Journal
APPLIED PHYSICS LETTERS
Volume 121, Issue 16, Pages -Publisher
AIP Publishing
DOI: 10.1063/5.0101426
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Funding
- U.S. National Science Foundation [1608744, 1650406, 1828793, PHY-1734030]
- National Institutes of Health [1R01MH107238-01, 1R34NS126800A]
- Human Frontier Science Program [RGP0008/2017]
- Jet Propulsion Laboratory [1632330]
- Alfred E. Mann Doctoral Fellowship
- Robert H. Dicke Fellowship in Experimental Physics from Princeton University
- Direct For Biological Sciences
- Division Of Integrative Organismal Systems [1650406] Funding Source: National Science Foundation
- Division Of Physics
- Direct For Mathematical & Physical Scien [1828793] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1608744] Funding Source: National Science Foundation
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ExD-SPIM is an improved light-sheet microscopy strategy that solves the limitation of capturing fluorescence signals in large volumes by extending the depth of field (DOF) of high-NA detection objectives to match the thickness of the illumination light sheet. Compared to conventional light sheet imaging, ExD-SPIM improves the signal-to-noise ratio and reduces the rate of photobleaching. In whole-brain activity imaging, ExD-SPIM enhances signal sensitivity and volumetric coverage.
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. (C) 2022 Author(s).
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