A Bright Surprise: Live‐Cell Labeling with Negatively Charged Fluorescent Probes based on Disulfonated Rhodamines and HaloTag

Authors:

Dr. Dojin Kim, Dr. Stefan Stoldt, Dr. Michael Weber, Prof. Stefan Jakobs, Dr. Vladimir N. Belov, Prof. Stefan W. Hell

Keywords:

Fluorescent Dyes, Rhodamines, Halo-Tag, Live Cell Imaging

Abstract:

Disulfonated rhodamines are photostable and bright dyes widely used in life science and optical microscopy. However, di-sulfonated dyes were considered cell impermeable and not applicable in living cells. We challenged this assumption with 5 most popular rhodamines (Rho) having two carboxylic acid residues, versatile sulfonation patterns and emitting green (AS488), yellow (Rho530), orange (Rho565) and red (Rho590 and STAR RED) light. The probes comprising one rhodamine entity and a HaloTag^TM amine (O2) ligand (x) were prepared and applied for labeling of living, Vimentin-Halo (VIM-Halo) expressing U-2 OS cells. Surprisingly, we observed specific and bright staining with simplest compounds Rho590-x, Rho565-x and Rho530-x bearing two negative charges; they performed well also in stimulated emission depletion (STED) microscopy. Specific staining and red shifts in absorption and emission bands were observed with other probes having one negative charge; they were prepared by native chemical ligation and esterification.

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