Conspectus
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 toolsfluorescent
probesfor 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 frontierplasma
membranes (PM)toward intracellular membrane compartmentsorganelles.
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 disting...