Christine Cecala , Stanislav S. Rubakhin , Jennifer M. Mitchell , Martha U. Gillette and Jonathan Sweedler
Single-cell measurements allow a unique glimpse into cell to cell heterogeneity; even small changes in selected cells can have a profound impact on an organism’s physiology. Here an integrated approach to single-cell sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively-fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydooxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10-14 mol to 10-16 mol, which is an order of magnitude lower than observed in untreated pinealocytes.
DOI
Single-cell measurements allow a unique glimpse into cell to cell heterogeneity; even small changes in selected cells can have a profound impact on an organism’s physiology. Here an integrated approach to single-cell sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively-fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydooxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10-14 mol to 10-16 mol, which is an order of magnitude lower than observed in untreated pinealocytes.
DOI
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