Focus-ISM: A universal tool to enhance optical sectioning in super-resolution microscopy.

Image Scanning Microscopy (ISM) is the evolution of Confocal Laser Scanning Microscopy (CLSM), enabling both super-resolution and excellent Signal-to-Noise Ratio (SNR). In ISM, an excitation beam scans the sample, and a detector array collects the fluorescence light, generating a micro-image for each scan point. However, although maximizing the SNR, the lack of a conventional pinhole degrades optical sectioning. Here, we present a new approach that fully exploits the information provided by the detector array, enabling optical sectioning in ISM without sacrificing the SNR. In detail, each element of the detector array generates a shifted CLSM image. First, we register the images with our adaptive pixel reassignment (APR) algorithm. The modified micro-images depend uniquely on the axial position of the fluorophores. Then, we fit each micro-image to a simple two-component model, enabling the removal of the out-of-focus background. The final image contains only the signal stemming from the focal plane. Notably, the same procedure is well suited to any laser scanning technique, such as STED or multi-photon microscopy, opening new paths for super-resolution imaging of thick samples.