Fluorescence-intensity distribution analysis and its application in biomolecular detection technology

Abstract

A methodology, fluorescence-intensity distribution analysis, has been developed for confocal microscopy studies in which the fluorescence intensity of a sample with a heterogeneous brightness profile is monitored. An adjustable formula, modeling the spatial brightness distribution, and the technique of generating functions for calculation of theoretical photon count number distributions serve as the two cornerstones of the methodology. The method permits the simultaneous determination of concentrations and specific brightness values of a number of individual fluorescent species in solution. Accordingly, we present an extremely sensitive tool to monitor the interaction of fluorescently labeled molecules or other microparticles with their respective biological counterparts that should find a wide application in life sciences, medicine, and drug discovery. Its potential is demonstrated by studying the hybridization of 5-(6-carboxytetramethylrhodamine)-labeled and nonlabeled complementary oligonucleotides and the subsequent cleavage of the DNA hybrids by restriction enzymes. In modern fluorescence correlation experiments, with a confocal high-aperture microscope, continuous wave laser excitation, and avalanche photodiode detection, more than 10 5 photons per s are detected routinely from a single photostable dye molecule passing through the focus of the microscope, which is more than two orders of magnitude higher than the background count rate (1). In a similar manner as cells are studied in cell-sorting devices, the method allows one to study single molecules independently of one another. Single molecules diffuse randomly in all three dimensions within the sample; however, each time they become visible, they do not necessarily pass through the center of the focus. Therefore, an event in which a relatively bright molecule enters the periphery of the laser beam only briefly cannot be distinguished from an event in which a dark molecule passes through the focus, because they leave identical traces in terms of detectable photon counts.Fluorescent species with different specific brightnesses can be distinguished, however, by collecting a statistical distribution of the number of photon counts at time intervals of given length. (Specific brightness is a molecular quantity, expressed as the mean count rate per molecule. It is proportional to the molecular absorption cross section and to the fluorescence quantum yield.) The distribution of photon count numbers is used to determine concentrations of molecules of heterogeneous brightness in the sample. We expect this method of sample analysis to be a valuable tool in various disciplines from fundamental research to very specific applications, e.g., drug discovery and diagnostics.Fluorescence-intensity fluctuations caused by random movement of fluorescent molecules into and out of an illuminated sample volume have been studied since fluorescence correlation spectroscopy (FCS) was established 27 years ago (2-4). An initial kind of sample analysis based on determining moments ...

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“…Fluorescence Correlation Spectroscopy (FCS) 5,6 and related fluctuation techniques have since the early 1990's become widely used for analyzing biomolecular interactions and fluctuations, in solution and in living cells [7][8][9] . When it comes to analysis of Aβ oligomers in solution, FCS has shown some promise, but these studies report only on large aggregates containing several hundred monomers [10][11][12] …”

Heterospecies partition analysis reveals binding curve and stoichiometry of protein interactions in living cells

“…Data analysis by photon counting histogram (17,18) or momentrelated techniques (19)(20)(21) are feasible. Here we perform cumulant analysis of the photon counts, because it provides analytical results that are crucial for interpreting the fit parameters of the heterospecies model.…”

Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

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