Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy

Stearns, Rebecca C.; Katler, Marshall; Godleski, John J.

doi:10.1002/jemt.1070280207

Cited by 11 publications

(3 citation statements)

References 25 publications

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“…The material is glutaraldehyde-fixed, post-fixed with OsO 4 and embedded in Epon together with Fe-Chelex with the same specifications as described above. Stearns et al (1994) showed good iron preservation (90%) using such liquid fixation techniques. Ultrathin sections were subsequently obtained and collected on 200-mesh unfilmed copper grids.…”

Section: Methodsmentioning

confidence: 90%

Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application

Beckers¹,

Gelsema²,

Bruijn³

et al. 1996

Journal of Microscopy

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Electron spectroscopic imaging (ESI) with the energy-filtering transmission electron microscope enables the investigation of chemical elements in ultrathin biological sections. An analysis technique has been developed to calculate elemental maps and quantitative distributions from ESI sequences. Extensive experience has been obtained with a practical implementation of this technique. A procedure for more robust element detection has been investigated and optimized. With the use of Fe-loaded Chelex beads, the measurement system has been evaluated with respect to the linearity of the element concentration scale, the reproducibility of the measurements and the visual usage of image results. In liver specimens of a patient with an iron storage disease the detectability of iron was tested and we tried to characterize iron-containing components. The concentration measurement scale is approximately linear up to a relative section thickness of approximately equal to 0.5. Monitoring of this parameter is therefore considered to be important. The reproducibility was measured in an experiment with Fe-Chelex. The iron concentration differed by 6.4% between two serial measurements. Element distributions are in many applications interpreted visually. For this purpose the frequently used net-intensity distributions are regarded as unsuitable. For the quantification and visual interpretation of concentration differences mass thickness correction has to be performed. By contrast, for the detection of elements the signal-to-noise ratio is the appropriate criterion. Application of ESI analysis demonstrated the quantitative chemical capabilities of this technique in the investigation of iron storage diseases. Based on an assumed ferritin iron loading in vivo, different iron components can be discerned in liver parenchymal cells of an iron-overloaded patient.

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Section: Methodsmentioning

confidence: 90%

Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application

Beckers¹,

Gelsema²,

Bruijn³

et al. 1996

Journal of Microscopy

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“…The EFTEM took place with the following parameters constant: 80 kV; beam current at 60 A; 20-eV spectrometer slit resolution; 60-m objective aperture; 0.8-mm spectrometer aperture; in image mode. Background noise levels for the system used in these experiments were established by a modified, previously described method (28). A range of three images (428, 438, and 448 eV for Ti; and 514, 504, and 494 eV for O) was used to determine background noise.…”

Section: Examination By Esi and Eelsmentioning

confidence: 99%

Endocytosis of Ultrafine Particles by A549 Cells

Stearns¹,

Paulauskis²,

Godleski³

2001

Am J Respir Cell Mol Biol

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224

108

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Alveolar epithelium's capacity to ingest inhaled ultrafine particles is not well characterized. The objectives of this study were to use an in vitro model of type II lung epithelium and evaluate the cells' ability to take up ultrafine particles (titanium dioxide [TiO(2)], 50 nm diameter). The human epithelial cell line A549 was grown on aclar substrates and exposed to 40 microg/ml TiO(2) particles for 3, 6, and 24 h before imaging with energy-filtering transmission electron microscopy. Elemental mapping and electron energy loss spectroscopy were used to colocalize Ti/O with electron-dense particles. Particle endocytosis was compared in A549 cells with and without pretreatment with cytochalasin D (cyto D) (2 microg/ml). After 3 h of TiO(2) exposure, cells internalized aggregates of the ultrafine particles which were observed in cytosolic, membrane-bound vacuoles. After 24 h of exposure there were considerably more intracellular aggregates of membrane-bound particles, and aggregated particles were also enmeshed in loosely and tightly packed lamellar bodies. Throughout 24 h of exposure a preponderance of particles remained associated with the free surface of the cells and were not internalized. The majority of membrane-bound vacuoles contained aggregates of particles and only occasionally did they contain as few as two or three particles, despite the use of several different approaches to assure the possibility for individual particles to be ingested and detected. There was morphologic evidence of microfilament disturbance, but no evidence of a decrease in internalized particles in cells pretreated with cyto D. Thus, this model of type II epithelium is able to internalize aggregates of ultrafine particles.

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“…Ultrafine particles (UFPs), defined as particles with a diameter < 100 nm, have been hypothesized as contributors to cardiovascular effects of PM (Seaton et al 1995) because, compared with fine particles at similar mass concentrations, they have greater pulmonary deposition efficiency (Chalupa et al 2004; Daigle et al 2003), induce more pulmonary inflammation (Li et al 1999; Oberdörster et al 1995), have enhanced oxidant capacity (Brown et al 2001; Li et al 2003), have a higher propensity to penetrate the epithelium and reach interstitial sites (Stearns et al 1994), and may even enter the systemic circulation in humans (Nemmar et al 2002; Oberdörster et al 2002). …”

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confidence: 99%

Inhalation of Ultrafine Particles Alters Blood Leukocyte Expression of Adhesion Molecules in Humans

Frampton

Stewart²,

Oberdörster

et al. 2006

Environ Health Perspect

124

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Ultrafine particles (UFPs; aerodynamic diameter < 100 nm) may contribute to the respiratory and cardiovascular morbidity and mortality associated with particulate air pollution. We tested the hypothesis that inhalation of carbon UFPs has vascular effects in healthy and asthmatic subjects, detectable as alterations in blood leukocyte expression of adhesion molecules. Healthy subjects inhaled filtered air and freshly generated elemental carbon particles (count median diameter ~ 25 nm, geometric standard deviation ~ 1.6), for 2 hr, in three separate protocols: 10 μg/m3 at rest, 10 and 25 μg/m3 with exercise, and 50 μg/m3 with exercise. In a fourth protocol, subjects with asthma inhaled air and 10 μg/m3 UFPs with exercise. Peripheral venous blood was obtained before and at intervals after exposure, and leukocyte expression of surface markers was quantitated using multiparameter flow cytometry. In healthy subjects, particle exposure with exercise reduced expression of adhesion molecules CD54 and CD18 on monocytes and CD18 and CD49d on granulocytes. There were also concentration-related reductions in blood monocytes, basophils, and eosinophils and increased lymphocyte expression of the activation marker CD25. In subjects with asthma, exposure with exercise to 10 μg/m3 UFPs reduced expression of CD11b on monocytes and eosinophils and CD54 on granulocytes. Particle exposure also reduced the percentage of CD4+ T cells, basophils, and eosinophils. Inhalation of elemental carbon UFPs alters peripheral blood leukocyte distribution and expression of adhesion molecules, in a pattern consistent with increased retention of leukocytes in the pulmonary vascular bed.

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Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy

Cited by 11 publications

References 25 publications

Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application

Quantitative electron spectroscopic imaging in bio‐medicine: evaluation and application

Endocytosis of Ultrafine Particles by A549 Cells

Inhalation of Ultrafine Particles Alters Blood Leukocyte Expression of Adhesion Molecules in Humans

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