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bioRxiv · 10.1101/2020.03.06.981472

Converting lateral scanning into axial focusing to speed up 3D microscopy

Chakraborty, T.·Chang, B.-J.·Daetwyler, S.·Sapoznik, E.·Chen, B.·Dean, K. M.·Fiolka, R. P.
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Abstract

In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of 3D volumetric imaging. Recently, by conjugating either a movable-mirror to the image plane or an electrotuneable lens (ETL) to the back-focal plane respectively, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil based actuators), while ETLs introduce spherical and higher order aberrations, thereby preventing high-resolution imaging. Here, we introduce a novel optical design that can transform a lateral-scan motion into a spherical-aberration-free, high-resolution, rapid axial scan. 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 image space. We characterize the optical performance of this remote focusing technique and use it to accelerate axially swept light-sheet microscopy (ASLM) by one order of magnitude, allowing the quantification of rapid vesicular dynamics in 3D.

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Chakraborty, T., Chang, B.-J., Daetwyler, S., Sapoznik, E., Chen, B., Dean, K. M., Fiolka, R. P.. 2020-03-08. Converting lateral scanning into axial focusing to speed up 3D microscopy. https://doi.org/10.1101/2020.03.06.981472

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