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Light Sheet Fluorescence Microscopy using Incoherent Light Detection
* 1 , 2 , 1 , 1
1  University of Illinois at Chicago
2  Northern Arizona University
Academic Editor: VIJAYAKUMAR ANAND

Published: 13 March 2023 by MDPI in Holography Meets Advanced Manufacturing session Holography 2
Abstract:

We previously developed an incoherent holography technique for use in lattice light sheet (LLS) microscopes, that represents a specialized adaptation of light sheet microscopy. Light sheet instruments resolve 3D information by illuminating the sample at 90o to the imaging plane with a sheet of laser light that excites fluorophores in the sample only in a narrow plane. Imaging this plane and then moving it in the imaging z-axis allows construction of the sample volume. Among these types of instruments, LLS microscopy gives higher z-axis resolution and tissue depth penetration. It has a similar working principle to light sheet fluorescence microscopy, but uses a lattice configuration of Bessel beams, instead of Gaussian beams. Our incoherent light detection technique replaces the glass tube lens of the original LLS with a dual diffractive lens system to retrieve the axial depth of the sample. Here, we show that the system is applicable to the light sheet instruments. To make a direct comparison in the same emission light path, we can imitate the nature of non-Bessel light sheet systems by altering the mask annuli used to create Bessel beams in the LLS system. We change the diffractive mask annuli from a higher NA anulus to a smaller NA anulus. This generates a Gaussian excitation beam similar to conventional light sheet systems. Using this approach, we propose an incoherent light detection system for 3D imaging by choosing a variable NA and moving only the light sheet while keeping the sample stage and detection microscope objective stationary.

Keywords: Incoherent Holography; Light Sheet; Lattice Light Sheet; Fluorescence Microscopy; Diffractive Lenses
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