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Enabling spectrally resolved single-molecule localization microscopy at high emitter densities

View ORCID ProfileKoen J.A. Martens, View ORCID ProfileMartijn Gobes, Emmanouil Archontakis, Roger R. Brillas, Niels Zijlstra, View ORCID ProfileLorenzo Albertazzi, View ORCID ProfileJohannes Hohlbein
doi: https://doi.org/10.1101/2022.06.29.498127
Koen J.A. Martens
aLaboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands
bInstitute for Microbiology and Biotechnology, Rheinische-Friedrich-Wilhelms-Universität Bonn, Bonn, Germany
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  • For correspondence: koenjamartens@gmail.com Johannes.Hohlbein@wur.nl
Martijn Gobes
aLaboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands
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Emmanouil Archontakis
cDepartment of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands
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Roger R. Brillas
cDepartment of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands
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Niels Zijlstra
aLaboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands
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Lorenzo Albertazzi
cDepartment of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven, Netherlands
dNanoscopy for Nanomedicine, Institute for Bioengineering of Catalonia, Barcelona, Spain
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Johannes Hohlbein
aLaboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, the Netherlands
eMicrospectroscopy Research Facility, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands
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  • For correspondence: koenjamartens@gmail.com Johannes.Hohlbein@wur.nl
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Abstract

Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common subpixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Competing Interest Statement

Wageningen University filed a patent application describing this method (WO2022053649).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 02, 2022.
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Enabling spectrally resolved single-molecule localization microscopy at high emitter densities
Koen J.A. Martens, Martijn Gobes, Emmanouil Archontakis, Roger R. Brillas, Niels Zijlstra, Lorenzo Albertazzi, Johannes Hohlbein
bioRxiv 2022.06.29.498127; doi: https://doi.org/10.1101/2022.06.29.498127
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Enabling spectrally resolved single-molecule localization microscopy at high emitter densities
Koen J.A. Martens, Martijn Gobes, Emmanouil Archontakis, Roger R. Brillas, Niels Zijlstra, Lorenzo Albertazzi, Johannes Hohlbein
bioRxiv 2022.06.29.498127; doi: https://doi.org/10.1101/2022.06.29.498127

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