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

Tonmoy Chakraborty,Oliver Vanderpoorten,Kevin M. Dean,Etai Sapoznik,Reto Fiolka,Bo-Jui Chang,Stephan Daetwyler,Bingying Chen,Clemens F. Kaminski,Tuomas P. J. Knowles

doi:10.1038/s41377-020-00401-9

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.

Highlights

The ability to rapidly change the focal plane of an optical imaging system is of great interest in microscopy, computer vision, and laser machining
Refocusing has been achieved by either mechanically moving the microscope objective or the sample under study. Because these objects have a high mass, axial scanning becomes prohibitively rate limiting, which is especially problematic in fields such as neuroscience, which requires high-speed volumetric imaging of dynamic biological processes, including membrane voltage activity[1] and cerebral blood flow
Wavefront modifications can be introduced in Fourier space with a tuneable acoustic gradient index of refraction (TAG) lens[4,5], an electrically tuneable lens (ETL)[6], or a deformable mirror (DM)[7]

Summary

Introduction

The ability to rapidly change the focal plane of an optical imaging system is of great interest in microscopy, computer vision, and laser machining. Refocusing has been achieved by either mechanically moving the microscope objective or the sample under study Because these objects have a high mass, axial scanning becomes prohibitively rate limiting, which is especially problematic in fields such as neuroscience, which requires high-speed volumetric imaging of dynamic biological processes, including membrane voltage activity (with dynamics on the timescale of 1 ms or less)[1] and cerebral blood flow. A potential way to alleviate this problem is through remote focusing, which is an emerging technique where the position of the optical focus is adjusted without moving the primary objective or the sample[2,3]. DMs with large stroke actuators that are capable of large focus changes are typically much slower

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Conclusion