Context (archaeology)

Biology

Photochemistry

Materials science

Biological imaging

Microscopy

Biochemistry

Computer science

Fluorophore

Super-resolution microscopy

Tetrazine

Paleontology

Quenching (fluorescence)

Physics

Nanotechnology

Organic chemistry

Optics

Image (mathematics)

Fluorescence

Artificial intelligence

Chemistry

Cell

Fluorescence microscope

Superresolution

Biophysics

Live cell imaging

Biomolecule

STED microscopy

ACS Central Science

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine–dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine–dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy