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Two-photon microscope achieves in vivo calcium imaging with near-millisecond precision

Neural circuits in the brain fire on the millisecond time scale. Although two-photon calcium imaging can image this in vivo, its temporal resolution isn’t as high as electrical recordings. Now, researchers from the Brain Research Institute at the University of Zurich have designed a two-photon microscope that makes use of an acousto-optic deflector (AOD) to image at rates up to 500 Hz.

Standard two-photon setups use galvanometric mirrors for scanning and can typically sample at up to 10 to 15 Hz. Using an AOD instead allows movement of the laser focus between any two positions in a field of view in milliseconds and also lets the imaging be concentrated on the structures of interest. However, AODs require correction of spatial and temporal laser beam distortions, which the researchers accomplished by modifying a single-prism compensation method to improve transmission and dispersion compensation over a large field of view.

Using this setup they were able to measure fluorescence from neurons in the mouse neocortex at a 180 to 490 Hz sampling rate, detect single action potential evoked calcium transients with signal-to-noise ratios of 2 to 5, and calculate spike times with near-millisecond precision.



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