Dirk-Peter Herten: Let the molecules do it! - Chemically controlled fluorescence probes in Microscopy

Abstract

Recent advances in fluorescence microscopy are yielding access to quantitative and structural information in fixed and living cells in an unprecedented manner. Imaging of biomolecular structures with a resolution better than 20 nm with single-molecule-based super-resolution microscopy and other nanoscopy techniques has already become routine in many labs. Most approaches, like PALM and STORM make use of photo-physical processes to specifically switch fluorescent probes between their bright and dark states to create only small subpopulations of fluorescent markers that can be individually resolved and localized with high-precision for reconstruction of a super-resolved fluorescence microscopy image. Recently, we have shown that also reversible chemical reactions can be used for stochastic activation and deactivation of fluorescent probes [1]. The experimental probe system is based on a reversible complexation of copper(II) cations which allows controlling the proportion of molecules in the off/on-state by changing the copper(II) concentration. Currently, we investigate alternative molecules and reactions to create different reliable probes that can be used for super-resolution microscopy. More recently, we could show that chemical switching of fluorescent probes can also be used to implement more channels for multiplexing in fluorescence microscopy which are orthognal to existing multiplexing modes, like color or fluorescence lifetime [2]. We envision that this principle will allow to dramatically the number of simultaneously imaged structures/proteins with fluorescence micoscopy when more switching reactions can be implemented. In this senes, the variety of chemical reactions that could be used for controling fluroescent probes adds a new dimension to fluorescence microscopy microscopy.