Abstract
Investigation of the transient processes integral to neuronal function demands rapid and high-resolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Here we describe a compact light sheet fluorescence microscope, featuring a 45° inverted geometry and an integrated photolysis laser, that is optimized for applications in neuroscience, in particular fast imaging of sub-neuronal structures in mammalian brain slices. We demonstrate the utility of this design for three-dimensional morphological reconstruction, activation of a single synapse with localized photolysis, and fast imaging of neuronal Ca2+ signalling across a large field of view. The developed system opens up a host of novel applications for the neuroscience community.
Highlights
Investigation of the transient processes integral to neuronal function demands rapid and highresolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes
We demonstrated photolytic release of the excitatory neurotransmitter glutamate using the 405 nm photolysis laser, which is integrated into our prototype Light sheet fluorescence microscopy (LSFM)
This example demonstrates an important advantage of LSFM over the conventional method of recording neuronal Ca2+ transients with an XT line-scan on a confocal microscope – with LSFM there is no need to decide ahead of time exactly where to set up the line scan
Summary
Investigation of the transient processes integral to neuronal function demands rapid and highresolution imaging techniques over a large field of view, which cannot be achieved with conventional scanning microscopes. Light sheet fluorescence microscopy (LSFM) uses a thin sheet of light to illuminate the sample, with the fluorescent signal detected perpendicular to the illuminated plane[1] This simple geometry dramatically enhances the acquisition speed, and reduces photo-bleaching and photo-damage in the specimen being imaged. These advantages make LSFM suitable for repeated imaging of living tissue, for example in studies of neuronal development or plasticity. Generating a light sheet requires independent illumination and detection arms, which is classically accomplished by a pair of objectives perpendicular to each other These two objectives can be on either a horizontal plane or a vertical plane, depending on the specimen to be imaged. We describe a novel design (based on openSPIM) which uses a modified vertical configuration (iSPIM) with both objectives oriented at 45° to the horizontal, and its use for imaging in living brain slices
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