A simple new approach is described and demonstrated for measuring the number of independent emitters along with the fluorescence intensity, lifetime, and emission wavelength for trajectories and images of single molecules and multichromophoric systems using a single PC plug-in card for time-correlated single-photon counting. The number of independent emitters present in the detection volume can be determined using the interphoton times in a manner similar to classical antibunching experiments. In contrast to traditional coincidence analysis based on pulsed laser excitation and direct measurement of coincident photon pairs using a time-to-amplitude converter, the interphoton distances are retrieved afterward by recording the absolute arrival time of each photon with nanosecond time resolution on two spectrally separated detectors. Intensity changes that result from fluctuations of a photophysical parameter can be distinguished from fluctuations due to changes in the number of emitters (e.g., photobleaching) in single chromophore and multichromophore intensity trajectories. This is the first report to demonstrate imaging with contrast based on the number of independently emitting species within the detection volume.

The association of neuropeptide Y (NPY) at the air/water interface and with phospholipid monolayers on water as subphase has been investigated using external infrared reflection absorption spectroscopy (IRRAS). Studies of the conformation and orientation of NPY suggest that it adopts an α-helical structure and is oriented parallel to the air/water interface in neat peptide monolayers. Both secondary structure and orientation are preserved in mixed lipid/NPY monolayers. Comparison of NPY associated with zwitterionic DPPC and with anionic DMPS suggests that electrostatic attraction plays a major role for peptide binding to the membrane surface.

The association of neuropeptide Y (NPY) with air/water interfaces and with phospholipid monolayers on water subphases and on physiological buffer has been investigated. Surface pressure (π) vs. molecular area (A) relations of the peptide at water surfaces depend on the concentration of the spreading solutions. Independent of that concentration, they show a transition from a low-density state to a high-density state at π ∼ 12 mN/m. Similar features are observed in the NPY adsorption to preformed monolayers (Δπ(t→∞) as a function of π i = π(t=0) where t = 0 signifies the time of peptide injection). The transition is also observed in cospread lipid/NPY monolayers and is interpreted as the exclusion of the peptide from the surface layer. The reproducibility of the isotherms after expansion suggests that cospread lipid/peptide monolayers are thermodynamically stable and that the peptide remains associated with the monolayer after exclusion from the lipid surface.A comparison of NPY association with zwitterionic and with anionic lipids, as well as a comparison of the interactions on pure water and on physiological buffer, suggests that electrostatic attraction plays a major role in the energetics of peptide binding to the membrane surface. Dual label fluorescence microscopy demonstrates that the peptide associates preferentially with the disordered liquid-condensed (LC) monolayer phase and also suggests that it self-aggregates upon exceeding a critical surface concentration. A NPY variant with a distorted α-helix interacts with the surface as strongly as the natural NPY but expands the monolayers more. This suggests that the helix motif in the peptide is more important for the interaction with the receptor than for binding of the peptide to the membrane surface. In context, these observations attribute a specific role to the membrane in funneling the signal peptide to its membrane receptor.