Volumetric Lissajous Confocal Microscopy

Deguchi, T.; Bianchini, P.; Palazzolo, G.; Oneto, M.; Diaspro, A.; Duocastella, M.

doi:10.1101/735654

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bioRxiv · 10.1101/735654

Volumetric Lissajous Confocal Microscopy

Deguchi, T.·Bianchini, P.·Palazzolo, G.·Oneto, M.·Diaspro, A.·Duocastella, M.

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Abstract

Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are based on rigid architectures, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Here, we developed volumetric Lissajous confocal microscopy to achieve unsurpassed 3D scanning speed with a tunable sampling rate. The system combines an acoustic liquid lens for continuous axial focus translation with a resonant scanning mirror. Accordingly, the excitation beam follows a dynamic Lissajous trajectory enabling sub-millisecond acquisitions of image series containing 3D information at a sub-Nyquist sampling rate. By temporal accumulation and/or advanced interpolation algorithms, volumetric imaging rate is selectable using a post-processing step at the desired spatiotemporal resolution for events of interest. We demonstrate multicolor and calcium imaging over volumes of tens of cubic microns with acquisition speeds up to 5 kHz.

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Deguchi, T., Bianchini, P., Palazzolo, G., Oneto, M., Diaspro, A., Duocastella, M.. 2019-08-15. Volumetric Lissajous Confocal Microscopy. https://doi.org/10.1101/735654

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