期刊
ACS PHOTONICS
卷 8, 期 9, 页码 2666-2673出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsphotonics.1c00685
关键词
fluorescence; nonlinear; reversibly photoswitchable fluorescent proteins; visible-wavelength two-photon excitation; confocal detection; super-resolution microscopy; cell imaging
类别
资金
- Japan Science and Technology Agency [JPMJCR15N3]
- JST COI-NEXT [JPMJPF2009]
- Japan Society for the Promotion of Science [18H05410]
This study utilized two-photon excitation to activate negatively switching reversibly photoswitchable fluorescent proteins, introducing nonlinear relationships to enhance spatial resolution in laser scanning microscopy. Experimental confirmation showed that certain RSFPs can be activated within a specific wavelength range for observation using confocal scanning microscopy on HeLa cells.
Nonlinear optical effects modify the point spread function (PSF) in laser scanning microscopy and have been utilized to enhance the spatial resolution in three dimensions. In this paper, we propose the use of visible-wavelength two-photon excitation to activate negatively switching reversibly photoswitchable fluorescent proteins (RSFPs) to introduce nonlinear relationships between the distribution of excitation light and the fluorescence emission. Single-photon excitation following the two-photon induced photoactivation provides a PSF corresponding to the cube of the excitation intensity distribution and achieves imaging properties equivalent to that using third-order nonlinear optical effects. We experimentally confirmed several species of negatively switching RSFPs can be activated by two-photon excitation in the wavelength range of 530-560 nm. We applied the cubic PSF imaging to the observation of HeLa cells stained by Skylan-NS to confirm the improvement of the spatial resolution and the image contrast in confocal scanning microscopy.
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