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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.
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Correcting anisotropic intensity in light sheet images using dehazing and image morphology
Light-sheet fluorescence microscopy (LSFM) provides access to multi-dimensional and multi-scale in vivo imaging of animal models with highly coherent volumetric reconstruction of the tissue morphology, via a focused laser light sheet. The orthogonal illumination and detection LSFM pathways account for minimal photobleaching and deep tissue optical sectioning through different perspective views. Although rotation of the sample and deep tissue scanning constitutes major advantages of LSFM, images may suffer from intrinsic problems within the modality, such as light mismatch of refractive indices between the sample and mounting media and varying quantum efficiency across different depths. To overcome these challenges, we hereby introduce an illumination correction technique integrated with depth detail amelioration to achieve symmetric contrast in large field-of-view images acquired using a low power objective lens. Due to an increase in angular dispersion of emitted light flux with the depth, we combined the dehazing algorithm with morphological operations to enhance poorly separated overlapping structures with subdued intensity. The proposed method was tested on different LSFM modalities to illustrate its applicability on correcting anisotropic illumination affecting the volumetric reconstruction of the fluorescently tagged region of interest.
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- Award ID(s):
- 1936519
- PAR ID:
- 10584166
- Publisher / Repository:
- American Institute of Physics
- Date Published:
- Journal Name:
- APL Bioengineering
- Volume:
- 4
- Issue:
- 3
- ISSN:
- 2473-2877
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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