Swept confocally-aligned planar excitation (SCAPE) microscopy for high-speed volumetric imaging of behaving organisms

Abstract

We report a three-dimensional microscopy technique—swept, confocally-aligned planar excitation (SCAPE) microscopy—that allows volumetric imaging of living samples at ultrahigh speeds. Although confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image three-dimensional volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light sheet in a single-objective, en face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless three-dimensional imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae. A swept light-sheet microscopy scheme allows volumetric imaging of living samples at high speed.