Nonlinear structured illumination microscopy (nSIM) is an effective approach for super-resolution wide-field fluorescence microscopy with a theoretically unlimited resolution. In nSIM, carefully designed, highly-contrasted illumination patterns are combined with the saturation of an optical transition to enable sub-diffraction imaging. While the technique proved useful for two-dimensional imaging, extending it to three-dimensions is challenging due to the fading of organic fluorophores under intense cycling conditions. Here, we present a compressed sensing approach that allows 3D sub-diffraction nSIM of cultured cells by saturating fluorescence excitation. Exploiting the natural orthogonality of speckles at different axial planes, 3D probing of the sample is achieved by a single two-dimensional scan. Fluorescence contrast under saturated excitation is ensured by the inherent high density of intensity minima associated with optical vortices in polarized speckle patterns. Compressed speckle microscopy is thus a simple approach that enables 3D super-resolved nSIM imaging with potentially considerably reduced acquisition time and photobleaching.
Bibliographical noteFunding Information:
The authors thank Isabelle Fanget for preparing the HeLa cells, Julien Pernier for providing the actin filaments and the corresponding staining protocol, and Laura Caccianini, Madjouline Abou Ghali, and Dany Khamsing for helping with samples. The authors also acknowledge Grace Kuo and Laura Waller for their support with the FISTA algorithm, and Marcel Lauterbach for careful reading of the manuscript. This work was supported by grants from the Région Ile-de-France (PhD program) and the French-Israeli Laboratory ImagiNano. It has received funding from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation program (Grants no. 677909) and from the French National Agency (ANR-10-INBS-04-01). This research was also supported by the ISRAEL SCIENCE FOUNDATION (Grant no. 1361/ 18), the Azrieli Foundation, the Centre National de la Recherche Scientifique and the France Bio-Imaging infrastructure.
© 2019, The Author(s).