Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array

Kayvan Samimi; Danielle E. Desa; Wei Lin; Kurt Weiss; Joe Li; Jan Huisken; Veronika Miskolci; Anna Huttenlocher; Jenu V. Chacko; Andreas Velten; Jeremy D. Rogers; Kevin W. Eliceiri; Melissa C. Skala

doi:10.1117/1.JBO.28.6.066502

21 June 2023 Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array

Kayvan Samimi, Danielle E. Desa, Wei Lin, Kurt Weiss, Joe Li, Jan Huisken, Veronika Miskolci, Anna Huttenlocher, Jenu V. Chacko, Andreas Velten, Jeremy D. Rogers, Kevin W. Eliceiri, Melissa C. Skala

Author Affiliations +

Journal of Biomedical Optics, Vol. 28, Issue 6, 066502 (June 2023). https://doi.org/10.1117/1.JBO.28.6.066502

Abstract

Significance

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.

Aim

We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.

Approach

A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.

Results

An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.

Conclusions

FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Kayvan Samimi, Danielle E. Desa, Wei Lin, Kurt Weiss, Joe Li, Jan Huisken, Veronika Miskolci, Anna Huttenlocher, Jenu V. Chacko, Andreas Velten, Jeremy D. Rogers, Kevin W. Eliceiri, and Melissa C. Skala "Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array," Journal of Biomedical Optics 28(6), 066502 (21 June 2023). https://doi.org/10.1117/1.JBO.28.6.066502

Received: 3 February 2023; Accepted: 6 June 2023; Published: 21 June 2023

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