Turn-on Fluorene Push-Pull Probes with High Brightness and Photostability for Visualizing Lipid Order in Biomembranes

The rational design of environmentally sensitive dyes with superior properties is critical for elucidating the fundamental biological processes and understanding the biophysical behavior of cell membranes. In this study, a novel group of fluorene-based push-pull probes was developed for imaging membrane lipids. The design of these fluorogenic conjugates is based on a propioloyl linker to preserve the required spectroscopic features of the core dye. This versatile linker allowed the introduction of a polar deoxyribosyl head, a lipophilic chain, and an amphiphilic/anchoring group to tune the cell membrane binding and internalization. It was found that the deoxyribosyl head favored cell internalization and staining of intracellular membranes, whereas an amphiphilic anchor group ensured specific plasma membrane staining. The optimized fluorene probes presented a set of improvements as compared to commonly used environmentally sensitive membrane probe Laurdan such as red-shifted absorption matching the 405 nm diode laser excitation, a blue-green emission range complementary to the red fluorescent proteins, enhanced brightness and photostability, as well as preserved sensitivity to lipid order, as shown in model membranes and living cells.