Spatiotemporal rank filtering improves image quality compared to frame averaging in 2-photon laser scanning microscopy

Abstract

Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Twophoton laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.