Rapid Electro-optic Two-photon Bessel Light Sheet Fluorescence Lifetime Imaging for Tissue Volume Interrogation
- 1Department of Physics, Stanford University, Stanford, CA 94305, USA
Abstract
We report a two-photon light sheet electro-optic fluorescence lifetime imaging microscopy (TPLS-EO-FLIM) technique based on an electro-optic (EO) modulation detection module. Scanning two-photon Bessel light sheet excitation was used to achieve in-depth volumetric imaging. The low cross-section of multiphoton excitation requires high detection efficiency, and electro-optic detection enables fast wide-field FLIM with a scientific complementary metal-oxide-semiconductor (sCMOS) detector. Phototoxicity is minimized by utilizing signal from the entire illuminated sample area in a wide field camera exposure, and time-resolved volume acquisitions are feasible in minutes, with cross-sectional field of view and light sheet length over 300 microns. The method has sub-nanosecond lifetime resolution and is capable of accurate FLIM images in 10 ms exposures with as low as a few hundred photons per pixel. The technique was demonstrated through volumetric FLIM of a green fluorescent protein (GFP) labeled zebrafish embryo heart, of multi-fluorescent protein (multi-FP) labeled plant root structures and of dynamic cardiac cycles in live zebrafish embryos. For the latter, we recorded the cardiac cycle dynamics of a zebrafish embryo at 50 frames per second and measured biomechanical parameters such as heart rate, ventricle/atrium areas, atrioventricular valve dynamics, and fractional area change. Taken together, these studies show the capability of the system for rapid, high resolution, and functional imaging in diverse biological contexts.
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BibTeX
@misc{kumar2026rapid,
title = {{Rapid Electro-optic Two-photon Bessel Light Sheet Fluorescence Lifetime Imaging for Tissue Volume Interrogation}},
author = {P. Kumar and V. R. Knight and D. E. Lysko and J.-G. Kim and W. S. Talbot and A. Bowman and M. B. Mudgett and S. Wakatsuki and M. Kasevich},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.03.763887},
url = {https://www.biorxiv.org/content/10.64898/2026.11.03.763887v1},
note = {CC BY-ND},
}
RIS
TY - UNPB
TI - Rapid Electro-optic Two-photon Bessel Light Sheet Fluorescence Lifetime Imaging for Tissue Volume Interrogation
AU - P. Kumar
AU - V. R. Knight
AU - D. E. Lysko
AU - J.-G. Kim
AU - W. S. Talbot
AU - A. Bowman
AU - M. B. Mudgett
AU - S. Wakatsuki
AU - M. Kasevich
PY - 2026
DA - 2026/11/03
PB - bioRxiv
DO - 10.64898/2026.11.03.763887
UR - https://www.biorxiv.org/content/10.64898/2026.11.03.763887v1
AB - We report a two-photon light sheet electro-optic fluorescence lifetime imaging microscopy (TPLS-EO-FLIM) technique based on an electro-optic (EO) modulation detection module. Scanning two-photon Bessel light sheet excitation was used to achieve in-depth volumetric imaging. The low cross-section of multiphoton excitation requires high detection efficiency, and electro-optic detection enables fast wide-field FLIM with a scientific complementary metal-oxide-semiconductor (sCMOS) detector. Phototoxicity is minimized by utilizing signal from the entire illuminated sample area in a wide field camera exposure, and time-resolved volume acquisitions are feasible in minutes, with cross-sectional field of view and light sheet length over 300 microns. The method has sub-nanosecond lifetime resolution and is capable of accurate FLIM images in 10 ms exposures with as low as a few hundred photons per pixel. The technique was demonstrated through volumetric FLIM of a green fluorescent protein (GFP) labeled zebrafish embryo heart, of multi-fluorescent protein (multi-FP) labeled plant root structures and of dynamic cardiac cycles in live zebrafish embryos. For the latter, we recorded the cardiac cycle dynamics of a zebrafish embryo at 50 frames per second and measured biomechanical parameters such as heart rate, ventricle/atrium areas, atrioventricular valve dynamics, and fractional area change. Taken together, these studies show the capability of the system for rapid, high resolution, and functional imaging in diverse biological contexts.
ER -