We report on the linear electro-optical scattering response from individual ferroelectric (KTiOPO4) nano-crystals. The newly developed Pockels Linear Electro-Optical Microscopy (PLEOM)1-3 is used in this context to map the second-order susceptibility χ<sup>(2)</sup> of non-centrosymmetric materials with a high sensitivity due to a stabilized interferometric homodyne detection. The random spatial orientation of single nano-crystals (with an average size of 150 nm), together with the orientation of the electric dipole moment of ferroelectric domains can be jointly inferred from the intensity polarization plots together with phase of the linear electro-optical response. Down- scaling the electro-optic response to nano-crystals opens-up new applications towards sub-diffraction electro-optic nano-labels for nonlinear microscopy with applications to nano-sciences and biophotonics. By using a low power He-Ne laser source and a low intensity illumination beam, PLEOM bears the potential of a new low-cost non-imaging method in biology, especially relevant for sensitive samples.
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.