Open-top axially swept light-sheet microscopy.

Bumju Kim,Jung-Hoon Park,Ki Hean Kim,Soohyun Park,Euiheon Chung,Kitae Kim,Sunghoe Chang,Myeongsu Na

doi:10.1364/boe.419030

Abstract

Open-top light-sheet microscopy (OT-LSM) is a specialized microscopic technique for high throughput cellular imaging of large tissue specimens including optically cleared tissues by having the entire optical setup below the sample stage. Current OT-LSM systems had relatively low axial resolutions by using weakly focused light sheets to cover the imaging field of view (FOV). In this report, open-top axially swept LSM (OTAS-LSM) was developed for high-throughput cellular imaging with improved axial resolution. OTAS-LSM swept a tightly focused excitation light sheet across the imaging FOV using an electro tunable lens (ETL) and collected emission light at the focus of the light sheet with a camera in the rolling shutter mode. OTAS-LSM was developed by using air objective lenses and a liquid prism and it had on-axis optical aberration associated with the mismatch of refractive indices between air and immersion medium. The effects of optical aberration were analyzed by both simulation and experiment, and the image resolutions were under 1.6µm in all directions. The newly developed OTAS-LSM was applied to the imaging of optically cleared mouse brain and small intestine, and it demonstrated the single-cell resolution imaging of neuronal networks. OTAS-LSM might be useful for the high-throughput cellular examination of optically cleared large tissues.

Highlights

High-speed optical microscopy methods, which can do the cell-level imaging of large tissue specimens, are useful for both the biological study of neuronal networks and the cellular examination of clinical tissues together with optical clearing
OTAS-Light-sheet microscopy (LSM) was developed for the high-throughput cellular imaging of optically cleared large tissues at the improved axial resolution
OTAS-LSM was implemented by using an electro tunable lens (ETL) for the axial sweeping, and air objective lenses and the liquid prism for illumination and imaging

Summary

Introduction

High-speed optical microscopy methods, which can do the cell-level imaging of large tissue specimens, are useful for both the biological study of neuronal networks and the cellular examination of clinical tissues together with optical clearing. LSM was later applied to the imaging of the larger samples such as optically cleared mouse brain [4]. AS-LSM used a tightly focused light sheet and axially swept the light sheet across the imaging FOV. AS-LSM methods have been applied to the imaging of large samples such as mouse brain [7,9], bone marrow [7], spinal cord [8], and chicken embryo [9].

Methods

Results

Discussion

Conclusion