Imaging of Freeze-Fractured Cells with in Situ Fluorescence and Time-of-Flight Secondary Ion Mass Spectrometry

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• 作者：Thomas P. Roddy ; Donald M. Cannon ; Jr. ; Chad A. Meserole ; Nicholas Winograd ; and Andrew G. Ewing
• 刊名：Analytical Chemistry
• 出版年：2002
• 出版时间：August 15, 2002
• 年：2002
• 卷：74
• 期：16
• 页码：4011 - 4019
• 全文大小：884K
• 年卷期：v.74,no.16(August 15, 2002)
• ISSN：1520-6882

文摘

Bioanalytical imaging techniques have been employed toinvestigate cellular composition at the single-cell andsubcellular regimes. Four imaging modes have beenperformed sequentially in situ to demonstrate the utilityof a more integrated approach to imaging cells. Thecombination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging ofnative biomolecules. Bright-field microscopy provides ablurred visualization of cells in frozen-hydrated samples,while scanning ion imaging provides a morphological viewof freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels,fluorescence microscopy allows single mammalian cellsto be located in the complex ice matrix of freeze-fracturedsamples, a task that has not been routine with eitherbright-field or TOF-SIMS. A fluorescent label, DiI (m/z834), that does not interfere with the mass spectra ofmembrane phosphatidylcholine, has been chosen forfluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopyallows the distinction of single cells from ice and othersample debris, previously not possible with bright-fieldor scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids,even in the membrane of a single 15-m rat pheochromocytoma cell. The utility of mapping lipids in themembranes of single cells using this integrated approachwill provide more understanding of the functional role ofspecific lipids in functions of cellular membranes.