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

Abstract

Light-sheet microscopes must compromise among field of view, optical sectioning, resolution, and detection efficiency. High-numerical-aperture (NA) detection objective lenses provide higher resolution, but their narrow depth of field inefficiently captures the fluorescence signal generated throughout the thickness of the illumination light sheet when imaging large volumes. Here, we present ExD-SPIM (extended depth-of-field selective-plane illumination microscopy), an improved light-sheet microscopy strategy that solves this limitation by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. This extension of the DOF uses a phase mask to axially stretch the point-spread function of the objective lens while largely preserving lateral resolution. This matching of the detection DOF to the illumination-sheet thickness increases the total fluorescence collection, reduces the background, and improves the overall signal-to-noise ratio (SNR), as shown by numerical simulations, imaging of bead phantoms, and imaging living animals. In comparison to conventional light sheet imaging with low-NA detection that yields equivalent DOF, the results show that ExD-SPIM increases the SNR by more than threefold and dramatically reduces the rate of photobleaching. Compared to conventional high-NA detection, ExD-SPIM improves the signal sensitivity and volumetric coverage of whole-brain activity imaging, increasing the number of detected neurons by over a third.

Extended Depth of Focus Two-Photon Light-Sheet Microscopy for In Vivo Fluorescence Imaging of Large Multicellular Organisms at Cellular Resolution

“…An application of the stair-step device in light-sheet microscopy has been reported [ 20 ]. They applied this extended DOF method to the detection of the high NA (0.8) and high magnification (40×) DO to match the thicker light-sheet (~8 μm) volume, and improved a way to axially elongate the large volume detection, although information on the axial position was compromised.…”