Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Suchr elay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells.These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows.S ubdiffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopyis also presented.

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Suchr elay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells.These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows.S ubdiffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopyis also presented.