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

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

^*Corresponding author for this work

Research output: Contribution to journal › Conference paper

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.

Original language	English
Journal	Progress in Biomedical Optics and Imaging - Proceedings of SPIE
DOIs	https://doi.org/10.1117/12.3001710
Publication status	Published - 12 Mar 2024
Event	Multiphoton Microscopy in the Biomedical Sciences XXIV 2024 - San Francisco, United States Duration: 28 Jan 2024 → 30 Jan 2024

Keywords

fluorescence lifetime imaging microscopy (FLIM)
intracellular diffusion
live-cell imaging
microviscosity
single-photon avalanche diode (SPAD) array
time-correlated single photon counting (TCSPC)
Time-resolved fluorescence anisotropy imaging (TR-FAIM)

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10.1117/12.3001710

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Mogridge, L. O., Nedbal, J., Awale, S., Tallis, J., Johnston, E. J., Della Rocca, F. M., Rosser, S. J., Henderson, R. K., & Suhling, K. (2024). Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array. Progress in Biomedical Optics and Imaging - Proceedings of SPIE. https://doi.org/10.1117/12.3001710

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

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.",

keywords = "fluorescence lifetime imaging microscopy (FLIM), intracellular diffusion, live-cell imaging, microviscosity, single-photon avalanche diode (SPAD) array, time-correlated single photon counting (TCSPC), Time-resolved fluorescence anisotropy imaging (TR-FAIM)",

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

note = "Publisher Copyright: {\textcopyright} 2024 SPIE.; Multiphoton Microscopy in the Biomedical Sciences XXIV 2024 ; Conference date: 28-01-2024 Through 30-01-2024",

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T1 - Development of widefield time-resolved fluorescence anisotropy imaging (TR-FAIM) using a single photon avalanche diode (SPAD) array

AU - Mogridge, Louis Obeid

AU - Nedbal, Jakub

AU - Awale, Shirwa

AU - Tallis, Jess

AU - Johnston, Emily J.

AU - Della Rocca, Francesco Mattioli

AU - Rosser, Susan J.

AU - Henderson, Robert K.

AU - Suhling, Klaus

PY - 2024/3/12

Y1 - 2024/3/12

N2 - 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.

AB - 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.

KW - fluorescence lifetime imaging microscopy (FLIM)

KW - intracellular diffusion

KW - live-cell imaging

KW - microviscosity

KW - single-photon avalanche diode (SPAD) array

KW - time-correlated single photon counting (TCSPC)

KW - Time-resolved fluorescence anisotropy imaging (TR-FAIM)

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U2 - 10.1117/12.3001710

DO - 10.1117/12.3001710

M3 - Conference paper

AN - SCOPUS:85194390919

SN - 1605-7422

JO - Progress in Biomedical Optics and Imaging - Proceedings of SPIE

JF - Progress in Biomedical Optics and Imaging - Proceedings of SPIE

T2 - Multiphoton Microscopy in the Biomedical Sciences XXIV 2024

Y2 - 28 January 2024 through 30 January 2024

ER -

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

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