions of water in frozen-hydrated biological cryosections. Previously, methods for water determination have been indirect in that they have required the cryosections to be dehydrated first. The new approach makes use of spectrum-imaging, where EELS data are collected in parallel at each pixel. Several operations are required to process the spectra including: subtraction of the detector dark current, deconvolution by the detector point-spread function, removal of plural inelastic scattering and correction for the support film. The resulting single scattering distributions are fitted to standard reference spectra at each pixel, and water content can be determined from the fitting coefficients. Although the darkfield or brightfield image from a hydrated cryosection shows minimal structure, the processed EELS image reveals strong contrast due to variations in water content. Reference spectra have been recorded from the major biomolecules (protein, lipid, carbohydrate, nucleic acid) as well as from vitrified water and crystalline ice. It has been found that quantitative results can be obtained for the majority of subcellular compartments by fitting only water and protein reference spectra, and the accuracy of the method for these compartments has been estimated as +/- 3.5%. With the present instrumentation the maximum allowed dose of 2 x 10(3) e/nm2 limits the useful spatial resolution to around 80 nm for +/- 5% precision at a single pixel. By averaging pixel intensities a value of 56.8% with a precision of +/- 2.0% has been determined for the water content of liver mitochondria. The water mapping technique may prove useful for applications to cell physiology and pathophysiology.

Animals↗

PubMed1994

Quantitative dark-field mass analysis of ultrathin cryosections in the field-emission scanning transmission electron microscope.

The availability of a cryotransfer stage, highly efficient electron energy loss spectrometers, and ultra-thin-window energy-dispersive x-ray spectrometers for the VG Microscopes HB501 field-emission scanning transmission electron microscope (STEM) provides this instrument with the potential for high resolution biological microanalysis. Recent technical advances offer cryosections that are thin enough to take advantage of the analytical capabilities of this microscope. This paper first discusses the quantitative characterization of freeze-dried, ultrathin cryosections of directly frozen liver and brain by low-dose dark-field STEM imaging. Such images reveal high-quality sections with good structural detail, mainly due to reduced preparation artifacts and electron beam damage. These sections are thin enough for dark-field mass analysis, so that the mass of individual organelles can be measured in situ, and their water content deduced. This permits the measurement of mass loss-corrected subcellular elemental concentrations. The results suggest several new applications for cryosections as illustrated by data on synaptic activity-dependent calcium regulation in Purkinje cells of mouse cerebellum. Low-dose mass analysis of cryosections in combination with x-ray and electron spectroscopy is a promising approach to quantitating physiological changes in mass distribution and elemental composition.

Animals↗

PubMed1994

Biological electron energy loss spectroscopy in the field-emission scanning transmission electron microscope.

The dedicated scanning transmission electron microscope (STEM) combined with parallel electron energy loss spectroscopy (EELS) provides a very sensitive means of detecting specific elements in small structures. EELS is more sensitive than optimized energy-dispersive X-ray spectroscopy by a factor of about three for calcium. Measurement of such low concentrations requires special processing methods such as difference-acquisition techniques and multiple least squares procedures for fitting reference spectra. By analyzing data recorded at each pixel in a spectrum-image it is possible to map quantitatively the elemental distributions in a specimen. It is possible to prepare cryosections that are sufficiently thin to avoid excessive plural inelastic scattering so analysis can be performed at 100 keV beam energy. Under optimal conditions, a resolution of 10 nm and detection limits of a few atoms are achievable for elements such as calcium, phosphorus and iron. In the field emission STEM certain types of chemical information can be extracted from biological specimens. Valence EELS has been exploited to measure water distributions in frozen hydrated cryosections.

Animals↗

PubMed1993

Trace elemental analysis at nanometer spatial resolution by parallel-detection electron energy loss spectroscopy.

Parallel-detection electron energy loss spectroscopy (EELS) combined with scanning transmission electron microscopy (STEM) and a field emission source provides an unprecedented sensitivity for elemental microanalysis. By deflecting the energy loss spectrum across a parallel detector and computing the difference spectrum from sequentially collected energy-shifted spectra, the effects due to detector pattern noise are nearly eliminated so that signals less than 0.1% of the background can be readily detected. Measurements on a series of glass standard reference materials show that EELS provides both high spatial resolution and trace sensitivity at the 10 atomic ppm level for a wide range of elements including the alkaline earths, 3-d transition metals, and the lanthanides. For analytical volumes with dimensions of the order of 10 nm, this translates into near-single atom detectability.

Electrons↗

PubMed1993

Single kinesin molecules crossbridge microtubules in vitro.

Kinesin is a cytoplasmic motor protein that moves along microtubules and can induce microtubule bundling and sliding in vitro. To determine how kinesin mediates microtubule interactions, we determined the shapes and mass distributions of squid brain kinesin, taxol-stabilized microtubules (squid and bovine), and adenosine 5'-[beta, gamma-imido]triphosphate-stabilized kinesin-microtubule complexes by high-resolution metal replication and by low-temperature, low-dose dark-field scanning transmission electron microscopy of unfixed, directly frozen preparations. Mass mapping by electron microscopy revealed kinesins loosely attached to the carbon support as asymmetrical dumbbell-shaped molecules, 40-52 nm long, with a mass of 379 +/- 15 kDa. The mass distribution and shape of these molecules suggest that these images represent kinesin in a shortened conformation. Kinesin-microtubule complexes were organized as bundles of linearly arrayed microtubules, stitched together at irregular intervals by cross-bridges typically < or = 25 nm long. The crossbridges had a mass of 360 +/- 15 kDa, consistent with one kinesin per crossbridge. These results suggest that kinesin has a second microtubule binding site in addition to the known site on the motor domain of the heavy chain; this second site may be located near the C terminus of the heavy chains or on the associated light chains. Thus, kinesin could play a role in either crosslinking or sliding microtubules.

Animals↗

PubMed1993

Radiation damage of fluorinated organic compounds measured by parallel electron energy loss spectroscopy.

The sensitivity of fluorinated organic compounds to radiation damage in the electron microscope was measured by electron energy loss spectroscopy (EELS). Five classes of molecules were investigated with fluorine atoms situated on: (1) an aliphatic chain, (2) an aromatic ring, (3) a trifluoromethyl group on an aromatic ring, (4) a trifluoromethyl group on a heterocyclic ring, and (5) a trifluoromethyl group next to a carbonyl group. The damage dose for fluorine loss was found to depend strongly on the position of the fluorine atoms and on specimen temperature. For poly-substituted fluorine on an aliphatic chain, approximately half of the fluorine was retained at doses in excess of 10(7) e/nm2. At room temperature molecules containing trifluoromethyl groups on aromatic or heterocyclic rings were much more sensitive to fluorine loss than compounds having fluorine substituted directly on the rings. This behavior is consistent with the relatively low chemical stability of the trifluoromethyl group in these structures. The rapid loss of fluorine at low electron doses (approximately 10(4) e/nm2) in the trifluoromethyl aromatic compounds was reduced several orders of magnitude by cooling the specimen to liquid-nitrogen temperatures. An understanding of how specific types of fluorinated compounds suffer damage under the electron beam will be important in their potential application as biochemical or pharmacological probes.

Hydrocarbons, Fluorinated↗

PubMed1993

Low-dose thickness measurement of glucose-embedded protein crystals by electron energy loss spectroscopy and STEM dark-field imaging.

Electron energy loss spectroscopy and dark-field imaging in a scanning transmission electron microscope were used to determine the thickness of glucose-embedded crotoxin complex crystals. The results demonstrate the feasibility of identifying protein crystals with a thickness of half a unit cell (12.8 nm) under low-dose and low-temperature conditions. The accuracy of this method is limited by the amount of surface coating of the crystal's embedding glucose used for preserving the high-resolution structure of the protein. The histogram of the crystal thickness distribution and the spread of the anticipated crystal thickness allow us to make an estimate of the uncertainty in the glucose layer thickness. This approach can be incorporated as part of the experimental procedure in the three-dimensional data collection for structure determination of protein crystals with variable thicknesses. The measurement can be done on areas approximately 200 nm in diameter so that crystals of suitable thickness can be pre-selected before the high-resolution data is recorded. Accurate determination of the crystal thickness will optimize the data collection efficiency by avoiding the collection and subsequent analysis of unmatchable data for the three-dimensional reconstruction.

Crotoxin↗

PubMed1993

Measurement of low calcium concentrations in cryosectioned cells by parallel-EELS mapping.

Electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope provides a high sensitivity for microanalysis of certain important biological elements such as calcium whose physiological concentrations in cells are rather low. Application of parallel-EELS mapping to the analysis of freeze-dried cryosections of rapidly frozen tissue provides a means of detecting small amounts of calcium in structures with diameter approximately 50 nm. Detector pattern noise due to channel gain variations can be reduced by acquiring difference spectra at each pixel. By segmenting nitrogen maps that reflect the structure through the protein distribution it is possible to sum spectra from specific compartments. These are then processed by fitting reference spectra for the Ca L23-edge and the carbon background. It has been found that useful data can be collected at 100 keV beam energy from freeze-dried cryosections of cerebellar cortex cut to nominal thickness of 100 nm. The analysis results in a sensitivity of +/- 0.4 mmol Ca/kg dry weight with a total acquisition time of 400 s, a significant improvement over that achievable with energy-dispersive X-ray spectroscopy.

Animals↗

PubMed1993

Elemental composition of secretory granules in pancreatic islets of Langerhans.

We have characterized, by electron probe microanalysis, rapidly frozen cultured rat islets at the level of individual secretory granules. Elemental analysis of thin, dried cryosections showed that beta granules could be distinguished by high Zn, Ca, and S, whereas non-beta (mainly alpha) granules contained elevated P and Mg. Although a single granule type predominated in a particular cell, some rebel granules were found in A cells that had the compositional fingerprint of B cell granules. Zn, which was found in millimolar concentrations in B cell granules, was considered a marker for the insulin storage complex. The data indicate that non-B islet cells in the adult pancreas may produce insulin-containing organelles and that, when glucagon and insulin are coexpressed, these hormones are packaged in separate granules.

Animals↗

PubMed1992

Characterization of biological macromolecules by combined mass mapping and electron energy-loss spectroscopy.

The combination of scanning transmission electron microscopy (STEM) and parallel-detection energy-loss spectroscopy (EELS) was used to detect specific bound elements within macromolecules and macromolecular assemblies prepared by direct freezing. After cryotransferring and freeze-drying in situ, samples were re-cooled to liquid nitrogen temperature and low-dose (about 10(3) e/nm2) digital dark-field images were obtained with single-electron sensitivity using a beam energy of approximately 100 keV and a probe current of approximately 5 pA. These maps provided a means of characterizing the molecular weights of the structures at low dose. The probe current was subsequently increased to about 5 nA in order to perform elemental analysis. The 320 copper atoms in a keyhole limpet haemocyanin molecule (mol.wt = 8 MDa) were detected with a sensitivity of +/- 30 atoms in an acquisition time of 200 s. Phosphorus was detected in an approximately 10-nm length of single-stranded RNA contained in a tobacco mosaic virus particle (mol.wt = 130 kDa/nm) with a sensitivity of +/- 25 atoms. Near single-atom sensitivity was achieved for the detection of iron in one haemoglobin molecule (mol.wt = 65 kDa, containing four Fe atoms). Such detection limits are only feasible if special processing methods are employed, as is demonstrated by the use of the second-difference acquisition technique and multiple least-squares fitting of reference spectra. Moreover, an extremely high electron dose (about 10(10) e/nm2) is required resulting in mass loss that may be attributable to 'knock-on' radiation damage.

Animals↗

PubMed1991

Analysis of directly frozen macromolecules and tissues in the field-emission STEM.

A VG Microscopes HB501 field-emission high-resolution scanning transmission electron microscope (STEM) was used to image and analyse rapidly frozen, isolated macromolecules and small organelles in tissue cryosections. Dark-field images were obtained from frozen-hydrated microtubules demonstrating that sufficient contrast is available to reveal structural information. The samples were subsequently freeze-dried in the STEM and low-dose (approximately 10(3) e/nm2) dark-field mass maps were recorded with single electron sensitivity. Elemental analysis of individual macromolecules was achievable at high dose using parallel-detection electron energy-loss spectroscopy, albeit with some structural degradation. Detection of copper (320 atoms) in di-decameric haemocyanin molecules was easily within the limits of sensitivity. Elemental analysis of hydrated cryosections is limited by radiation damage to a resolution of approximately 1 micron2. For freeze-dried sections, however, the high probe current and stable cold stage of the HB501 STEM allow energy-dispersive X-ray (EDX) microanalysis of low elemental concentrations in highly localized subcellular volumes. EDX spectra from cryosections of cerebellar cortex show that a 100-s analysis time is sufficient to quantify the calcium content of 400-nm2 regions within Purkinje cell dendrites with an uncertainty of +/- 2 mmol/kg dry weight, equivalent to +/- 12 atoms.

Animals↗

PubMed1988

Electron probe microanalysis of the subcellular compartments of bovine adrenal chromaffin cells. Comparison of chromaffin granules in situ and in vitro.

The elemental and water content of cultured bovine adrenal chromaffin cells and their secretory chromaffin granules have been measured and compared with isolated chromaffin granules using quick freezing, ultracryomicrotomy, and electron microprobe analysis methods. In units of millimole/kilogram dry weight (+/- S.E.) granules in situ contained: P, 523 +/- 32; K+, 124 +/- 9; S, 82 +/- 3; Cl-, 74 +/- 9; Ca2+, 13 +/- 2; Mg2+, 6 +/- 2; and Na+, -2 +/- 2. Following routine isolation in isotonic sucrose buffer, granule K and Cl- had decreased while granule Na+ increased. Cl- exhibited a consistent decrease to 35-40 mmol/kg dry weight. Granule Na+ and K+ concentrations ranged from 43 to 12 mmol/kg and 28 to 60 mmol/kg dry weight, respectively, depending on the Na+ and K+ content of the buffer. Despite the redistribution of monovalent ions, granule Ca2+, granule P, being in the form of ATP, and granule S, being in the form of protein, were not significantly changed. The stability of these elements is consistent with the existence of a stable storage complex for Ca2+, ATP, and protein. Using the granule as an internal standard with a water content of 66%, the water contents of external space, nucleus, cytoplasm, and mitochondria were estimated to be 89, 88, 82, and 70%, respectively. Wet weight concentrations for each element were calculated for granules and cytoplasm from which the transgranular concentration gradients for K+, Cl-, and Na+ were determined. Cl-, a permeant anion, was 2-fold higher in the granule than in the cytoplasm while K+, a slightly permeant cation, had an opposite distribution ratio slightly less than two. Together, the K+ and Cl- data suggest the presence of an inside-positive granule membrane potential of approximately 10-16 mV. The surprising lack of Na+ from the granule matrix suggests a hugh inward gradient for Na+ even though the Na+ content of chromaffin cell cytoplasm is low at 5 mmol/kg water. The lack of an outward Na+ gradient is important in that it indicates that the previously described electroneutral Na+-Ca2+ exchange system, by which isolated granules accumulate Ca2+, does not operate in mature granules in situ. Consequently, if chromaffin granules regulate internal calcium during stimulus secretion coupling, a mechanism other that Na+-Ca2+ exchange is necessary.

Adenosine Triphosphate↗

PubMed1988

Trypanosoma cruzi: elemental composition heterogeneity of cloned stocks.

Concentrates of the epimastigote stage of Trypanosoma cruzi stocks derived from single cell clones and cultured under identical conditions display a spectrum of 'colors' varying from dark brown to milk white. The color of the concentrate is reproducible for a parasite stock. An essential component of the culture medium for epimastigote growth is hemin, an iron-containing compound. Consequently, it seemed possible that the color spectrum of the epimastigote stocks reflected differences in the uptake, concentration or utilization of iron. This report describes the quantitative studies utilizing electron probe X-ray elemental mapping, energy dispersive X-ray microanalysis, and energy dispersive X-ray fluorescence spectrometry of the epimastigote stage of two T. cruzi stocks (CA-I/72 and HO-3/15) which display extreme color differences. Striking and statistically significant quantitative differences were found in the levels of Fe, Zn, and K between the two stocks. On the basis of atomic ratios, the CA-I/72 stock contains approximately two-fold more Fe per cell than the HO-3/15 stock. However, in the case of Zn the ratio is reversed; the HO-3/15 stock contains approximately two-fold more Zn per cell than the CA-I/72 stock. The marked inter-stock differences which exist in the levels of several elements could modulate the pathogenicity, survival, or adaptability of T. cruzi and, consequently, be important factors in our understanding of the complex problem of Chagas' disease.

Animals↗

PubMed1988

Separation of overlapping core edges in electron energy loss spectra by multiple-least-squares fitting.

A multiple-least-squares fitting procedure for quantitating electron energy loss spectra is demonstrated on some strongly overlapping core edges. The method, first applied by Shuman and Somlyo [Ultramicroscopy 21 (1987) 23], takes into account plural inelastic scattering and can be applied under conditions of non-uniform sample thickness where Fourier deconvolution techniques are invalid. By using appropriate reference spectra generated from pure compounds, quantitation of potassium and calcium (L23 edges) is possible in the presence of carbon (K edge), and sulfur in the presence of phosphorus (L23 edges). Some of the advantages and limitations of the multiple-least-squares approach are discussed.

Algorithms↗

PubMed1988

Quantitative X-ray mapping of biological cryosections.

The potential for applying X-ray mapping to the elemental microanalysis of biological cryosections is discussed. Methods are described for acquiring and processing data, including use of the top-hat digital filter to remove the average effects of the background contribution. Practical considerations for X-ray mapping are discussed in terms of typical counts per pixel and minimum detectability which depends on the number of pixels chosen to integrate the signal. These aspects are illustrated with elemental maps (Na, P, K, Ca and Fe) from freeze-dried cryosections of mouse cerebellar cortex. A calcium sensitivity in the range 0.5 to 2.5 mmol/kg wet weight of tissue is demonstrated. The correction for overlap of potassium K beta and calcium K alpha is demonstrated with X-ray maps from cryosectioned synaptosomes of squid optic lobe. Quantitative results obtained using internal standards to determine wet weight concentrations are in reasonable agreement with expected values. Alternate schemes applicable to X-ray maps for determining the dry mass concentration, such as the peak/continuum (Hall method), are also discussed.

Electron Probe Microanalysis↗

PubMed1988

Quantitative electron energy loss spectroscopy in biology.

The potential for applying electron energy loss spectroscopy (EELS) in biology is assessed. Some recent developments in instrumentation, spectrometer design, parallel detection and elemental mapping are discussed. Quantitation is demonstrated by means of the spectrum from DNA which gives an elemental ratio for N:P close to the expected value. A range of biologically important elements that can be usefully analyzed by EELS is tabulated and some possible applications for each are indicated. Detection limits and the effects of radiation damage are illustrated by spectra from the protein, insulin, and from the fluorinated amino-acid, histidine. Calcium detectability under optimum conditions may be as low as 1 mmol/kg dry weight. The application of EELS to analysis of cryosectioned adrenomedullary (chromaffin) cells is described in order to help determine the composition of the secretory granule. Water content can be determined from the amount of inelastic scattering as measured by the low-loss spectrum. The nitrogen/phosphorus ratio can be measured to provide information about the relative concentrations of ATP, chromogranin, and catecholamines. Quantitative EELS elemental maps are obtained in the STEM mode from chromaffin cells in order to measure the distribution of light elements.

Body Water↗

PubMed1988

Activity-dependent accumulation of calcium in Purkinje cell dendritic spines.

The calcium content of synapses of parallel fibers on Purkinje cell dendritic spines was determined by electron probe x-ray microanalysis of freeze-dried cryosections from directly frozen slices of mouse cerebellar cortex. In fresh slices frozen within 20-30 sec of excision, calcium concentrations ranging from 0.8 to 18.6 mmol/kg of dry weight were measured in cisterns of smooth endoplasmic reticulum within Purkinje cell dendritic spines. The average calcium content of spine cisterns in rapidly excised slices (6.7 +/- 0.6 mmol/kg of dry weight +/- SEM) was higher than the average calcium content of spine cisterns in brain slices incubated without stimulation for 1-2 hr before direct freezing (2.5 +/- 0.4 mmol/kg of dry weight). Depolarization of incubated cerebellar slices by isotonic 55 mM KCl resulted in the accumulation within spine cisterns of very high amounts of calcium or isotonically substituted strontium, both derived from the extracellular fluid. These results suggest that one function of spine cisterns is to sequester free calcium that enters the spine through ligand-gated or voltage-gated channels during synaptic transmission.

Animals↗

PubMed1987

Distribution of calcium and potassium in presynaptic nerve terminals from cerebellar cortex.

The elemental composition of the presynaptic nerve terminals in rapidly frozen synapses of the cerebellar molecular layer was determined by electron probe x-ray microanalysis and elemental imaging of characteristic x-rays. Elemental imaging of thin freeze-dried cryosections from fresh cerebellar slices frozen within 20 sec of removal from the brain showed normal concentrations of potassium (95 +/- 6 mmol/liter wet tissue +/- SEM) and calcium (0.8 +/- 0.4 mmol/liter) in whole presynaptic terminals, even though mitochondrial and nonmitochondrial sites containing up to 30 mmol of calcium per liter were present elsewhere in the neuropil. Quantitative electron probe analysis of synaptic vesicle clusters and intraterminal mitochondria indicated that their calcium concentrations were 0.4 +/- 0.1 and 1.2 +/- 0.2 mmol/liter, respectively. The low calcium content of presynaptic organelles was confirmed by the absence of detectable deposits in preparations freeze-substituted so as to stabilize calcium content. Similar experiments were carried out on cerebellar slices rapidly frozen after incubation in vitro. The distribution of potassium and calcium in presynaptic terminals of resting and depolarized (55 mM potassium) slices was qualitatively and quantitatively similar to that in freshly excised cortex, although resting slices lacked the few calcium-rich sites that appeared in other areas of the neuropil after stimulation. The calcium concentrations in whole terminals, synaptic vesicles, and mitochondria of resting slices were 1.4 +/- 0.7, 0.7 +/- 0.2, and 0.9 +/- 0.2 mmol/liter, respectively. Thus, amounts of calcium typical of storage organelles in other tissues are not present within cerebellar synaptic vesicles, suggesting that they have a limited role in calcium storage and release.

Animals↗