Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yasushi; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

doi:10.1016/j.jsb.2010.01.005

Cited by 171 publications

(151 citation statements)

References 43 publications

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“…The ASEM enables direct observation of subcellular structures and the localization of proteins of interest in wet cells at SEM resolution (Murai et al, 2011). The observable specimen thickness is 2-3 mm from the silicon nitride (SiN) film (Nishiyama et al, 2010). We labeled CD44 on the cell surface of MbCD-treated or untreated cells with Nanogold, followed by gold enhancement.…”

Section: Mbcd Upregulates the Ha-binding Ability Of Cd44mentioning

confidence: 99%

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Murai

Sato

et al. 2013

Journal of Cell Science

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SummaryThe adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-b-cyclodextrin (MbCD), which disintegrates lipid rafts, i.e. cholesterol-and sphingolipid-enriched membrane microdomains. Increasing concentrations of MbCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.

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Section: Mbcd Upregulates the Ha-binding Ability Of Cd44mentioning

confidence: 99%

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Murai

Sato

et al. 2013

Journal of Cell Science

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“…A new approach was introduced recently, to image whole cells with labeled proteins in liquid state using STEM [1][2][3] , or correlative fluorescence microscopy and STEM [4][5][6][7] . This methodology is capable of studying the spatial distribution of membrane proteins and protein complexes including their stoichiometry at the single molecule level in the intact plasma membranes of whole cells.…”

Section: Introductionmentioning

confidence: 99%

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

Peckys

Jonge

2015

JoVE

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This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

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“…In situ ESEM will probably be used to overcome technical and fundamental challenges in many scientific domains. The recent developments of a high temperature stage in the FIB (Fielden, 2008), a new tomography mode in the ESEM (Jornsanoh et al, 2011) and of the atmospheric scanning electron microscope (Nishiyama et al, 2010 ;Suga et al, 2011) can be cited as examples for this future.…”

Section: Discussionmentioning

confidence: 99%

In Situ Experiments in the Scanning Electron Microscope Chamber

Podor¹,

Ravaux²,

Brau³

2012

Scanning Electron Microscopy

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Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film

Cited by 171 publications

References 43 publications

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Membrane cholesterol modulates the hyaluronan-binding ability of CD44 in T lymphocytes and controls rolling under shear flow

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

In Situ Experiments in the Scanning Electron Microscope Chamber

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