Probing single molecules in single living cells.
Single-molecule detection in single living cells has been achieved by using confocal fluorescence microscopy and externally tagged probe molecules. The intracellular background fluorescence is substantially higher than that in aqueous buffer, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecule photon bursts. As a result, single-molecule data have been obtained on three types of fluorescent probes at spatially resolved locations (e.g., cytoplasm and nucleus) inside human HeLa cells. First, the iron transport protein transferrin labeled with tetramethylrhodamine undergoes rapid receptor-mediated endocytosis, and single transferrin molecules are detected inside living cells. Second, the cationic dye rhodamine 6G (R6G) enters cultured cells by a potential-driven process, and single R6G molecules are observed as intense photon bursts when they move in and out of the intracellular laser beam. Third, we report results on synthetic oligonucleotides that are tagged with a fluorescent dye and are taken up by living cells via a passive, nonendocytic pathway.
Byassee
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Department of Chemistry, Indiana University, Bloomington 47405, USA.
Chan
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Nie
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D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D015335:Molecular Probes
D015335:Molecular Probes