Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.

A novel class of ZnSalens (ZnL₁1_–10–10) with lipophilic and cationic conjugates as optical probes in single and two-photon fluorescence microscopy images of living cells were prepared, which exhibited chemo- and photostability, low cytotoxicity and high subcellular selectivity.

Construction of a colour palette based on modular “core” dyes with tuneable emission and tailor-made intracellular localization is of importance to visualize and distinguish different organelles and even observe their intracellular cross-talking. However, due to lack of structural information linking photophysical properties and the specificity of subcellular localizations, the modification of photophysical properties cannot always enable tailor-made specificity. In this work, we report the construction of a ZnSalen/Salophen library (48 examples) and study the one- and two-photon optical properties, lipophilicity and subcellular distribution of the complexes. Experimental and theoretical studies demonstrate that changes of the electronic states of the diamine moieties are effective for modulation of the photophysical properties of the ZnSalen/Salophen complexes. On the other hand, the subcellular localization is highly related to the lipophilicity of the ZnSalen/ZnSalophen complexes, which is correlated to the functionalization of the N-substitutients at the 4-position. Thus, the orthogonality between the photophysical properties and the subcellular localizations of the ZnSalen/Salophen complexes, regulated by discrete moieties (diamines and salicylaldehydes), renders them suitable fluorophores for the modular design of bioprobes in live cell imaging. More importantly, to demonstrate their potential application, we applied these Zn complexes as a colour palette for multicolour imaging in live cells using one- and two-photon fluorescence microscopes.

Rational design of effective photoactivatable/photoswitchable fluorophores, by introducing appropriate photoreactions to tune the electron transfer processes of the ground or/and excited states and switch fluorescence off/on, is crucial to achieve high temporal and spatial resolution in live cell (organism) imaging. Besides one photon activatable fluorophores, it is highly desirable to develop two photon activatable fluorophores using light in the NIR or IR region, which reduces photodamage and allows deep penetration into cells or tissues. In this work, we describe the design of one and two photon activatable ZnSalen by incorporating thioether moieties in the 3,3′-positions which quenches the fluorescence as a result of a PET process. Through one or two photon irradiation, the thioethers can be oxidized to sulfoxides and the fluorescence of ZnSalen “switched on”, due to the electron-withdrawing sulfoxides, which perturbs the PET process. We further demonstrate the application of this ZnSalen as a photoactivatable fluorophore in living cells using one and two photon fluorescence microscopies. In two photon microscopy, a high signal to noise contrast was achieved by irradiation with an 840 nm laser. Moreover, this photoactivatable ZnSalen was successfully applied in bioimaging in a model living organism Caenorhabditis elegans (C. elegans), and ca. 5 times fluorescence intensity increase was observed after one and two photon irradiation. This paradigm by modulation of the PET process in ZnSalen provides a promising methodology for the design of photoactivatable fluorophores in further applications in super resolution molecular imaging.