μSPIM Toolset: A software platform for selective plane illumination microscopy

Highlights

• μSPIM Toolset is open-source software for Selective Plane Illumination Microscopy.

• μSPIM synchronizes two scanned laser beams with various hardware components.

• Built around MicroManager software to integrate with range of cameras and hardware.

• μSPIM calibration procedures make it independent of microscope optical design.

• μSPIM images 100 planes through the brain of larval zebrafish at 1 Hz.

Abstract

Background

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 using a scanned laser beam is the control and synchronization of the various hardware components.

New Method

We present an open-source software, μSPIM Toolset, built around the widely adopted MicroManager platform, that provides control and acquisition functionality for a SPIM. A key advantage of μSPIM Toolset is a series of calibration procedures that optimize acquisition for a given set-up, making it relatively independent of the optical design of the microscope or the hardware used to build it.

Results

μSPIM Toolset allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution.

Comparison with Existing Methods

Several designs of SPIM have been published but are focused on imaging of developmental processes using a slower setup with a moving stage and therefore have limited use for functional imaging. In comparison, μSPIM Toolset uses a scanned beam to allow imaging at higher acquisition frequencies while minimizing disturbance of the sample.

Conclusions

The μSPIM Toolset provides a flexible solution for the control of SPIM microscopes and demonstrated its utility for brain-wide imaging of neural activity in larval zebrafish.