Bioothogonally applicable, π-extended rhodamines for super-resolution microscopy imaging for intracellular proteins.

A set of new, bioorthogonally applicable tetrazine and polarity modulated double fluorogenic π-extended rhodamine probes were synthesized. Fluorogenicity and cell labeling experiments suggest that combination of the two quenching mechanisms allows low background labeling schemes even for probes with poor reactivity based fluorogenicity. Two of the new probes were tested in biological labeling schemes of intracellular proteins both in fixed and live cells. The labeled cells were subsequently subjected to confocal and STED imaging. These studies revealed that the rhodaindanes tested are membrane permeable, can stand the challenging environment of live cells and suitable for bioorthogonal, site-specific labeling of intracellular proteins. Furthermore, we found that both probes are suitable for subdiffraction imaging of the labeled structures using STED microscopy.