Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics.

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

Photophysical characterization of interchromophoric interactions between rhodamine dyes conjugated to proteins.

Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E. coli sliding clamp (ß clamp) with emphasis on the properties of the H-dimers that form when the dimeric protein is labeled with one dye at each side of the dimer interface. Overall, results are consistent with an equilibrium between non-emissive dimers and unstacked monomers that experience efficient dynamic quenching Protein constructs labeled with tetramethylrhodamine show the characteristic features of H-dimers in their absorption spectra and a c.a. 40-fold quenching of fluorescence intensity. The degree of quenching decreases when samples are labeled with a tetramethylrhodamine derivative bearing a six-carbon linker. H-dimers do not form in samples labeled with Alexa 488 and A546, but fluorescence is still quenched in these samples through a dynamic mechanism. These results should help researchers design and interpret fluorescence experiments that take advantage of the properties of rhodamine dimers in protein research.

Structural Implications on the Properties of Self-Assembling Supramolecular Hosts for Fluorescent Guests.

Nine amphiphilic macromolecules with decyl and oligo(ethylene glycol) side chains, randomly distributed along a common poly(methacrylate) backbone, were synthesized from the radical copolymerization of appropriate methacrylate monomers. The resulting amphiphilic constructs differ in (1) the ratio between their hydrophobic and hydrophilic components, (2) the length of their oligo(ethylene glycol) chains, and/or (3) the molecular weight. When the ratio between hydrophobic and hydrophilic segments is comprised between 6:1 and 1:2, the macromolecules assemble spontaneously into particles with nanoscaled dimensions in neutral buffer and capture hydrophobic borondipyrromethene chromophores in their interior. However, the critical concentration required for the assembly of these supramolecular hosts as well as their hydrodynamic diameter, supramolecular weight, and number of constituent macromolecular building blocks all vary monotonically with the ratio between hydrophobic and hydrophilic components. Specifically, the critical concentration decreases and the other three parameters increase as the relative hydrophobic content raises. Furthermore, an increase in the relative hydrophobic content also discourages interchromophoric interactions between entrapped guests in both ground and excited states as well as delays access of potential quenchers. In fact, these observations demonstrate that the hydrophobic components must be in excess over their hydrophilic counterparts for optimal supramolecular hosts to assemble. Indeed, a ratio of 6:1 between the numbers of decyl and oligo(ethylene glycol) side chains appears to be ideal for this particular structural design. Under these conditions, supramolecular hosts assemble spontaneously even at relatively low polymer concentrations and their fluorescent guests do not escape into the bulk aqueous solution, despite the reversibility of the noncovalent interactions holding the supramolecular container together. Thus, these systematic investigations provide invaluable structural guidelines to design self-assembling supramolecular hosts with optimal composition for the effective encapsulation of fluorescent guests and can lead to ideal delivery vehicles for the transport of imaging probes to target locations in biological samples.