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

Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon 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.利用光学显微镜对生物样本进行活体成像，其性能受限于空间分辨率、时间分辨率、完整样本成像深度与光毒性之间的多重权衡。双光子激光扫描显微镜（Two-photon laser scanning microscopy, 2P-LSM）是体内浑浊样本成像的金标准，传统上通过对焦平面执行连续光栅扫描，并对采集信号进行时间积分，来构建具备足够信噪比（signal-to-noise ratio, SNR）的图像。本文提出了时空秩滤波——一种可替代时间积分的非线性方法，其可在双光子激光扫描显微镜图像的采集过程中，通过选择性抑制噪声来更高效地利用采集到的光子。此举可在保留图像边缘与细微细节的同时，大幅提升信噪比。实际应用中，该方法可使采集时长至少缩短至原时长的1/4，这对生物系统的时序成像而言是一项重大性能提升。