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.
🔬 Techniques
✨ Fluorophores
🏛️ Research Organizations (ROR)
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📊 Figures
Figure 1.
Design of fluorogenic dyes (top). Thiocarbonyl substitution at the carbonyl group of fluorophores results in very weak fluorescence via a PET-quenching mechanism. Upon irradiation with light, the thio...
Figure 2.
(A) Overlay of 1 HNMR spectra (6.2u20138.8 ppm) of SNile Red taken at the indicated light irradiation times (615 nm, 0.4 u03bc W cm u22122 ). (B) Normalized absorbance spectra of Nile Red, SNile Red, ...
Figure 3.
(A) Signals from photoactivated SNile Red colocalize with BODIPY 493/503. Adipocytes were incubated with SNile Red, BODIPY 493/503, and Hoechst 33342, followed by photoactivation using the 561 nm lase...
Figure 4.
(A) Differential interference contrast (DIC), widefield, and PALM images of an adipocyte labeled with SNile Red (2 u03bc M). (B) Magnified region featuring SNile Red labeled lipid droplets by super-re...
Figure 5.
(A) Scheme of the labeling of the protein of interest (POI) with the SDMAP-Halo ligand for fluorescence imaging. (B) A plot of the changes in relative intracellular fluorescence intensity of SDMAP-Hal...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
💬 Discussion
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