Evaluation of resonant scanning as a high-speed imaging technique for two-photon imaging of cortical vasculature

Abstract

We demonstrate a simple, low-cost two-photon microscope design with both galvo-galvo and resonant-galvo scanning capabilities. We quantify and compare the signal-to-noise ratios and imaging speeds of the galvo-galvo and resonant-galvo scanning modes when used for murine neurovascular imaging. The two scanning modes perform as expected under shot-noise limited detection and are found to achieve comparable signal-to-noise ratios. Resonant-galvo scanning is capable of reaching desired signal-to-noise ratios using less acquisition time when higher excitation power can be used. Given equal excitation power and total pixel dwell time between the two methods, galvo-galvo scanning outperforms resonant-galvo scanning in image quality when detection deviates from being shot-noise limited.

A deep learning approach for improving two-photon vascular imaging speeds

“…We hypothesize that this is a result of the denoising that occurs in the PSSR process. Since all upscaled images had higher intensity thresholding accuracy compared to the original image, we further postulate that upscaled images have less noise overall because fewer pixels are physically captured-all pixels created during upscaling are interpolated from neighboring pixel values and thus free of noise from the image acquisition process 21 . For performing vectorization, however, the diminished noise does not offset accuracy losses from the upscaling process.…”

“…All images were acquired using a custom-built two-photon microscope, previously described 21 . The excitation source was an ytterbium fiber amplifier with an output beam of 1050 nm wavelength, 120 fs pulse width, and 80 MHz repetition rate 29 .…”

“…Image stacks were acquired with 3 μm axial spacing. A resonant-galvanometer scanning system was used 21 , with average pixel dwell times of 87.8 ns and 20-frame averaging. Power at the sample did not exceed 170 mW and was identical between low- and high-resolution pairs.…”

“…Image stacks were acquired with 3 µm axial spacing. A resonant-galvanometer scanning system was used 21 , with average pixel dwell times of 87.8 ns and 20-frame averaging.…”

Interpreting Inverse Correlation Time: from Blood flow to Vascular Network

“…For 2P imaging, images were acquired with a custom microscope and laser system 42,43 . The same anesthesia procedure as above-mentioned was used.…”