Multimodal single-molecule microscopy with continuously controlled spectral resolution

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number100013
JournalBiophysical Reports
Volume1
Issue number1
DOIs
StatePublished - 8 Sep 2021

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