Single-Atom Fluorescence Switch: A General Approach toward Visible-Light-Activated Dyes for Biological Imaging

Abstract

Photoactivatable fluorophores afford powerful molecular tools to improve the spatial and temporal resolution of subcellular structures and dynamics. By performing a single sulfur-for-oxygen atom replacement within common fluorophores, we have developed a facile and general strategy to obtain photoactivatable fluorogenic dyes across a broad spectral range. Thiocarbonyl substitution within fluorophores results in significant loss of fluorescence via a photoinduced electron transfer-quenching mechanism as suggested by theoretical calculations. Significantly, upon exposure to air and visible light residing in their absorption regime (365–630 nm), thio-caged fluorophores can be efficiently desulfurized to their oxo derivatives, thus restoring strong emission of the fluorophores. The effective photoactivation makes thio-caged fluorophores promising candidates for super-resolution imaging, which was realized by photoactivated localization microscopy (PALM) with low-power activation light under physiological conditions in the absence of cytotoxic additives (e.g., thiols, oxygen scavengers), a feature superior to traditional PALM probes. The versatility of this thio-caging strategy was further demonstrated by multicolor super-resolution imaging of lipid droplets and proteins of interest.

Single-atom replacement as a general approach towards visible-light/near-infrared heavy-atom-free photosensitizers for photodynamic therapy

“…More importantly, upon excitation with visible light, singlet oxygen species are generated by these thio-based fluorophores, indicating the likelihood that they can serve as PSs for PDT applications. 42 During our preparation of this manuscript, Nguyen et al reported the preparation of thio-based naphthalimide dyes and their utility for photodynamic studies, 43 reinforcing the potential utility of thio-based fluorophores for PDT. Taking all of the above into account, we envision that thiocarbonyl substitution within common biocompatible fluorophores of any wavelength represents a general approach toward yielding heavy-atom-free PSs with excellent potential for use in PDT ( Fig.…”

“… 39 – 41 In 2019, we developed a general approach for preparing thio-based fluorophores across a broad emission range (294–626 nm) for use in biological imaging. 42 We found that these thio-based fluorophores exhibited significant bathochromic shifts in absorption maxima (more than 170 nm), as well as enhanced extinction coefficients compared to corresponding carbonyl compounds. These properties improve the tissue penetration and light absorbance efficiency, respectively, of the thio-based compounds.…”

Thionated organic compounds as emerging heavy-atom-free photodynamic therapy agents

“…This chemistry was further extended through the incorporation of a dual-functional morpholine group (i.e., electron-donating and lysosome-targeting) into the thionaphthalimide, which was shown to generate ROS in the lysosomes of HeLa cells and to cause cell death upon light irradiation. 79 Xiao and co-workers 80,81 have recently generalized the thionation approach to design heavy-atom-free photosensitizers that can be photoactivated by visible or near-infrared light. Thionation of the carbonyl groups leads to near-unity singletoxygen quantum yields and large molar absorption coefficients at wavelengths as long as 760 nm (Fig.…”

“…Collectively, these results indicate the promising value of thionated photosensitizers as heavy-atom-free photodynamic agents for PDT applications based on intracellular generation of ROS. 78,79,81…”

Tetrazine as a general phototrigger to turn on fluorophores

“…Photoactivatable uorophores, also called turn-on uorophores, [1][2][3][4][5][6][7] have numerous advanced biological applications, including detection and release of ions [8][9][10][11] and metabolites, [12][13][14][15] monitoring of enzyme activity, [16][17][18][19] and multiple types of specialized microscopy. 7,[20][21][22][23][24][25] Most of these photoactivatable uorophores are masked by a reactive "cage" group intimately attached to the uorophore to alter its photophysical properties or to serve as an energy-transfer agent for use in a uorescence resonance energy transfer (FRET) pair. 26 Upon light or chemical treatment, these "caged" uorophores undergo chemical reactions that release the "cage" groups to regenerate the uorophores in their active forms.…”