Light Sheet Fluorescence Microscopy Using Incoherent Light Detection

Engineering Proceedings

article

Mariana Potcoava¹  Christopher Mann²  Jonathan Art¹  Simon Alford¹ 

[1] Department of Anatomy and Cell Biology, University of Illinois at Chicago;Department of Applied Physics and Materials Science, Northern Arizona University;Center for Materials Interfaces in Research and Development, Northern Arizona University

关键词: incoherent holography;    light sheet;    fluorescent microscopy;   

DOI  :  10.3390/HMAM2-14156

来源: mdpi

【 摘 要 】

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 90° 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 all 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 light sheet 3D imaging by choosing a variable NA and moving only the light sheet while keeping the sample stage and detection microscope objective stationary.

【 授权许可】

Unknown