Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells.
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.
Marriott
Gerard
G
Department of Physiology, University of Wisconsin, 1300 University Avenue, Madison, WI 53705, USA. marriott@physiology.wisc.edu
Mao
Shu
S
Sakata
Tomoyo
T
Ran
Jing
J
Jackson
David K
DK
Petchprayoon
Chutima
C
Gomez
Timothy J
TJ
Warp
Erica
E
Tulyathan
Orapim
O
Aaron
Holly L
HL
Isacoff
Ehud Y
EY
Yan
Yuling
Y
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
D000199:Actins; D005456:Fluorescent Dyes