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Proc Natl Acad Sci U S A. November 18, 2008; 105 (46): 17789-94.

Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells.

Marriott G , Mao S , Sakata T , Ran J , Jackson DK , Petchprayoon C , Gomez TJ , Warp E , Tulyathan O , Aaron HL , Isacoff EY , Yan Y .

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

PubMed ID: 19004775
PMC ID: PMC2584753
Article link: Proc Natl Acad Sci U S A.
Grant support: 5PN2EY018241 NEI NIH HHS , R01EB005217 NIBIB NIH HHS , R01NS050833 NINDS NIH HHS , PN2 EY018241-05 NEI NIH HHS , PN2 EY018241 NEI NIH HHS , R01 NS041564 NINDS NIH HHS

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