Axially overlapped multi-focus light sheet with enlarged field of view

Hongjin Li,Karl Zhanghao,Zihan Wu,Dayong Jin,Zhichao Yang,Peng Xi

doi:10.1063/5.0049013

Abstract

Light sheet fluorescence microscopy provides optical sectioning and is widely used in volumetric imaging of large specimens. However, the axial resolution and the lateral Field of View (FoV) of the system, defined by the light sheet, typically limit each other due to the spatial band product of the excitation objective. Here, we develop a simple multi-focus scheme to extend the FoV, where a Gaussian light sheet can be focused at three or more consecutive positions. Axially overlapped multiple light sheets significantly enlarge the FoV with improved uniformity and negligible loss in axial resolution. By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a FoV of 450 μm and a maximum axial FWHM of 7.5 μm. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish.

Highlights

The self-reconstructing beams, such as Bessel beams,7,8 Airy beams,9 and attenuation-compensated propagation-invariant beams,10 have been used to maintain the thin sheet over a long propagation distance
By measuring the point spread function of fluorescent beads, we demonstrated that the obtained light sheet has a Field of View (FoV) of 450 lm and a maximum axial FWHM of 7.5 lm
The trade-off between N.A. and FoV seems to be the bottleneck for large-scale volumetric imaging with high resolution

Summary

Introduction

The self-reconstructing beams, such as Bessel beams,7,8 Airy beams,9 and attenuation-compensated propagation-invariant beams,10 have been used to maintain the thin sheet over a long propagation distance. Compared with the conventional single-focus one, the multi-focus Gaussian light sheet displays a significantly improved optical sectioning ability over the full FoV when imaging cells and zebrafish. There are still different opinions doubting the superiority of self-reconstructing beams over Gaussian beams in light sheet microscopy.23,24 Besides, the trade-off between N.A. and FoV seems to be the bottleneck for large-scale volumetric imaging with high resolution.

Results

Conclusion