Sampling efficiency of a single-cell capillary electrophoresis system

被引:10
作者
Brown, Robert B.
Audet, Julie
机构
[1] Univ Toronto, Inst Biomat & Biomed Engn, Toronto, ON M5S 3G9, Canada
[2] Univ Toronto, Terrence Donnelly Ctr Cellular & Biomol Res CCBR, Toronto, ON, Canada
关键词
capillary electrophoresis; single-cell analysis; cell-sampling efficiency; cell lysis; pulsed lasers; green fluorescent protein;
D O I
10.1002/cyto.a.20448
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Capillary electrophoresis (CE) combined with a laser-induced fluorescence (LIF) detection scheme is a powerful approach for single-cell analysis. For measurements requiring a high temporal resolution, CE-LIF is often combined with cell lysis systems based on pulsed lasers. Although extremely rapid, laser lysis has raised some concerns about the efficiency at which the cell contents are sampled. We have assembled a single-cell CE-LIF mounted on the stage of a microscope. This system was coupled with a nanosecond pulsed laser for cell lysis. We have analyzed green fluorescent protein (GFP) expressed in single mammalian cells and developed a novel approach to estimate the cell sampling efficiency (SE) based on the use of fluorescent calibration microspheres and flow cytometry. A significant advantage of this method is that it does not require any knowledge or assumption regarding the cell volume. We have evaluated the SE for different laser pulse energies (from 2 to 9 mu J) and two different pulse focal positions in the xy plane (0-10 mu m from the center of the cell). We found the maximum SE at the lowest energy (2 mu J), with the pulse focused directly on the cell. We have demonstrated the utility of a novel method to measure the SE of a single-cell CE system. The measurements presented in this study indicate that rapid cell lysis with nanosecond lasers requires careful optimization of pulse parameters for maximum sampling of the cell contents. (C) 2007 International Society for Analytical Cytology.
引用
收藏
页码:882 / 888
页数:7
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