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Super-resolution imaging of multiple cells by optimized flat-field epi-illumination


Biological processes are inherently multi-scale, and supramolecular complexes at the nanoscale determine changes at the cellular scale and beyond. Single-molecule localization microscopy (SMLM)1,2,3 techniques have been established as important tools for studying cellular features with resolutions of the order of around 10 nm. However, in their current form these modalities are limited by a highly constrained field of view (FOV) and field-dependent image resolution. Here, we develop a low-cost microlens array (MLA)-based epi-illumination system—flat illumination for field-independent imaging (FIFI)—that can efficiently and homogeneously perform simultaneous imaging of multiple cells with nanoscale resolution. The optical principle of FIFI, which is an extension of the Köhler integrator, is further elucidated and modelled with a new, free simulation package. We demonstrate FIFI's capabilities by imaging multiple COS-7 and bacteria cells in 100 × 100 μm2 SMLM images—more than quadrupling the size of a typical FOV and producing near-gigapixel-sized images of uniformly high quality.

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Figure 1: Wide-field epi-illumination is not optimized for SMLM imaging of large FOVs.
Figure 2: Layout and design parameters for FIFI.
Figure 3: Simulated 1D sample plane irradiance profiles through the centre of the illuminated region demonstrate the strong dependence of the illumination homogeneity on the design parameters.
Figure 4: Uniform, large FOV STORM imaging of multiple eukaryotic cells or hundreds of bacterial cells in a single image.


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The authors would like to thank K. Bellvé for providing the pgFocus hardware, F. Huang for his help in troubleshooting the sCMOS localization software, D. G. Voelz for providing code examples for modelling partially coherent optical fields, R. Völkel for discussions about the Köhler integrator and N. Berliner, D. Ott, M. Weidman and P. Ramdya for useful discussions. Research in S.M.'s laboratory is supported by the National Centre of Competence in Research Chemical Biology and by the European Research Council (ERC 243016-PALMassembly). K.M.D. is supported by a Transition Postdoc Fellowship (2014/227).

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Authors and Affiliations



S.M. and K.M.D. conceived the idea. K.M.D. designed the epi-illumination system, built the microscope and wrote the simulation code. C.S. and A.L. performed the cell preparation and imaging. C.S. and A.A. assisted K.M.D. with building and testing the microscope. K.M.D. wrote the manuscript with contributions from all authors.

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Correspondence to Kyle M. Douglass or Suliana Manley.

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The authors declare no competing financial interests.

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Douglass, K., Sieben, C., Archetti, A. et al. Super-resolution imaging of multiple cells by optimized flat-field epi-illumination. Nature Photon 10, 705–708 (2016).

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