Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

Hassan Dibaji, Ali Kazemi Nasaban Shotorban, Rachel M. Grattan, Shayna Lucero, David J. Schodt, Keith A. Lidke, Jonathan Petruccelli, Diane S. Lidke, Sheng Liu and Tonmoy Chakraborty ()
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Hassan Dibaji: University of New Mexico
Ali Kazemi Nasaban Shotorban: University of New Mexico
Rachel M. Grattan: University of New Mexico Health Sciences Center
Shayna Lucero: University of New Mexico Health Sciences Center
David J. Schodt: University of New Mexico
Keith A. Lidke: University of New Mexico
Jonathan Petruccelli: University at Albany–State University of NewYork
Diane S. Lidke: University of New Mexico Health Sciences Center
Sheng Liu: University of New Mexico
Tonmoy Chakraborty: University of New Mexico

Nature Communications, 2024, vol. 15, issue 1, 1-11

Abstract: Abstract Rapid, high-resolution volumetric imaging without moving heavy objectives or disturbing delicate samples remains challenging. Pupil-matched remote focusing offers a promising solution for high NA systems, but the fluorescence signal’s incoherent and unpolarized nature complicates its application. Thus, remote focusing is mainly used in the illumination arm with polarized laser light to improve optical coupling. Here, we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light, lets them pass through the remote focusing module separately, and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks with an axial range of 70 μm and camera-limited acquisition speed. Owing to its general nature, we believe this technique will find its application in many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging, such as confocal, 2-photon, and light sheet variants.

Date: 2024
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DOI: 10.1038/s41467-024-49291-0

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