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

Abstract

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 µm 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.

Bessel-droplet foci enable high-resolution and high-contrast volumetric imaging of synapses and circulation in the brainin vivo

“…S6). Previous investigation by us indicated that Bessel foci are degraded by Zernike aberration modes with |m| > 1 (e.g., astigmatism and trefoil) (24). In contrast, astigmatism and trefoil degraded Bessel-droplet PSFs much less than Bessel PSFs in simulation.…”

“…Z r with |m| > 1 (e.g., astigmatism and trefoil) (24). In contrast, astigmatism and trefoil degraded Bessel-droplet PSFs much less than Bessel PSFs in simulation.…”

“…All PSFs measured using 0.2-µm-diameter beads with the same post-objective power and displayed in scales normalized to their own maximum signal, with optical system aberrations corrected following Ref. [24]. (which was not certified by peer review) is the author/funder.…”

“…However, the same aberration only degraded the peak signal of the Bessel-droplet PSFs by 1.03-1.08×, without any perceivable enhancement of side-ring contamination. Throughout the manuscript, system-induced aberrations were corrected for all experiments, using a novel adaptive optics procedure (24), to ensure an accurate comparison between Bessel-droplet foci and conventional Bessel foci.…”

“…(g) Axial profiles of Bessel PSFs in a and Bessel-dropletPSFs in f. Dashed lines: profiles of Bessel-droplet foci with ∆φ = 0, π. All PSFs measured using 0.2-µm-diameter beads with the same post-objective power and displayed in scales normalized to their own maximum signal, with optical system aberrations corrected following Ref [24]…”