Open AccessVolumetric Lissajous confocal microscopy with tunable spatiotemporal resolution
Author(s) -
Takahiro Deguchi,
Paolo Bianchini,
Gemma Palazzolo,
Michele Oneto,
Alberto Diaspro,
Martí Duocastella
Publication year - 2020
Publication title -
biomedical optics express
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 1.362
H-Index - 86
ISSN - 2156-7085
DOI - 10.1364/boe.400777
Subject(s) - lissajous curve , optics , frame rate , light sheet fluorescence microscopy , temporal resolution , sampling (signal processing) , confocal , image resolution , microscope , computer science , refresh rate , microscopy , computer vision , materials science , physics , scanning confocal electron microscopy , detector
Dynamic biological systems present challenges to existing three-dimensional (3D) optical microscopes because of their continuous temporal and spatial changes. Most techniques are rigid in adapting the acquisition parameters over time, as in confocal microscopy, where a laser beam is sequentially scanned at a predefined spatial sampling rate and pixel dwell time. Such lack of tunability forces a user to provide scan parameters, which may not be optimal, based on the best assumption before an acquisition starts. 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, the 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 3D acquisition speeds of 30 Hz and frame rates up to 5 kHz.