Light-sheet microscopy (LSM) is an emergent fluorescence microscopy technique showing great promise for biomedical research. LSM enables rapid, high-contrast imaging of large specimens with high spatiotemporal resolution and minimal photo-damage. When imaging large specimens, the intensity of the light-sheet reduces across the field-of-view (FOV) due to absorption. This results in an image with spatially-variant intensity, affecting quantitative measurements, and ultimately limits the penetration depth of the illumination. Some existing approaches to alleviate this issue involve illuminating the sample from multiple directions or rotating the sample. These approaches are not always practical and restrict specimen choice.
Separately, propagation-invariant light modes have been used to develop high-resolution LSM techniques as they can overcome the natural divergence of a Gaussian beam, producing a thin and uniform light-sheet over long distances. Most notably, Bessel and Airy beam-based LSM techniques have been implemented.
For propagation-invariant beams, there exists a mapping between the transverse coordinate in the pupil plane of a lens, and the axial propagation in the focal plane. Spatially-variant amplitude modulation therefore offers control of the intensity of the beam with propagation.
In this paper, we report that such amplitude modulation in the pupil plane of an Airy LSM can yield a system which counteracts absorption of the light-sheet and gives uniform intensity across the FOV with a single acquisition and without restricting specimen choice. This technique is an alternative to, and may be complimented by, wavefront correction. We demonstrate this technique through numerical simulations and experimental validation in absorbing tissue phantoms.
Jonathan Nylk, Miguel A. Preciado, Michael Mazilu, and Kishan Dholakia, "New depths for Airy light-sheet microscopy: attenuation compensation techniques to increase imaging depths within absorbing samples (Conference Presentation)," Proc. SPIE 10073, Adaptive Optics and Wavefront Control for Biological Systems III, 1007306 (Presented at SPIE BiOS: January 28, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2251934.5380600119001.
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