Bioorthogonal Ligation-Activated Fluorogenic FRET Dyads.

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. Such relay 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.

Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes.

A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes O generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.