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14 October 2024 |
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Article overview
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Extended depth-of-field light-sheet microscopy improves imaging of large volumes at high numerical aperture | | Kevin Keomanee-Dizon
; Matt Jones
; Peter Luu
; Scott E. Fraser
; Thai V. Truong
; | | Date: |
1 Jun 2022 | | Abstract: | Light-sheet microscopes must compromise between field of view, optical
sectioning, resolution, and detection efficiency. High-numerical-aperture (NA)
detection objective lenses provide high resolution but their narrow depth of
field fails to capture effectively the fluorescence signal generated by the
illumination light sheets, in 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 total fluorescence collection, reduces
background, and improves the overall signal-to-noise ratio (SNR). We
demonstrate, through numerical simulations and imaging of bead phantoms as well
as living animals, that ExD-SPIM increases the SNR by more than three-fold and
dramatically reduces the rate of fluorescence photobleaching, when compared to
a low-NA system with an equivalent depth of field. 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. | | Source: | arXiv, 2206.00141 | | Services: | Forum | Review | PDF | Favorites | |
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