Incoherent Holographic Lattice Light-Sheet Microscopy for Low-Signal Volumetric Imaging

Abstract Imaging activity dependent signaling in live tissue such as evoked Ca 2+ imaging often requires stimulation electrodes or perfusion lines at the sample, which can conflict with moving the detection objective or the sample during a volume scan. Incoherent holographic lattice light-sheet (IHLLS) microscopy avoids both, because only the light sheet moves while a self-interference hologram recorded through the stationary detection objective refocuses each plane numerically. We describe a shortened IHLLS detection arm (z h = 277.8 mm, with interleaved diffractive lenses of 262 and 296 mm at the 520 nm emission wavelength) and a paraxial two-lens model of how the refocus distance depends on depth. Near the matched condition the model predicts a refocus distance that is almost even in depth, and two planes, at − 5.70 and + 5.81 µm, where one lens images the emitter directly and the hologram collapses toward a spot; these spot planes locate the focal plane in every stack without relying on the piezo position. For 0.1 µm beads, recorded at 200 ms per phase frame, the refocused IHLLS amplitude has a lateral FWHM of 0.330 µm, comparable with the resolution of the conventional LLS in dithering mode, without deconvolution. For 0.2 µm beads, recorded at 10 ms per phase frame, we track the signal and depth of 10 beads over 200 s, with a 25.7 +- 11.1% coefficient of variation. In NSC-34 cells labeled with MitoTracker Green FM, fluorescence is detected and the cellular structure is resolved; the fringe visibility was only 6%, consistent with overlap of the holograms of densely packed emitters. Because each phase is recorded as a full galvo sweep, the volume interval and the separation of the phase frames, not the exposure, set the temporal resolution. Calcium transients and neurotransmitter release are not measured in this study.