Caging-group-free photoactivatable fluorescent dyes

• Caging-group-free activation: Reduces molecular complexity, simplifies chemical synthesis, and improves solubility and cell permeability.
• Rapid and efficient photoactivation: Quantitative conversion to the fluorescent state with low UV (one photon) or NIR (two photon) light doses compatible with the complex cellular environment.
• Enhanced photostability: Reduced photobleaching enables longer observation times and single-molecule-level sensitivity.
• Live-cell labeling: Cell membrane permeability allows for rapid labeling with state- of the art technologies such as HaloTag, SNAP-tag, and bioorthogonal click chemistry.
• No toxic or reactive byproducts: Ensures cell viability and integrity during long-term imaging processes.
• Compatible with all microscopes: Suitable for a variety of conventional (confocal, wide-field) and super-resolution microscopy techniques, including PALM/STORM, STED and MINFLUX.
• Additive-free imaging: No dedicated media/buffers enables detailed observation of cellular processes without compromising cell health.
• Broad color options: Combinable yellow-green, orange, and red emission options with a single dedicated photoactivation wavelength.
• Large Stokes shift option: Expands color-channel selection, facilitates multiplexing, and reduces spectral overlap.

• Fluorescence microcopy and nanoscopy: Photoactivatable bright and photostable labels for live-cell labeling with protein tags, as organelle specific markers, and immunostaining, in conventional and super-resolution methods.
• Biomedical research: Studying cellular processes, such as organelle or protein dynamics, and protein–protein interactions.
• Pharmaceutical development: High-throughput imaging screening for drug discovery, target validation and pathway analysis.
• Clinical diagnostic: Enhanced labelling for diagnostic probe and sensor development research.
• Material science: Tracing and mapping of materials, surfaces and interfaces at the nanoscale, flow and diffusion studies.
• Neuroscience: Observation of neuronal structure and activity with high precision.

Traditional photoactivatable dyes rely on caging groups that add to the molecular mass of the fluorescent label, decrease solubility and can release toxic photoproducts. These new caging-group-free fluorescent labels have been designed with live-cell applications in mind. These labels provide an optimized tool for researchers, with significant improvements over prior art in terms of biocompatibility, efficiency, and imaging quality.

The technology leverages a novel photoactivation mechanism for a new class of photoactivatable xanthone-based dyes that can be precisely controlled by light without the need for traditional caging groups nor media additives. Upon irradiation, these dyes transition from a non-fluorescent to a bright and photostable fluorescent state, enabling high-resolution imaging of live cells. The dyes cover a wide spectrum of wavelengths and they are compatible with various labeling techniques and state of the art super-resolution microscopy methods.

PCT application (WO 2023284968 A1), entered the national phase in the USA and Europe

R. Lincoln et al., Nature Chemistry 2022, 14, 1013-1020. A general design of caging-group-free photoactivatable fluorophores for live-cell nanoscopy, https://doi.org/10.1038/s41557-022-00995-0.

A. Aktalay et al., ACS Central Science 2023, 9, 1581-1590. Bioorthogonal caging-group-free photoactivatable probes for minimal-linkage-error nanoscopy, https://doi.org/10.1021/acscentsci.3c00746.

Likhotkin et al., Journal of the American Chemical Society 2023, 145, 1530-1534. Photoactivatable large Stokes shift fluorophores for multicolor nanoscopy, https://doi.org/10.1021/jacs.2c12567.

Remmel et al. Small Methods 2024, 2301497. Photoactivatable xanthone (PaX) dyes enable quantitative, dual color, and live-cell MINFLUX nanoscopy, https://doi.org/10.1002/smtd.202301497.