Journal
ANNUAL REVIEW OF PHYSICAL CHEMISTRY, VOL 63
Volume 63, Issue -, Pages 595-617Publisher
ANNUAL REVIEWS
DOI: 10.1146/annurev-physchem-032210-103340
Keywords
FRET; single-particle tracking; single-molecule stoichiometry; triplet state; redox blinking; photochromic blinking
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Funding
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [U19AI083025] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM065367] Funding Source: NIH RePORTER
- NIAID NIH HHS [U19 AI083025, AI083025] Funding Source: Medline
- NIGMS NIH HHS [GM065367, R01 GM065367] Funding Source: Medline
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Single-molecule fluorescence spectroscopy and super-resolution microscopy are important elements of the ongoing technical revolution to reveal biochemical and cellular processes in unprecedented clarity and precision. Demands placed on the photophysical properties of the fluorophores are stringent and drive the choice of appropriate probes. Such fluorophores are not simple light bulbs of a certain color and brightness but instead have their own personalities regarding spectroscopic parameters, redox properties, size, water solubility, photostability, and several other factors. Here, we review the photophysics of fluorescent probes, both organic fluorophores and fluorescent proteins, used in applications such as particle tracking, single-molecule FRET, stoichiometry determination, and super-resolution imaging. Of particular interest is the thiol-induced blinking of Cy5, a curse for single-molecule biophysical studies that was later overcome using Trolox through a reducing/oxidizing system but a boon for super-resolution imaging owing to the controllable photoswitching. Understanding photophysics is critical in the design and interpretation of single-molecule experiments.
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