Combining multi-site FRAP and HILO-TIRF microscopy using a spatial light modulator
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Date
2025
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Upadhya, A.
Lim, Y.J.
Lee, W.M.
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Optics Express, 2025; 33(21):43676-43686
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Avinash Upadhya, Yean Jin Lim, and Woei Ming Lee
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Abstract
Fluorescence recovery after photobleaching (FRAP) has remained a powerful tool to probe intracellular dynamics. FRAP relies on two aspects: (1) localized excitation resulting in photobleaching, and (2) fluorescence recovery of the bleached volume which provides insight into kinetics. Existing FRAP systems are limited by a trade-off between time and spatial multiplexing due to galvanometric scanning methods, precluding the study of multiple independent positions simultaneously as well as advanced widefield imaging modes. Hence, they may not capture the dynamic and non-isotropic environments in biological studies. In this paper, we utilize phase profiles corresponding to an array of Fresnel lenses and diffractive masks to switch between simultaneous FRAP of independent spatial positions whilst maintaining epifluorescence, highly inclined laminated optical sheet (HILO), and total internal reflection fluorescence (TIRF) microscopy modalities. Our approach bridges high contrast fluorescence imaging (HILO and TIRF) and a multi-position FRAP technique using a single spatial light modulator. As such, this technique enables high contrast bleaching and screening across volumes, which we envision will be of value to areas such as single particle tracking and single molecule imaging where dynamic photobleaching is necessary to measure fast events across the field of view in a single versatile instrument.
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© 2025 Optica Publishing Group (formerly OSA). All rights reserved. Published by Optica Publishing Group under the terms of the Creative Commons Attribution 4.0 License. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI.