Fluorescent Probes for Lipid Membranes: From the Cell Surface to Organelles

Biomembranes are ubiquitous lipid structures that delimit the cell surface and organelles and operate as platforms for a multitude of biomolecular processes. The development of chemical tools─fluorescent probes─for the sensing and imaging of biomembranes is a rapidly growing research direction, stimulated by a high demand from cell biologists and biophysicists. This Account focuses on advances in these smart molecules, providing a voyage from the cell frontier─plasma membranes (PM)─toward intracellular membrane compartments─organelles. General classification of the membrane probes can be based on targeting principles, sensing profile, and optical response. Probes for PM and organelle membranes are designed based on multiple targeting principles: conjugation with natural lipids or synthetic targeting ligands and in situ cell labeling by bio-orthogonal chemistry, conjugation to protein tags, and receptor-ligand interactions. Thus, to obtain membrane probes targeting PM with selectivity to one leaflet, we designed membrane anchor ligands based on a charged group and an alkyl chain. According to the sensing profile, we define basic membrane markers with constant emission and probes for biophysical and chemical sensing. The markers are built from classical fluorophores, exemplified by a series of bright cyanines and BODIPY dyes bearing the PM anchors (MemBright). Membrane probes for biophysical sensing are based on environment-sensitive fluorophores: (1) polarity-sensitive solvatochromic dyes; (2) viscosity-sensitive fluorescent molecular rotors; (3) mechanosensitive fluorescent flippers; and (4) voltage-sensitive electrochromic dyes. Our solvatochromic probes based on Nile Red (NR12S, NR12A, NR4A), Laurdan (Pro12A), and 3-hydroxyflavone (F2N12S) through polarity-sensing can visualize liquid ordered and disordered phases of lipid membranes, sense lipid order and its heterogeneity in cell PM, detect apoptosis, etc. Chemically sensitive probes, combining a dye, membrane-targeting ligand, and molecular recognition unit, enable the detection of pH, ions, redox species, lipids, and proteins at the biomembrane surface. In terms of the optical response profile, we can identify (1) fluorogenic (turn-on) probes, allowing background-free imaging; (2) ratiometric probes, e.g., solvatochromic probes, which enable ratiometric imaging by changing their emission/excitation color; (3) fluorescence lifetime-responsive probes, e.g., fluorescence molecular rotors and flippers, suitable for fluorescence lifetime imaging (FLIM); and (4) switchable probes, important for single-molecule localization microscopy. We showed that combining solvatochromic probes with on-off switching through a reversible binding specifically to cell PM enables the mapping of their biophysical properties with superior resolution. While the majority of efforts have been focused on PM, the probes for cellular organelles, such as endoplasmic reticulum, mitochondria, Golgi apparatus, etc., emerge rapidly. Thus, nontargeted solvatochromic probes can distinguish organelles by the emission color. Targeted solvatochromic probes based on Nile Red revealed unique signatures of polarity and lipid order of individual organelles and their different sensitivities to oxidative or mechanical stress. Lipid droplets, which are membraneless lipidic structures, constitute another interesting organelle target for probing the cell stress. Currently, we stand at the beginning of a long route with big challenges ahead, in particular (1) to achieve superior organelle specificity; (2) to label specific biomembrane leaflets, notably the inner leaflet of PM; (3) to detect lipid organization in a proximity of specific proteins; and (4) to probe biomembranes in tissues and animals.