Spatial Phasor Analysis for Optical Sectioning Nanoscopy

Super-resolution microscopy has broken the traditional resolution barrier of optical microscopy. However, its application in imaging live and thick specimens has been limited. To date, optical sectioning in super-resolution microscopy either rely on inaccurate background estimation or been hindered in live-cell imaging by excessive complexity and cost. Here, we report spatial phasor image scanning microscopy (spISM), which aims to enhance the optical sectioning by a factor of ∼2 without drawbacks for any microscope equipped with a detector array. By incorporating spatial-domain phasor analysis into image scanning microscopy, spISM decodes information about the axial position, thus accurately identifying in-focus and out-of-focus signals. We demonstrate that this approach is automatic, adaptive, and robust to specimen and microscope setups. It has a rapid processing speed, enabling multicolor imaging in live-cell. The performance of the reported approach is validated by imaging up to eight subcellular structures. As spISM is fully compatible with laser scanning microscopy, it holds great potential to become a turn-key solution for biological research.