Multimodal single-molecule microscopy with continuously controlled spectral resolution

Jonathan Jeffet; Ariel Ionescu; Yael Michaeli; Dmitry Torchinsky; Eran Perlson; Timothy D Craggs; Yuval Ebenstein

doi:10.1016/j.bpr.2021.100013

Multimodal single-molecule microscopy with continuously controlled spectral resolution

Biophys Rep (N Y). 2021 Aug 6;1(1):100013. doi: 10.1016/j.bpr.2021.100013. eCollection 2021 Sep 8.

Authors

Jonathan Jeffet^{1

2

3}, Ariel Ionescu^{4

5}, Yael Michaeli^{1

2}, Dmitry Torchinsky^{1

2

3}, Eran Perlson^{4

5}, Timothy D Craggs⁶, Yuval Ebenstein^{1

2

3}

Affiliations

¹ Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel.
² Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel.
³ Center for Light Matter Interaction, Tel Aviv University, Tel Aviv, Israel.
⁴ Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
⁵ Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.
⁶ Sheffield Institute for Nucleic Acids, Department of Chemistry, University of Sheffield, Sheffield, United Kingdom.

Abstract

Color is a fundamental contrast mechanism in fluorescence microscopy, providing the basis for numerous imaging and spectroscopy techniques. Building on spectral imaging schemes that encode color into a fixed spatial intensity distribution, here, we introduce continuously controlled spectral-resolution (CoCoS) microscopy, which allows the spectral resolution of the system to be adjusted in real-time. By optimizing the spectral resolution for each experiment, we achieve maximal sensitivity and throughput, allowing for single-frame acquisition of multiple color channels with single-molecule sensitivity and 140-fold larger fields of view compared with previous super-resolution spectral imaging techniques. Here, we demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer, and multicolor single-particle tracking in live neurons, using a range of samples and 12 distinct fluorescent markers. A simple add-on allows CoCoS to be integrated into existing fluorescence microscopes, rendering spectral imaging accessible to the wider scientific community.