Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

Abstract

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 and 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 superresolution SIM methods, and with newer Bayesian restoration approaches which we are developing. ConclusionVarious methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments is not typically published. Publicly 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 was processed with SIMToolbox, an open source and freely available software solution for SIM.

Imaging Membrane Curvature inside a Model Immunological Synapse in RBL-2H3 Cells using TIRF Microscopy with Polarized Excitation

“…In the future, to help reveal the nature of the curvature features we detect, live cell superresolution microscopy will be valuable. Structured illumination microscopy [31,32] would allow imaging of receptor patch formation with super-resolution, and single molecule localization microscopy methods [33,34] would allow evaluation of nanoscopic membrane features in relation to…”

Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction

“…The imaging rate of processed result frames was therefore 3.6 Hz. The full image sequence is available at [53 and also available at GigaDB [54]. We further analyzed these data as shown in the Supplementary Figs.…”

Flexible multiplane structured illumination microscope with a four-camera detector

“…This project uses a home-built SIM set-up based on the same design as described in our previous publications [8,12,29,30]. The SIM system is based on an IX83 microscope (Olympus, Tokyo, Japan) with our 4-camera setup serving as a detector.…”