ISM 2022 (Microscopy)


Yuval Ebenstein 1 Jonathan Jeffet 1 Ariel Ionescu 1 Eran Perlson 1 Timothy Craggs 2
1Physical Chemistry, Tel Aviv University, Tel Aviv, Israel
2Department of Chemistry, University of Sheffield, Sheffield, UK

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 to previous super-resolution spectral imaging technics. We demonstrate the utility of CoCoS in three experimental formats, single-molecule spectroscopy, single-molecule Förster resonance energy transfer (smFRET) 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.