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Probing Single Molecules in Single Living Cells

Published online by Cambridge University Press:  02 July 2020

Tyler A. Byassee
Affiliation:
Dept. of Chemistry, Indiana University, Bloomington, IN, 47405
Warren C. W. Chan
Affiliation:
Dept. of Chemistry, Indiana University, Bloomington, IN, 47405
Shuming Nie
Affiliation:
Dept. of Chemistry, Indiana University, Bloomington, IN, 47405
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Abstract

Direct observation of single molecules and single molecular events inside living cells could dramatically improve our understanding of basic cellular processes (e.g., signal transduction and gene transcription) as well as improving our knowledge on the intracellular transport and fate of therapeutic agents (e.g., antisense RNA and gene therapy vectors). However, a key remaining question is whether single-molecule methodologies could be developed to study complex molecular processes in living cells. in contrast to clean and well-controlled conditions in-vitro, the intracellular environment contains a broad collection of biological macromolecules and fluorescent materials such as porphyrins and flavins. This complex environment is known to produce intense background fluorescence, commonly known as autofluorescence. Thus, a major concern is that this intracellular background could overwhelm the relatively weak signals arising from single molecules.

We demonstrate that fluorescence detection of single molecules can be achieved by tightly focusing a laser beam into a living cell (see Figure 1). The observed background fluorescence is indeed higher than that in-vitro (e.g., pure biological buffer), but this background is continuous and stable, and does not significantly interfere with the measurement of single-molecule photon bursts. Specifically, we report single-molecule results on three types of extrinsic fluorescent molecules in cultured human HeLa cells (a cervical cancer cell line).

Type
Bridging the Gap Between Structural and Molecular Biology (Organized by B. Herman)
Copyright
Copyright © Microscopy Society of America 2001

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References

references

(1)Taylor, D. L. and Wang, Y. L. (Eds), Fluorescence Microscopy of Living Cells and Cultures, Parts A and B, Academic Press, New York (1989).Google Scholar
(2)Femino, A. M.et al., Science 280 (1998) 585.CrossRefGoogle Scholar
(3)Carrington, W. A.et al., Science 268 (1995) 1483.CrossRefGoogle Scholar
(4)Brock, R.et al., Proc. Natl. Acad. Sci. 96 (1999) 10123.CrossRefGoogle Scholar
(5)Sokol, D. L.et al., Proc. Natl. Acad. Sci. USA 95 (1998) 11538.CrossRefGoogle Scholar
(6)Schutz, G. J.et al., EMBO J. 19 (2000), 892.CrossRefGoogle Scholar
(7)Sako, Y., Minoghchi, S., and Yanagida, T.. Nat. Cell Biol. 2 (2000) 168.CrossRefGoogle Scholar