A Novel Viewpoint to Analyze Structured Illumination Microscopy (Sim) Data

Structured Illumination Microscopy (SIM) is an important tool among the Super Resolution Microscopy techniques [1]. SIM stands out for its capability to perform optical sectioning, multicolor channel acquisition and live cell imaging, thanks to its low phototoxicity. In SIM, the specimen is illuminated with a high-frequency grating pattern, instead of a uniform field. The multiplication of sample features and this pattern gives rise to Moiré fringes.The obtained interference pattern allows encoding of sub-diffraction sample's features into lower frequencies collected by the objective lens. The reconstruction process then decodes otherwise unresolvable high-frequency information creating the super-resolved image.Here we aim to analyze SIM data from a rather different standpoint, based on the idea of Separation of Photons by LIfetime Tuning (SPLIT)[2]. In SPLIT the sub-diffraction spatial information is encoded into an additional channel of the microscope. The information encoded within this channel is visualized and analyzed in the phasor plot and allows separating a fractional image component of higher spatial resolution corresponding to the center of the PSF.In the context of Stimulated Emission Depletion (STED) microscopy, we recently demonstrated that SPLIT can be used to decode spatial information encoded in the nanosecond fluorescence lifetime [2] or variations of the depletion power [3]. We show here that in the case of SIM it is possible to use SPLIT to analyze the information encoded into the images acquired at different illumination patterns. In particular an important aspect of SPLIT is that the Phasor Plot provides a visual, intuitive and direct evaluation of the acquired data [3].We demonstrate that knowledge of the illumination pattern can be used to perform SPLIT analysis and to reconstruct a super-resolved image bypassing the traditional Fourier reconstruction.As an application, we use the SPLIT-SIM approach to perform super-resolution imaging of chromatin-related structures.