Light sheet fluorescence microscopy using axi-symmetric binary phase filters

Abstract

Light sheet fluorescence microscopy (LSFM) has become an indispensable tool in biomedical studies owing to its depth-sectioning capability and low photo-bleaching. The axial resolution in LSFM is determined mainly by the thickness of the illumination sheet, and a high numerical-aperture lens is thus preferred in the illumination to increase the axial resolution. However, a rapid divergence of the illumination beam limits the effective field-of-view (FoV), that provides high-resolution images. Several strategies have been demonstrated for FoV enhancement, which involve the use of Bessel or Airy beams, for example. However, the generation of these beams requires complicated optical setup or phase filters with continuous phase distributions, which are difficult to manufacture. In contrast, a binary phase filter (BPF) comprising concentric rings with 0 or π phases produces a response similar to its continuous original and is easy to realize. Here, we present a novel form of LSFM that integrates BPFs derived from two representative axi-symmetric aberrations, including phase axicon and spherical aberrations, to improve the imaging performance. We demonstrate that these BPFs significantly increase the FoV, and those derived from axicon generate self-reconstructing beams, which are highly desirable in imaging through scattering specimens. We validate its high-contrast imaging capability over extended FoV by presenting three-dimensional images of microspheres, imaginal disc of Drosophila larva, and Arabidopsis.

E2E-BPF microscope: extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution

“…In terms of applications, one of the potential applications is its utility in light sheet fluorescence microscopy, which calls for large-DoF and high light efficiency. A BPF can be designed to generate sharp and elongated excitation light sheet or focus on the illumination path 51 . The resultant fluorescence emission from a large 3D sample can be detected though our E2E-BPF platform, allowing for high light-throughput, high-resolution volumetric imaging of fluorophores without re-focusing.…”

E2E-BPF microscope: Extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution

“…In terms of applications, one of the potential applications is its utility in light sheet uorescence microscopy, which calls for large-DoF and high light e ciency. A BPF can be designed to generate sharp and elongated excitation light sheet or focus on the illumination path 43 . The resultant uorescence emission from a large 3D sample can be detected though our E2E-BPF platform, allowing for high lightthroughput, high-resolution volumetric imaging of uorophores without re-focusing.…”

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

“…Other microscopy techniques utilizing the elongated PSF for volumetric imaging suffer from compromised lateral and axial resolution [13] , or limited axial detection range [30], even the aberration caused by the phase mask along with the pinhole which not compatible slits [31][32][33]. These aberrations could be reduced by adaptive optics [34,35].…”