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

Wei Chen,Ryan G Natan,Na Ji,Yuhan Yang,Shih-Wei Chou,Ehud Y Isacoff,Qinrong Zhang

doi:10.1038/s41467-021-26965-7

Wei Chen, Ryan G Natan + Show 5 more

Open Access

https://doi.org/10.1038/s41467-021-26965-7

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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.

Highlights

Studying neuronal activity at synapses requires high spatiotemporal resolution
Maintaining high spatial resolution at depth in vivo, is only possible via adaptive optics (AO), which measures and corrects for the optical aberrations accumulated on the wavefront of image-forming light while it passes through optically heterogeneous specimen[2,3]
We find that AO is essential for the accurate characterization of synaptic calcium and glutamate activity measured with Bessel focus scanning 2PFM

Summary

Introduction

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. 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. Of how Bessel focus quality is affected by optical aberrations and how we can correct these aberrations in order to recover diffraction-limited performance at depth

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Conclusion