Thickness measurement of hydrated and dehydrated cryosections by EELS

Shi, Shanling; Sun, Songquan; Andrews, S. Brian; Leapman, Richard D.

doi:10.1002/(sici)1097-0029(19960215)33:3<241::aid-jemt1>3.0.co;2-t

Cited by 26 publications

(11 citation statements)

References 27 publications

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“…Under optimal conditions the EELS system can detect about 2.2 mmol/kg dry weight Ca in a 730--thick biological specimen (Ho et al 1999). Although the absolute thickness of the sections was not available in our study, the relative thickness t/l, where l is a function of the specimen composition, especially of density (Yang and Egerton 1995;Shi et al 1996), was maintained below 0.5, the recommended limit for analytical purposes (Beckers et al 1996). Taking these precautions, scarce calcium signals were recorded from osteoblasts maintained in buffer, as reported by other investigators.…”

Section: Discussionsupporting

confidence: 62%

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Bréchot¹,

Guerquin‐Kern

Lieberherr³

et al. 2004

Histochemistry and Cell Biology

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Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca(2+)](i)) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca(2+)](i). Cryomethods, quick-freezing and freeze-drying, and OsO(4) vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.

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Section: Discussionsupporting

confidence: 62%

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Bréchot¹,

Guerquin‐Kern

Lieberherr³

et al. 2004

Histochemistry and Cell Biology

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“…Figure 2 shows representative images from sections prepared with nominal thicknesses ranging from 60 to 20 nm. We use the term ‘nominal thickness’ to refer to the fact that we have not yet determined the thickness of these sections independently, and expect that there will be some difference between the values set on the microtome and the true thickness (Shi et al ., 1996). Several trends are evident in this set of images.…”

Section: Resultsmentioning

confidence: 99%

Direct visualization of receptor arrays in frozen‐hydrated sections and plunge‐frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

Zhang

Bos

Heymann

et al. 2004

Journal of Microscopy

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SummaryWe have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60-kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween-80 display hexagonally close-packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge-freezing, and (ii) frozen-hydrated sections obtained from cells frozen under high pressure. The frozenhydrated sections were generated by sectioning at − 150 ° C using a diamond knife with a 25 ° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen-hydrated sections and the arrangements found in negatively stained membrane extracts and plunge-frozen cells, highlighting the potential of using frozen-hydrated sections for the study of macromolecular assemblies within cells under near-native conditions.

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“…The degree of compression that we observed in sections of frozen-hydrated rat liver tissue cut with a 35° knife falls in the range 30-50%, with the greatest compression observed in sections thinner than 100 nm. Reports in the literature for specimens such as liver tissue, plant cells, yeast, bacteria, and skin Chang et al, 1983;Michel et al, 1992;Richter, 1994;Shi et al, 1996) indicate a similar range of compression for each of these specimens. Al-Amoudi and colleagues (2003) found that compression in frozen-hydrated sections may be reduced by the use of an oscillating diamond knife, but only for a small fraction of sections.…”

Section: Section Compressionmentioning

confidence: 99%

“…Key technical contributions have come from several laboratories, including those of Dubochet Chang et al, 1983), Mueller (Michel et al, 1991(Michel et al, , 1992, Lepault (Erk et al, 1998), Somlyo (Somlyo et al, 1977(Somlyo et al, , 1985, and Leapman (Shi et al, 1996). This work is detailed in a comprehensive review by Sitte (1996).…”

Section: Introductionmentioning

confidence: 99%

Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections

Hsieh

Leith

Mannella

et al. 2006

Journal of Structural Biology

131

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Thickness measurement of hydrated and dehydrated cryosections by EELS

Cited by 26 publications

References 27 publications

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Direct visualization of receptor arrays in frozen‐hydrated sections and plunge‐frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections

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