PT  - JOURNAL ARTICLE
AU  - Dora Mahecic
AU  - Davide Gambarotto
AU  - Kyle M. Douglass
AU  - Denis Fortun
AU  - Niccoló Banterle
AU  - Maeva Le Guennec
AU  - Khalid Ibrahim
AU  - Pierre Gönczy
AU  - Virginie Hamel
AU  - Paul Guichard
AU  - Suliana Manley
TI  - Homogeneous multifocal excitation for high-throughput super-resolution imaging
AID  - 10.1101/2020.01.08.895565
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.08.895565
4099  - http://biorxiv.org/content/early/2020/01/09/2020.01.08.895565.short
4100  - http://biorxiv.org/content/early/2020/01/09/2020.01.08.895565.full
AB  - Super-resolution microscopies, which allow features below the diffraction limit to be resolved, have become an established tool in biological research. However, imaging throughput remains a major bottleneck in using them for quantitative biology, which requires large datasets to overcome the noise of the imaging itself and to capture the variability inherent to biological processes. Here, we develop a multi-focal flat illumination for field independent imaging (mfFIFI) module, and integrate it into an instant structured illumination microscope (iSIM). Our instrument extends the field of view (FOV) to >100×100 µm2 without compromising image quality, and maintains high-speed (100 Hz), multi-color, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching multiple adjacent images together to perform fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM setup with ultrastructure expansion microscopy (U-ExM) to collect 3D images of hundreds of centrioles in human cells, as well as of thousands of purified Chlamydomonas reinhardtii centrioles per hour at an effective resolution of ∼35 nm. We apply classification and particle averaging to these large datasets, allowing us to map the 3D organization of post-translational modifications of centriolar microtubules, revealing differences in their coverage and positioning.