RESUMEN
Photon counting statistics in 3D photon counting histogram analysis for one-photon excitation is a function of the number of molecules of particular brightness in the excitation-detection volume of a confocal microscope. In mathematical form that volume is approximated by a three-dimensional Gaussian function which is embedded in the PCH theoretical equations. PCH theory assumes that a molecule can be found anywhere inside the excitation-detection volume with equal probability. However, one can easily imagine systems in which this assumption is violated because molecules are constrained by the geometry of the sample. For example, molecules on a surface or in a membrane would be constrained to two dimensions. To enable the analysis of such systems by PCH, the theoretical framework requires modification. Herein, we present an extension of the PCH analysis to systems where molecules exist in thin structures that are effectively two-dimensional. The method, aptly called two-dimensional photon counting histogram (2D PCH), recovers the number of fluorescent particles per unit area and their molecular brightness. Both theoretical background and experimental results are presented. The theory was tested using computer-simulated and experimental 2D PCHs obtained from confocal experiments. We demonstrate that this modification of the theoretical framework provides a tool to extract data that reveal states of aggregation, surface photophysics, and reactivity.
RESUMEN
Photon counting histogram (PCH) analysis is a statistical method that provides independent information about the relative brightness and the number of molecular entities in the system. This suggests that it could be very advantageous in the analysis of intermolecular interactions in multicomponent systems. In this paper we employ PCH to study the interaction of Rhodamine 6G (Rh-6G) with sodium dodecyl sulfate (SDS) micelles and show how the method can unveil the mechanism of quenching of Rh-6G by methyl viologen (MV).
RESUMEN
Fluorescence resonance energy transfer (FRET) measurements offer a reliable and noninvasive approach to studying protein and lipid colocalization in cells. We have considered systems in which FRET occurs as intramolecular and/or intermolecular process. The proposed dynamic FRET model shows that in the case of intermolecular process the degree of aggregation only slightly affects the energy transfer efficiency. The theory was tested on a set of donor-acceptor pairs in which energy transfer occurs intramolecularly, intermolecularly, or both. The obtained experimental results are in a good agreement with the proposed model. It is well known that the energy transfer efficiency depends both on the distance between the donor and acceptor molecules and the relative orientation of their respective transition dipole moments. This dual dependence often leads to ambiguity. In this article, we show how FRET efficiency can be significantly reduced even in highly coupled system through conformational restrictions in the donor-acceptor pair. Importantly, such restrictions can be imposed on the system by cell fixation, a procedure routinely used when conducting FRET measurements.
