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Light-sheet microscopes must compromise between field of view, opticalsectioning, resolution, and detection efficiency. High-numerical-aperture (NA)detection objective lenses provide high resolution but their narrow depth offield fails to capture effectively the fluorescence signal generated by theillumination light sheets, in imaging large volumes. Here, we present ExD-SPIM(extended depth-of-field selective-plane illumination microscopy), an improvedlight-sheet microscopy strategy that solves this limitation by extending thedepth of field (DOF) of high-NA detection objectives to match the thickness ofthe illumination light sheet. This extension of the DOF uses a phase mask toaxially stretch the point-spread function of the objective lens while largelypreserving lateral resolution. This matching of the detection DOF to theillumination-sheet thickness increases total fluorescence collection, reducesbackground, and improves the overall signal-to-noise ratio (SNR). Wedemonstrate, through numerical simulations and imaging of bead phantoms as wellas living animals, that ExD-SPIM increases the SNR by more than three-fold anddramatically reduces the rate of fluorescence photobleaching, when compared toa low-NA system with an equivalent depth of field. Compared to conventionalhigh-NA detection, ExD-SPIM improves the signal sensitivity and volumetriccoverage of whole-brain activity imaging, increasing the number of detectedneurons by over a third.

Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture