Strategic and practical guidelines for successful structured illumination microscopy.

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d.