Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics in vivo

Significance: The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device. Aim: We introduce a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD). This microscope has a maximum line scan frequency of 400 kHz and a maximum frame rate of 10,000 frames per second (fps) at 250 x 40 pixels. For stepwise magnification from population view to subcellular view with high spatial and temporal resolution, we combined the AOD with resonance-galvo (RS) scanning. Approach: With this combinatorial device that supports both large-view navigation and smallview high-speed imaging, we measured dendritic calcium propagation velocity and the velocity of single red blood cells (RBCs). Results: We measured dendritic calcium propagation velocity (80/62.5 - 116.7 mu m/ms) in OGB-1-labeled single cortical neurons in mice in vivo. To benchmark the spatial precision and detection sensitivity of measurement in vivo, we also visualized the trajectories of single RBCs and found that their movement speed follows Poiseuille's law of laminar flow. Conclusions: This proof-of-concept methodological development shows that the combination of AOD and RS scanning two-photon microscopy provides both versatility and precision for quantitative analysis of single neuronal activities and hemodynamics in vivo. (c) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Automated summary

This study introduces a high-speed two-photon microscope based on a custom-built acousto-optic deflector (AOD) that provides high temporal resolution and versatility. By combining AOD with resonance-galvo (RS) scanning, the microscope allows for stepwise magnification from population view to subcellular view with high spatial and temporal resolution. The results demonstrate that this method can effectively measure the velocity of neuronal activities and blood flow.