Anionic Cyanine Membrane Probes for Live Cells and In Vivo Fluorescence Imaging

Molecular probes for cell plasma membranes are indispensable for fluorescence imaging. Herein, we present an array of five anionic cyanine-based turn-on plasma membrane probes with emission spanning from green to near infrared. They are analogous to the commonly used MemBright probe family, where two zwitterionic anchor groups are replaced with anionic sulfonates with dodecyl chains. The developed probes provide selective wash-free staining of plasma membranes of live cells in vitro, featuring improved brightness and slower internalization inside the cells. In comparison to protein-based (wheat germ agglutinin) membrane markers, new membrane probes provide better staining in poorly accessible cell-cell contacts. A key challenge is to stain cell plasma membranes directly <i>in vivo</i>. During <i>in vivo</i> brain tissue imaging in living mice by two-photon microscopy, the anionic cyanine probes allowed us to visualize in detail the pyramidal neurons with high image quality, clearly resolving neuron soma, dendrites with dendritic spines, and axons with axonal boutons. The developed anionic cyanine-based plasma membrane probes constitute an important extension of the toolbox for plasma membrane research.