Abstract
.Significance: To expand our understanding of the roles of astrocytes in neural circuits, there is a need to develop optical tools tailored specifically to capture their complex spatiotemporal dynamics. This interest is not limited to 2D, but to multiple depths.Aim: The focus of our work was to design and evaluate the optical performance of an enhanced version of a two-photon (2P) microscope with the addition of a deformable mirror (DM)-based axial scanning system for live mammalian brain imaging.Approach: We used a DM to manipulate the beam wavefront by applying different defocus terms to cause a controlled axial shift of the image plane. The optical design and performance were evaluated by an analysis of the optical model, followed by an experimental characterization of the implemented instrument.Results: Key questions related to this instrument were addressed, including impact of the DM curvature change on vignetting, field of view size, image plane flatness, wavefront error, and point spread function. The instrument was used for imaging several neurobiological samples at different depths, including fixed brain slices and in vivo mouse cerebral cortex.Conclusions: Our implemented instrument was capable of recording -stacks of in depth with a fine step size, parameters that make it useful for astrocyte biology research. Future work includes adaptive optics and intensity normalization.
Highlights
Two-photon (2P) microscopy[1,2] is a widely used technique for imaging in high-scattering media, such as the mammalian brain
Our 2P microscope is a custom-built instrument which combines a traditional upright fluorescence microscope (BX51WI, Olympus) with three additional modules to enable 2P microscopy: the illumination module composed of the pulsed laser and the relay optics, the scanning module where the deformable mirror (DM) and scanning galvanometer mirrors (GMs) are housed, and a detection system that uses photomultiplier tubes (PMTs)
While we were not able to test this hypothesis in the optical model, we have identified the fraction of the axial scanning range in which the point spread function (PSF) performance is preserved (0 < A < 0.5), so the user can trade axial range for PSF quality if the entire range is needed
Summary
Key questions related to this instrument were addressed, including impact of the DM curvature change on vignetting, field of view size, image plane flatness, wavefront error, and point spread function. The instrument was used for imaging several neurobiological samples at different depths, including fixed brain slices and in vivo mouse cerebral cortex
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