Imaging tissues and cells beyond the diffraction limit with structured illumination microscopy and Bayesian image reconstruction
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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 super-resolution SIM methods, and with newer Bayesian restoration approaches which we are developing.
Conclusion
Various 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.
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Now published in GigaScience doi: 10.1093/gigascience/giy126
Jakub Pospíšil 1Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague 6, Czech RepublicFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteTomáš Lukeš 1Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague 6, Czech Republic2Laboratory of Nanoscale Biology, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, SwitzerlandFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteJustin Bendesky 3UCCS center for the Biofrontiers Institute, University of Colorado at Colorado Springs, 1420 Austin Bluffs Parkway, Colorado Springs, Colorado, …
Now published in GigaScience doi: 10.1093/gigascience/giy126
Jakub Pospíšil 1Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague 6, Czech RepublicFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteTomáš Lukeš 1Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague 6, Czech Republic2Laboratory of Nanoscale Biology, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, SwitzerlandFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteJustin Bendesky 3UCCS center for the Biofrontiers Institute, University of Colorado at Colorado Springs, 1420 Austin Bluffs Parkway, Colorado Springs, Colorado, 80918, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteKarel Fliegel 1Department of Radioelectronics, Faculty of Electrical Engineering, Czech Technical University in Prague, Technická 2, 16627 Prague 6, Czech RepublicFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteKathrin Spendier 3UCCS center for the Biofrontiers Institute, University of Colorado at Colorado Springs, 1420 Austin Bluffs Parkway, Colorado Springs, Colorado, 80918, USA4Department of Physics and Energy Science, University of Colorado at Colorado Springs, 1420 Austin Bluffs Parkway, Colorado Springs, Colorado, 80918, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteGuy M. Hagen 3UCCS center for the Biofrontiers Institute, University of Colorado at Colorado Springs, 1420 Austin Bluffs Parkway, Colorado Springs, Colorado, 80918, USAFind this author on Google ScholarFind this author on PubMedSearch for this author on this siteORCID record for Guy M. Hagen
A version of this preprint has been published in the Open Access journal GigaScience (see paper https://doi.org/10.1093/gigascience/giy126 ), where the paper and peer reviews are published openly under a CC-BY 4.0 license.
These peer reviews were as follows:
Reviewer 1: http://dx.doi.org/10.5524/REVIEW.101401 Reviewer 2: http://dx.doi.org/10.5524/REVIEW.101402
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