Theory of Measuring the Selfsame Single Fluorescent Molecule in Solution Suited for Studying Individual Molecular Interactions by SPSM-FCS

Abstract

Abstract Three exact criteria are first derived for the probabilities that determine single molecule sensitivity in single-phases, for example in solutions or membranes. It is shown how the criteria can be used to decide whether single molecule sensitivity is obtained. Most straightforwardly, all we require is that we experimentally determine the Poisson probability for the absolute number of fluorescent molecules in the volume of observation. This is achieved by fluorescence correlation spectroscopy and allows identifying the selfsame, one single fluorescent molecule in single-phases. Further, we first provide the new and powerful analytical capability to fluorescencetagged inidividual molecules and reactions in in-vitro and living systems to follow their molecular interactions and function by means of spatial stochastic behavior. This fully stochastic modeling is derived which describes the influence of spatial separation on the reaction coordinate of one single individual molecule in single-phases within the observation volume. The theoretical concept is applied to experimental data 'at the single-molecule level' of photosensitive riboflavin and riboflavin-containing blue fluorescence protein. The data were taken from the literature. As simulation results of the elucidative examples, experiments are suggested that would measure individual, single molecules of flavins and flavoproteins m solution. They are of importance for experimentalists.