Volumetric Lissajous Confocal Microscopy

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.

Utilizing phase-modulated Lissajous to enhance spatial and temporal resolution of laser scanning microscopy

“…In some applications, such as fluorescence live cell imaging, it is important to decrease the imaging time in order to follow the behavior of cells. To reduce the imaging time, faster scanners can be chosen, such as resonant [11] and microelectromechanical Systems (MEMS) scanners [12,13], or acoustic scanners like tunable acousto-optic lenses [14][15][16]. One remarkable way is sparse sampling of the field of view (FOV) using compressed sensing methods to speed up the imaging and also reduce photobleaching [17,18], which is a substantial issue in fluorescence microscopy [19].…”

An inertia-free beam scanning device for single-wavelength 2PE-STED nanoscopy

“…Achieving fast imaging is crucial for the collection of large amounts of data. In many of the application where 2PE microscopy is advantageous, one solution for faster imaging is to avoid raster scanning [3,4], using custom scan patterns, and imaging only regions where fluorescent structures of interest are present [5,6]. One such custom scan pattern is random-access scanning.…”