Nonlinear confocal microscopy using visible-wavelength two-photon activation of fluorescent proteins

Toshiki Kubo; Kenta Temma; Kazunori Sugiura; Hajime Shinoda; Kai Lu; Nicholas I. Smith; Tomoki Matsuda; Takeharu Nagai; Katsumasa Fujita

doi:10.1117/12.2608548

7 March 2022 Nonlinear confocal microscopy using visible-wavelength two-photon activation of fluorescent proteins

Toshiki Kubo, Kenta Temma, Kazunori Sugiura, Hajime Shinoda, Kai Lu, Nicholas I. Smith, Tomoki Matsuda, Takeharu Nagai, Katsumasa Fujita

Author Affiliations +

Proceedings Volume PC11965, Multiphoton Microscopy in the Biomedical Sciences XXII; PC119650S (2022) https://doi.org/10.1117/12.2608548
Event: SPIE BiOS, 2022, San Francisco, California, United States

Abstract

We propose the use of visible-wavelength two-photon excitation (v2PE) for activation of reversibly photo-switchable fluorescent proteins (RSFPs) and successive confocal detection to achieve super-resolution imaging. In this method, three photons interact with the sample molecules in total, which provides imaging properties equivalent to using third-order nonlinearity in fluorescence response. Because this technique uses visible light, it can achieve higher spatial resolution than confocal microscopy. In this study, we performed experimental investigations to confirm the activation of negative RSFPs by v2PE and demonstrated super-resolution imaging of live cells.

Conference Presentation

Citation Download Citation

Toshiki Kubo, Kenta Temma, Kazunori Sugiura, Hajime Shinoda, Kai Lu, Nicholas I. Smith, Tomoki Matsuda, Takeharu Nagai, and Katsumasa Fujita "Nonlinear confocal microscopy using visible-wavelength two-photon activation of fluorescent proteins", Proc. SPIE PC11965, Multiphoton Microscopy in the Biomedical Sciences XXII, PC119650S (7 March 2022); https://doi.org/10.1117/12.2608548

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