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Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.

Published version
Peer-reviewed

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Authors

Chakraborty, Tonmoy  ORCID logo  https://orcid.org/0000-0001-7956-1932
Chen, Bingying 
Daetwyler, Stephan 
Vanderpoorten, Oliver 

Abstract

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

Description

Funder: MedImmune, and Infinitus (China) Ltd.

Keywords

Light-sheet microscopy, Microscopy

Journal Title

Light Sci Appl

Conference Name

Journal ISSN

2095-5545
2047-7538

Volume Title

9

Publisher

Springer Science and Business Media LLC
Sponsorship
Engineering and Physical Sciences Research Council (EP/H018301/1)
Wellcome Trust (089703/Z/09/Z)
Medical Research Council (MR/K015850/1)
Medical Research Council (MR/K02292X/1)
Engineering and Physical Sciences Research Council (EP/L015889/1)
Biotechnology and Biological Sciences Research Council (BB/H023917/1)
Medical Research Council (G0902243)
Wellcome Trust (203249/Z/16/Z)