Multimodal single-molecule microscopy with continuously controlled spectral resolution

• Published in: Biophysical Reports
• Main Authors: Jonathan Jeffet, Ariel Ionescu, Yael Michaeli, Dmitry Torchinsky, Eran Perlson, Timothy D. Craggs, Yuval Ebenstein
• Format: Article
• Language: English
• Published: Elsevier 2021-09-01
• Online Access: http://www.sciencedirect.com/science/article/pii/S2667074721000136

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.