Schematic of the light-sheet FLIM microscope (adapted with permission from Ref. 52). (a) Top view of the SPIM system. The right illumination arm uses a 375 nm ps-pulsed diode laser operated at 50 MHz repetition rate to excite NAD(P)H fluorescence via a 200μm wide light sheet, while the time-resolved SPAD camera images the NAD(P)H autofluorescence lifetime (after a 495 nm LP DM and a 440/80nm filter) and the sCMOS camera images the NAD(P)H intensity (using a 530/55nm filter). The left illumination arm uses a bank of CW lasers to excite other fluorophores via a 1.5 mm wide light sheet, while emissions are imaged on the sCMOS camera. A red LED provides trans-illumination for bright field imaging. (b) Side view of the beams in the illumination arms and the orientation of the sample tube. DM, dichroic mirror.
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