In vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution

Studying neuronal activity at synapses requires high spatiotemporal resolution. For high spatial resolution in vivo imaging at depth, adaptive optics (AO) is required to correct sample-induced aberrations. To improve temporal resolution, Bessel focus has been combined with two-photon fluorescence microscopy (2PFM) for fast volumetric imaging at subcellular lateral resolution. To achieve both high-spatial and high-temporal resolution at depth, we develop an efficient AO method that corrects the distorted wavefront of Bessel focus at the objective focal plane and recovers diffraction-limited imaging performance. Applying AO Bessel focus scanning 2PFM to volumetric imaging of zebrafish larval and mouse brains down to 500 mu m depth, we demonstrate substantial improvements in the sensitivity and resolution of structural and functional measurements of synapses in vivo. This enables volumetric measurements of synaptic calcium and glutamate activity at high accuracy, including the simultaneous recording of glutamate activity of apical and basal dendritic spines in the mouse cortex. Adaptive optics (AO) corrects sample aberrations and allows high spatial resolution at depth in vivo. Here the authors report an AO method for Bessel focus; they apply AO Bessel focus scanning fluorescence microscopy to volumetric imaging and measure synaptic calcium and glutamate activity in vivo.

Automated summary

Studying neuronal activity at synapses requires high spatiotemporal resolution, which can be achieved through adaptive optics to correct sample-induced aberrations at depth for high spatial resolution. The combination of Bessel focus with two-photon fluorescence microscopy allows for fast volumetric imaging at subcellular lateral resolution. The developed AO method corrects distorted wavefront of Bessel focus at the objective focal plane, demonstrating significant improvements in sensitivity and resolution of structural and functional measurements of synapses in vivo.