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Total internal reflection fluorescence microscopy with polarized excitation (P-TIRF) can be used to image nanoscale curvature phenomena in live cells. We used P-TIRF to visualize rat basophilic leukemia cells (RBL-2H3 cells) primed with fluorescent anti-dinitrophenyl (anti-DNP) immunoglobulin E (IgE) coming into contact with a supported lipid bilayer containing mobile, monovalent DNP, modeling an immunological synapse. The spatial relationship of the IgE-bound high affinity IgE receptor (FcεRI) to the ratio image of P-polarized excitation and S-polarized excitation was analyzed. These studies help correlate the dynamics of cell surface molecules with the mechanical properties of the plasma membrane during synapse formation. 

Background Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.