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Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination

View ORCID ProfileSheng Liu, Fang Huang
doi: https://doi.org/10.1101/586404
Sheng Liu
1Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA
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  • For correspondence: liu1840@purdue.edu fanghuang@purdue.edu
Fang Huang
1Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana, USA
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  • For correspondence: liu1840@purdue.edu fanghuang@purdue.edu
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Abstract

Over the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combining with single-molecule detection/localization and adaptive optics, iPALM/4PiSMS/W-4PiSMSN allowed 10-15 nm isotropic 3D resolution throughout the whole cell. However, further improving the achieved 3D resolution poses significantly challenges which, in part, is blocked by the complexity of single-molecule emission pattern generated by these systems rendering a large portion of information carrying photons unusable. Here we introduce a localization algorithm that achieves the theoretical information limit for 4Pi based single-molecule switching nanoscopy (4Pi-SMSN), and demonstrates improvements in resolution, accuracy as well as applicability comparing with the state of art 4Pi-SMSN methods. Further, with a novel 4Pi-compatible light-sheet illumination reducing the fluorescence background by >5-fold, we demonstrated the new system enables further improvement in the achievable resolution of 4Pi/interferometric single-molecule imaging systems.

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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 March 22, 2019.
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Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination
Sheng Liu, Fang Huang
bioRxiv 586404; doi: https://doi.org/10.1101/586404
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Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination
Sheng Liu, Fang Huang
bioRxiv 586404; doi: https://doi.org/10.1101/586404

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