title: Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells.
abstract: 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.
authors.affiliation: Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53705, USA. marriott@physiology.wisc.edu
authors.forename: Gerard
authors.initials: G
authors.lastname: Marriott
authors.forename: Shu
authors.initials: S
authors.lastname: Mao
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Sakata
authors.forename: Jing
authors.initials: J
authors.lastname: Ran
authors.forename: David K
authors.initials: DK
authors.lastname: Jackson
authors.forename: Chutima
authors.initials: C
authors.lastname: Petchprayoon
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Gomez
authors.forename: Erica
authors.initials: E
authors.lastname: Warp
authors.forename: Orapim
authors.initials: O
authors.lastname: Tulyathan
authors.forename: Holly L
authors.initials: HL
authors.lastname: Aaron
authors.forename: Ehud Y
authors.initials: EY
authors.lastname: Isacoff
authors.forename: Yuling
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000199:Actins; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast / Q000379:methods*; D009132:Muscles / Q000166:cytology; D041681:NIH 3T3 Cells; D009474:Neurons / Q000166:cytology; D051381:Rats; D014981:Xenopus; D015027:Zebrafish
chemical_list: D000199:Actins; D005456:Fluorescent Dyes