Abstract
The introduction of specific molecules into live cells is a widely used approach to probe cellular mechanisms. Recently, we have reported on the sustained dosing of molecules into single cells via a microscopic diffusion port. Here we describe temporal ratiometry, a method to reconstruct intracellular concentration distribution of the delivered molecules as it varies in time during dosing. To characterize this method, we analyzed fluorescence intensity maps obtained during delivery of Lucifer Yellow CH, LY, a polar fluorophore into A7r5 vascular smooth muscle cells, normal rat kidney epithelial cells (NRKE), and MCF-7 human breast cancer cells. Temporal ratiometry indicates a linear increase in concentration of LY in these cells with a nearly uniform distribution during 20 min of delivery. The method cancels the effects of varying cell height and variable accessible volume on the measured intensities at different locations within the cell. Temporal ratiometry will be useful to estimate dynamic changes in intracellular concentration distributions and thus, facilitate the understanding of transport, binding, sequestration, and efflux of molecules introduced into cells.
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Acknowledgments
We thank Drs. George Dubyak, Maria Hatzoglou, and Ruth Keri for the gift of A7r5, NRKE, and MCF-7 cells, respectively, and Dr. Sumitha Nair for critical comments and useful suggestions on the manuscript. This work was partly supported by the National Science Foundation, USA Grant 0352443.
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Oruganti, P., Gratzl, M. Temporal ratiometry to assess dynamic concentration distributions of fluorescent molecules in single live cells during continuous diffusional dosing. Anal Bioanal Chem 395, 449–456 (2009). https://doi.org/10.1007/s00216-009-2961-z
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DOI: https://doi.org/10.1007/s00216-009-2961-z