Few to single molecule imaging of fluorescent probe molecules can provide information on the distribution, dynamics, interactions and activity of specific fluorescently tagged proteins during cellular processes. Unfortunately, these imaging studies are made challenging in living cells because of fluorescence signals from endogenous cofactors. Moreover, related background signals within multi-cell systems and intact tissue are even higher and reduce signal contrast even for ensemble populations of probe molecules. High-contrast optical imaging within high-background environments will therefore require new ideas on the design of fluorescence probes, and the way their fluorescence signals are generated and analysed to form an image. To this end, in the present review we describe recent studies on a new family of fluorescent probe called optical switches, with descriptions of the mechanisms that underlie their ability to undergo rapid and reversible transitions between two distinct states. Optical manipulation of the fluorescent and non-fluorescent states of an optical switch probe generates a modulated fluorescence signal that can be isolated from a larger unmodulated background by using OLID (optical lock-in detection) techniques. The present review concludes with a discussion on select applications of synthetic and genetically encoded optical switch probes and OLID microscopy for high-contrast imaging of specific proteins and membrane structures within living systems.
Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems
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Yuling Yan, M. Emma Marriott, Chutima Petchprayoon, Gerard Marriott; Optical switch probes and optical lock-in detection (OLID) imaging microscopy: high-contrast fluorescence imaging within living systems. Biochem J 1 February 2011; 433 (3): 411–422. doi: https://doi.org/10.1042/BJ20100992
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