ABSTRACT
We report on the synthesis of two fluorescent probes which can be activated by ß-Galactosidase (ß-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with ß-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed ß-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.
Subject(s)
Fluorescent Dyes , beta-Galactosidase , beta-Galactosidase/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Humans , Microscopy, Fluorescence/methods , Staining and Labeling/methods , Microscopy, Confocal , Vimentin/metabolismABSTRACT
New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared and characterized. The dye core displays large Stokes shift (in H2O, abs./emission max. 480/550â nm; ϵ~19 000, τfl>4 ns), strong binding with the host (~560â nM Kd) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595â nm or 655â nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable.
ABSTRACT
The single-molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer-scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561 nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560 , PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single-molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561 nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive-independent nature of PaX photoactivation is showcased in fast live-cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561 nm MINFLUX imaging.
ABSTRACT
Our report describes the facile and scalable preparation of 9H-thioxanthen-9-one 10,10-dioxides via Pd-catalyzed sulfonylative homocoupling of the appropriately substituted benzophenones. This transformation provides a straightforward route to previously unreported sulfone-fluoresceins and -fluorones. Several examples of these red fluorescent dyes have been prepared, characterized, and evaluated as live-cell permeant labels compatible with super-resolution fluorescence microscopy with 775 nm stimulated emission depletion.