Computational NeurosciencePixel timing correction in time-lapsed calcium imaging using point scanning microscopy

•Point scanning introduces a delay between image pixels when used at frequencies below 10 Hz.•The time delays can be corrected by using temporal interpolation.•Real two-photon imaging data were produced at different scanning frequencies for comparison.•Simulations show that the interpolation methods present a similar performance and are able to correct the time delays introduced by point scanning.•Qualitative and quantitative results on the real imaging data show the performance of pixel timing correction for point scanning.

In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.