Fluorescent biomembrane probe for ratiometric detection of apoptosis.

Herein, we developed the first ratiometric fluorescent probe for apoptosis detection. This probe incorporates selectively into the outer leaflet of the cell plasma membrane and senses the loss of the plasma membrane asymmetry occurring during the early steps of apoptosis. The high specificity to the plasma membranes was achieved by introduction into the probe of a membrane anchor, composed of a zwitterionic group and a long (dodecyl) hydrophobic tail. The fluorescence reporter of this probe is 4'-(diethylamino)-3-hydroxyflavone, which exhibits excited-state intramolecular proton transfer (ESIPT), resulting in two-band emission highly sensitive to the lipid composition of the biomembranes. Fluorescence spectroscopy, flow cytometry, and microscopy measurements show that the ratio of the two emission bands of the probe changes dramatically in response to apoptosis. This response reflects the changes in the lipid composition of the outer leaflet of the cell plasma membrane because of the exposure of the anionic phospholipids from the inner leaflet at the early steps of apoptosis. Being ratiometric, the response of the new probe can be easily quantified on an absolute scale. This allows monitoring by laser scanning confocal microscopy the degree and spatial distribution of the apoptotic changes at the cell plasma membranes, a feature that can be hardly achieved with the commonly used fluorescently labeled annexin V assay.

Membrane dipole potential as measured by ratiometric 3-hydroxyflavone fluorescence probes: accounting for hydration effects.

We previously applied the electrochromic modulation of excited-state intramolecular proton-transfer (ESIPT) reaction for the design of novel 3-hydroxyflavone (3-HF) derivatives as fluorescent probes for measuring the dipole potential, Psi(D), in lipid bilayers (Klymchenko et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11219). In the present work, this method was revisited to take into account the influence of the bilayer hydration on the emission ratiometric response of 3-HF probes. For this reason, it was necessary to deconvolute the whole fluorescence spectra into three bands corresponding to the non H-bonded forms, normal N* and tautomer T* forms, both participating to the ESIPT reaction, and to the H-bonded H-N* form, excluded from this reaction. This allowed us to determine the pure N*/T* intensity ratio, without any contribution from the H-N* form emission depending essentially on the bilayer hydration. This new approach allowed us to confirm the correlation we obtained between the response of 3-HF probes on dipole potential modifications and the corresponding response of the reference fluorescent probe di-8-ANEPPS, thus further confirming the potency of 3-HF probes as excellent emission ratiometric probes to measure dipole potential in lipid membranes.

Two-color fluorescent probes for imaging the dipole potential of cell plasma membranes.

The dipole potential (Psi(d)) constitutes a large and functionally important part of the electrostatic potential of cell plasma membranes. However, its direct measurement is not possible. Herein, new 3-hydroxyflavone fluorescent probes were developed that respond strongly to Psi(d) changes by a variation of the intensity ratio of their two well-separated fluorescence bands. Using fluorescence spectroscopy with cell suspensions and confocal microscopy with adherent cells, we showed, for the first time, two-color fluorescence ratiometric measurement and visualization of Psi(d) in cell plasma membranes. Using this new tool, a heterogeneous distribution of this potential within the membrane was evidenced.