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.

Picosecond time-resolved fluorescence spectroscopy has been applied to the studies of excited-state intramolecular proton transfer (ESIPT) dynamics in two 4‘-(dialkylamino)-3-hydroxyflavone derivatives (unsubstituted and substituted at the 6-position) in ethyl acetate and dichloromethane. In all the studied cases, the fluorescence decay kinetics of both short-wavelength normal (N*) and long-wavelength tautomer (T*) bands can be characterized by the same two lifetime components, which are constant over the all wavelength range of the emission. In the meantime, the preexponential factor of the short-lifetime component changes its sign, being positive for the N* and negative for the T* emission band. Moreover, the two preexponential factors of the T* emission decay are the same in magnitude but opposite in sign. These features are characteristic of a fast reversible two-state ESIPT reaction. Reconstruction of time-resolved spectra allows observing the evolution of these spectra with the appearance, rapid growth, and stabilization (in less than 200 ps) of the relative intensities of the two emission bands. A detailed kinetic model was applied for the analysis of these data, which involved the determination of radiative and nonradiative decay rate constants of both N* and T* forms and of forward and reverse rate constants for transitions between them. We show that ESIPT reaction in the studied conditions occurs on the scale of tens of picoseconds and thus is uncoupled with dielectric relaxations in the solvent occurring at subpicosecond times. Moreover, the radiative and nonradiative deactivation processes were found to be much slower than the ESIPT reaction, suggesting that the relative intensities of the two emission bands are mainly governed by the ESIPT equilibrium. Therefore, both electrochromic and solvatochromic effects on the relative intensities of the two emission bands in 4‘-(dialkylamino)-3-hydroxyflavones result from the shifts in the ESIPT equilibrium.