Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells

Abstract

AbstractFluorogenic dyes with high brightness, large turn‐on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high‐resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N‐diethyl group with a pyrrole substituent. The resulting dye, Rh‐NH, exhibited doubled Stokes shifts (54 nm) and a red‐shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh‐NH preferentially exists in a non‐emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh‐NH with significant fluorescence turn‐on, making it a suitable candidate for wash‐free, high‐contrast lysosome tracking. Consequently, Rh‐NH enabled us to successfully explore stimulated emission depletion (STED) super‐resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.