Abstract
In situ labeling of cells within living biological tissues using photoconversion has provided valuable information on cellular physiology in their natural environments. However, current photoconvertible probes typically require seconds to minutes of light exposure, limiting their uses in rapid biological processes such as intracellular diffusion and circulating cells. Here, we report that two-photon photoconversion of cyanine-based dyes offers unprecedentedly rapid photoconversion down to millisecond timescales per cell. We demonstrate potential biological applications including measuring intracellular diffusion kinetics in a spinal nerve, labeling of rapidly flowing cells in a microfluidic channel, and photoconversion of a circulating cell in vivo.
Highlights
Advances in fluorescent probes for optical microscopy have revolutionized our understanding of complex biological systems [1,2,3]
Photoconvertible probes enable in situ labeling of cells of interest by spatially localized light exposure, altering the emission spectrum of the probes [4,5]
We describe the theoretical background of rapid photoconversion mediated by the two-photon process and demonstrate potential biological applications, including measuring intracellular diffusion kinetics in a spinal nerve, and labelling of rapidly flowing cells in a microfluidic channel and in blood circulation in vivo
Summary
Advances in fluorescent probes for optical microscopy have revolutionized our understanding of complex biological systems [1,2,3]. The photoconverted cells of interest can be readily distinguished from non-photoconverted cells for in vivo tracking over large spatiotemporal scales or isolated for further in vitro molecular analyses [6,7,8,9,10]. Because of their unique capabilities, photoconvertible probes are widely utilized. Most conventional photoconvertible probes have low photoconversion yields, usually requiring seconds to minutes of irradiation time to obtain detectable photoconversion [11,12]. The requirement of poorly penetrating UV-blue irradiation prevents efficient photoconversion of cells deep in scattering biological tissues. Applications of photoconvertible proteins have mostly been applicable to nearly stationary or slowly moving cells, such as in 3D cell culture [6], tumors [8], and lymph nodes [13]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
