Light-sheet Autofluorescence Lifetime Imaging with a Single-photon Avalanche Diode Array

Overview

Journal J Biomed Opt

Specialties Biomedical Engineering
Ophthalmology

Date 2023 Jun 23

PMID 37351197

Authors

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

Affiliations

Soon will be listed here.

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 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 to 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.

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