Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array

Louis Obeid Mogridge; Jakub Nedbal; Shirwa Awale; Jess Tallis; Emily J. Johnston; Francesco Mattioli Della Rocca; Susan J. Rosser; Robert K. Henderson; Klaus Suhling

doi:10.1117/12.3001710

12 March 2024 Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array

Louis Obeid Mogridge, Jakub Nedbal, Shirwa Awale, Jess Tallis, Emily J. Johnston, Francesco Mattioli Della Rocca, Susan J. Rosser, Robert K. Henderson, Klaus Suhling

Author Affiliations +

Proceedings Volume 12847, Multiphoton Microscopy in the Biomedical Sciences XXIV; 128470J (2024) https://doi.org/10.1117/12.3001710
Event: SPIE BiOS, 2024, San Francisco, California, United States

Abstract

The time-resolved fluorescence anisotropy of a biological sample can reveal rotational dynamics and structure of a fluorescent probe’s local environment. Here, methods are presented towards developing widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array (QuantICAM). Such detector allows for simultaneous time-correlated single photon counting (TCSPC) measurements in each pixel with single-photon sensitivity and picosecond time resolution. Our method integrates the SPAD array with a widefield microscope, and automated rotating polarizers in the excitation and emission pathways to demonstrate TR-FAIM. We have shown the robustness of the method through spectroscopic measurements of the fluorophore PM546, and we have demonstrated the usefulness of simultaneous FLIM and TR-FAIM for studying properties of plasma membranes in live yeast cells.

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(2024) Published by SPIE. Downloading of the abstract is permitted for personal use only.

Citation Download Citation

Louis Obeid Mogridge, Jakub Nedbal, Shirwa Awale, Jess Tallis, Emily J. Johnston, Francesco Mattioli Della Rocca, Susan J. Rosser, Robert K. Henderson, and Klaus Suhling "Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array", Proc. SPIE 12847, Multiphoton Microscopy in the Biomedical Sciences XXIV, 128470J (12 March 2024); https://doi.org/10.1117/12.3001710

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