Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.
The use of designed polymer coatings for specific applications such as drug delivery or modifying cell response is a critical aspect of medical device manufacturing. The chemical composition and physical characteristics of thin polymer coatings need to be analysed in-situ and this can present difficulties for traditional analytical methods. For example, changes in the polarity of polymer coatings are typically measured using the contact angle (CA) method. This is a simple process and gives good results however; it cannot be used to measure very hydrophilic polymers, or to analyse features smaller than a couple of mm in size. There is a need for a non-contact method for polarity measurement that is suitable for hydrophilic polymers on a macro- and microscopic scale. 4'-diethylamino-3-hydroxyflavone (FE), 5, 6-benzo-4'-diethylamino-3-hydroxyflavone (BFE), and 4'-diethylamino-3-hydroxy-7-methoxyflavone (MFE) are fluorescence probes based on 3-hydroxyflavone. They respond to environment perturbations by shift and changes in the relative intensity of two well-separated bands in the emission spectra. These bands originate from an excited state intramolecular proton transfer (ESIPT) reaction. We have incorporated FE, BFE, and MFE into a novel thermoresponsive hydrophilic/hydrophobic copolymer system (NIPAM-NtBA) and studied its fluorescence behaviour. The fluorescence emission spectra depend strongly on copolymer composition, with increasing hydrophobicity (greater NtBA fraction) leading to a decrease in the value of log (IN*/IT*). This allows for the non-contact, measurement of the exact composition and surface energy of the copolymer system.
Development of fluorescence microscopic methods is limited by the application of new dyes, the response of which could be sensitive to different functional states in the living cells, and, in particular, to electrostatic potentials on their plasma membranes. Recently, we showed that newly designed 3-hydroxyflavone fluorescence dyes are highly electrochromic and show a strong two-band ratiometric response to electric dipole potential in lipid membranes. In the present report we extend these observations and describe a new generation of these dyes as electrochromic probes in biomembrane research. Modification of the membrane dipole potential was achieved by addition of 6-ketocholestanol (6-KC), cholesterol and phloretin. The dipole potential was also estimated by the reference probe di-8-ANEPPS. As an example, we show that on addition of 6-KC there occurs a dramatic change of the intensity ratio of the two emission bands, which is easily detected as a change of color. We describe in detail the applications of one of these dyes, PPZ8, to the studies of cells in suspension or attached to the glass surface. Confocal microscopy demonstrates strong preference of the probe for the cell plasma membrane, which allows us to apply this dye for studying electrostatic and other biomembrane properties. We demonstrate that the two-color response provides a direct and convenient way to measure the dipole potential in the plasma membrane. Applying PPZ8 in confocal microcopy and two-photon microspectroscopy allowed us to provide two-color imaging of the membrane dipole potential on the level of a single cell.
The 4'-dialkylamino derivatives of 3-hydroxyflavone find many applications as molecular probes, since their two-band fluorescence spectra produce a strong response to different intermolecular interactions including H-bonding. The results of our steady-state and time-resolved studies in neat and mixed solvents reveal an important and probably unique property of these dyes: their ground-state equilibrium between H-bonded and non H-bonded forms is not changed significantly on excitation to the normal (N*) excited state. In the 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. These dyes do not change significantly the distribution between H-bonded and non H-bonded species in their environment and therefore they can provide a measure of the H-bonding potential of their environment. Due to this feature, they can serve as unique sensors of the H-bonding potential in unknown media. This sensing can be provided by the dramatic change of the relative intensities of their two separated emission bands.
We demonstrate a possibility for simultaneous probing of several microscopic physicochemical properties of supramolecular systems using the dye molecules existing in several forms of the ground and/or excited states. This idea is realized on a series of 3-hydroxychromone dyes, which exhibit the Excited-State Intramolecular Proton Transfer reaction resulting in two excited state tautomeric forms. Microenvironment of these dyes can be characterized by a number of characteristic energies of absorption and emission transitions (positions of the corresponding bands) and by redistribution between the forms (relative intensities of the bands). We demonstrate that the positions and relative intensities of fluorescence excitation and emission bands can serve as independent variables with different sensitivity to polarity, electronic polarizability, electric field effects, hydrogen bonding acidity and basicity, thus, allowing multiparametric description of probe environment. The results of numerous applications of this approach in the studies of neat solvents, solvent mixtures, proteins and phospholipid membranes are presented.