Rapid Electro-optic Two-photon Bessel Light Sheet Fluorescence Lifetime Imaging for Tissue Volume Interrogation

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.