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.
The spectroscopic behavior of the 4′-dialkylamino-3-hydroxyflavones in protic environments is very unusual. Previous studies showed that in contrast to other solvatochromic dyes containing carbonyl group (coumarins, Nile Red, PRODAN, etc.), their Stokes shift does not increase on the formation of intermolecular H-bonds with protic solvents. The present steady-state and time-resolved studies show that the ground-state equilibrium between the H-bonded and non-H-bonded forms of this derivative in mixed solvents is not changed significantly when the dye is excited to the normal (N*) excited state. New H-bonds do not form, but those already existing in the ground state can disrupt on a slow time scale. This last process is probably coupled with the slow excited-state intramolecular proton transfer (ESIPT) reaction of the H-bonded form of the dye. Therefore, the fluorescence spectra of the dye provide a measure of the ground state distribution between its H-bonded and non-H-bonded forms, which in turn reflects the H-bonding potential of the environment. Due to this feature, this dye can serve not only as a calibrator of solvent properties but also as a unique sensor of H-bonding potential in unknown media. This sensing can be provided by the relative intensities of the two separated emission bands in the fluorescence spectra.
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.
Nerve growth cones (GCs) are chemical sensors that convert graded extracellular cues into oriented axonal motion. To ensure a sensitive and robust response to directional signals in complex and dynamic chemical landscapes, GCs are presumably able to amplify and filter external information. How these processing tasks are performed remains however poorly known. Here, we probe the signal-processing capabilities of single GCs during γ-Aminobutyric acid (GABA) directional sensing with a shear-free microfluidic assay that enables systematic measurements of the GC output response to variable input gradients. By measuring at the single molecule level the polarization of GABA(A) chemoreceptors at the GC membrane, as a function of the external GABA gradient, we find that GCs act as i), signal amplifiers over a narrow range of concentrations, and ii), low-pass temporal filters with a cutoff frequency independent of stimuli conditions. With computational modeling, we determine that these systems-level properties arise at a molecular level from the saturable occupancy response and the lateral dynamics of GABA(A) receptors.
The unique optical properties of arrays of metallic nanoparticles are of great interest for many applications such as in optical data storage, sensing applications, optoelectronic devices or as platforms to increase the detection limit in spectroscopic measurements. Nonlinear optical phenomena can also be altered by metallic nanostructures opening new possible applications. In this work, arrays composed of non-centrosymmetric individual structures with three fold axial symmetry made of gold are designed and fabricated using electron beam lithography. The nonlinear optical properties of these structures are investigated using second-harmonic generation microscopy (SHGM) with a femtosecond excitation source set near the plasmon resonance frequency. Modeling of the electromagnetic field distribution around the metallic structures is performed using the Finite Difference Time Domain (FDTD) method, highlighting the confinement of the SHG signal and its polarization dependence. Polarization-resolved measurements are conducted to correlate the SHG signal with the structure and symmetry of the individual nanostructures. Since both two-photon induced photoluminescence (TPPL) and SHG signals are produced upon excitation of these structures, lifetime measurements are performed to further evaluate the magnitude of these two effects.
We show how a single gold nanorod can serve as a multifunctional probe in an organized DNA matrix. Polarization analysis of two-photon luminescence excited with a femtosecond laser enables imaging of the orientation of a single nanorod, which reports the orientation of DNA strands. Carefully controlled photoinduced heating by the same laser is able to degrade the DNA matrix in a highly localized volume.
3-Hydroxyflavones are characterized by an excited-state proton transfer reaction between two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Due to the high sensitivity of the relative intensities of the two emission bands to solvent polarity, hydrogen bonding, and the local electrical field, these dyes found numerous applications in biomembrane studies. In the present work, we evidenced a new band strongly shifted to the red in both absorption and excitation spectra for 4′-(dimethylamino)-3-hydroxyflavone (probe F) in the presence of anionic phosphatidylglycerol vesicles. Excitation of this new band provided a single-band emission spectrum with a maximum around 530 nm that differs from the two-band profile obtained at lower excitation wavelengths. Furthermore, an additional longlived lifetime (3.8 ns) was observed in the time-resolved decay only when the probe was excited in the wavelength range of the new excitation band. These spectroscopic features were observed at neutral pH in HEPES but not phosphate-citrate-borate buffer. From their pH dependence, these features could be unambiguously attributed to the anionic form of probe F bound at the interface of the lipid bilayer. This anion may form even at neutral pH due to the HEPES molecules that bind at the bilayer interface to the negatively charged polar heads of phosphatidylglycerol and deprotonate probe F. Consequently, HEPES can significantly modify the acid-base properties of the negatively charged lipid membrane and thus affect the properties of phenolic compounds and other weak acids bound to lipid vesicles or cell membranes. It follows that HEPES buffer should be used with care in biomembrane studies.
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