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 . 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 small-view 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 ( ) in OGB-1-labeled single cortical neurons in mice . To benchmark the spatial precision and detection sensitivity of measurement , 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 .