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

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

J Biomed Opt. 2023 Jun;28(6):066502. doi: 10.1117/1.JBO.28.6.066502. Epub 2023 Jun 21.

Authors

Kayvan Samimi¹, Danielle E Desa¹, Wei Lin², Kurt Weiss^{1

3}, Joe Li¹, Jan Huisken^{1

4}, Veronika Miskolci^{5

6

7}, Anna Huttenlocher^{5

8}, Jenu V Chacko⁹, Andreas Velten^{1

2

10

11}, Jeremy D Rogers^{1

11

12}, Kevin W Eliceiri^{1

9

10

11

13}, Melissa C Skala^{1

11

13}

Affiliations

¹ Morgridge Institute for Research, Madison, Wisconsin, United States.
² University of Wisconsin, Department of Electrical and Computer Engineering, Madison, Wisconsin, United States.
³ University of Wisconsin, Department of Biochemistry, Madison, Wisconsin, United States.
⁴ Georg-August-University Göttingen, Department of Biology and Psychology, Göttingen, Germany.
⁵ University of Wisconsin, Department of Medical Microbiology and Immunology, Madison, Wisconsin, United States.
⁶ Rutgers New Jersey Medical School, Center for Cell Signaling, Newark, New Jersey, United States.
⁷ Rutgers New Jersey Medical School, Department of Microbiology, Biochemistry and Molecular Genetics, Newark, New Jersey, United States.
⁸ University of Wisconsin, Department of Pediatrics, Madison, Wisconsin, United States.
⁹ University of Wisconsin, Laboratory for Optical and Computational Instrumentation, Madison, Wisconsin, United States.
¹⁰ University of Wisconsin, Department of Biostatistics and Medical Informatics, Madison, Wisconsin, United States.
¹¹ University of Wisconsin, McPherson Eye Research Institute, Madison, Wisconsin, United States.
¹² University of Wisconsin, Department of Ophthalmology and Visual Sciences, Madison, Wisconsin, United States.
¹³ University of Wisconsin, Department of Biomedical Engineering, Madison, Wisconsin, United States.

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 $30 \times$ to $6 \times$ 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.

Keywords: autofluorescence; fluorescence lifetime imaging microscopy; light-sheet microscopy; nicotinamide adenine dinucleotide (phosphate); single-photon avalanche diode.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Microscopy, Fluorescence / methods
NAD* / metabolism
Optical Imaging / methods
Pancreatic Neoplasms*
Photons
Zebrafish

Substances

NAD

Abstract

Publication types

MeSH terms

Substances

Grants and funding