Selective Plane Illumination Microscopy (SPIM) is a fluorescence imaging technique that allows volumetric imaging at high spatio-temporal resolution to monitor neural activity in live organisms such as larval zebrafish. A major challenge in the construction of a custom SPIM microscope is the control and synchronization of the various hardware components. Here we present a control toolset, μSPIM, built around the open-source MicroManager platform that has already been widely adopted for the control of microscopy hardware. Installation of μSPIM is relatively straightforward, involving a single C++ executable and a Java-based extension to Micro-Manager. Imaging protocols are defined through the μSPIM extension to Micro-Manager. The extension then synchronizes the camera shutter with the galvanometer mirrors to create a light-sheet that is scanned in the z-dimension, in synchrony with the imaging objective, to produce volumetric recordings. A key advantage of μSPIM is that a series of calibration procedures optimizes acquisition for a given set-up making it relatively independent of the optical design of the microscope, or the hardware used to build it. Two laser illumination arms can be used while also allowing for the introduction of illumination masks. μSPIM allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution as well as slower imaging to reconstruct cell populations, for example, in the cleared brains of mice. ### Competing Interest Statement The authors have declared no competing interest.
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