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Measurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data. By analyzing live-cell FCS measurements, we show that our new model can satisfactorily account for the binding interactions introduced by attaching a DNA binding domain to the dimerization domain derived from a site-specific transcription factor (the vitellogenin binding protein (VBP)). We find that our FCS estimates are quantitatively consistent with our fluorescence recovery after photobleaching (FRAP) measurements on the same VBP domains. However, due to the fast binding interactions introduced by the DNA binding domain, FCS generates independent estimates for the diffusion constant (6.7 +/- 2.4 microm2/s) and the association (2 +/- 1.2 s(-1)) and dissociation (19 +/- 7 s(-1)) rates, whereas FRAP produces only a single, but a consistent, estimate, the effective-diffusion constant (4.4 +/- 1.4 microm2/s), which depends on all three parameters. We apply this new FCS method to evaluate the efficacy of a potential anticancer drug that inhibits DNA binding of VBP in vitro and find that in vivo the drug inhibits DNA binding in only a subset of cells. In sum, we provide a straightforward approach to directly measure binding rates from FCS data.

Knowledge of the size and shape of the illuminated volume is critical when carrying out quantitative studies involving Fluorescent Correlation Microscopy (FCM). Determination of that volume may be difficult when FCM is applied to crowded systems such as cells, tissues, and congested polymer solutions. We thus have carried out Monte-Carlo simulations to address of the effects of multiple scattering on a focused laser beam, using a model of optically non-absorptive spherical scatterers to mimic a concentrated solution of non-fluorescent crowders. We find that as the concentration or size of the nanoparticles increases, the illumining beam spot is broadened in a non-uniform manner. Further, the focal point of the spot shifts towards the incoming beam source. This analysis has been performed for a selection of media conditions, where we vary both bead size and concentration.

Many techniques rely on the binding activity of surface-immobilized proteins, including antibody-based affinity biosensors for the detection of analytes, immunoassays, protein arrays, and surface plasmon resonance biosensors for the study of thermodynamic and kinetic aspects of protein interactions. To study the functional homogeneity of the surface sites and to characterize their binding properties, we have recently proposed a computational tool to determine the distribution of affinity and kinetic rate constants from surface binding progress curves. It is based on modeling the experimentally measured binding signal as a superposition of signals from binding to sites spanning a range of rate and equilibrium constants, with regularization providing the most parsimonious distribution consistent with the data. In the present work, we have expanded the scope of this approach to include a compartment-like transport step, which can describe competitive binding to different surface sites in a zone of depleted analyte close to the sensor surface. This approach addresses a major difficulty in the analysis of surface binding where both transport limitation as well as unknown surface site heterogeneity may be present. In addition to the kinetic binding parameters of the ensemble of surface sites, it can provide estimates for effective transport rate constants. Using antibody-antigen interactions as experimental model systems, we studied the effects of the immobilization matrix and of the analyte flow-rate on the effective transport rate constant. Both were experimentally observed to influence mass transport. The approximate description of mass transport by a compartment model becomes critical when applied to strongly transport-controlled data, and we examined the limitations of this model. In the presence of only moderate mass transport limitation the compartment model provides a good description, but this approximation breaks down for strongly transport-limited surface binding. In the latter regime, we report experimental evidence for the formation of gradients within the sensing volume of the evanescent field biosensor used.

Our kinetic studies have characterized the mechanisms of deoxyhemoglobin S polymer depolymerization when exposed to CO (used as a model for oxygen because rates can be controlled photolytically). Fibers dissolve slowly, losing monomers from their ends at low partial pressure, and very rapidly at higher partial pressures that induce fiber fracture and therefore many new ends. Slow dissolution that is not complete in the time red cells traverse the pulmonary microvasculature will generate residual arterial polymers (RAPs), enhancing pathogenesis by seeding nucleation of new polymers, accelerating repolymerization and increasing its extent.We now demonstrate, to the best of our knowledge for the first time by direct observation, that anaerobically drawn arterial blood of sickle patients shows birefringence in many red cells and therefore RAPs exist, which we confirm by EM observation of aligned polymers. RAPs exist not only under hypoxemic conditions, when they can be explained by limited solubility due to the presence of deoxyHbS, but also when hypoxemia is absent. RAPs without hypoxemia imply that slow depolymerization kinetics are responsible. One minute of voluntary hyperventilation and (separately) brief nasal oxygen greatly decrease RAPs. RAPs increase during sleep. We attribute these results to accelerated depolymerization at increasing levels of oxygen that cooperatively induce polymer fracture (fracture, using CO, exhibits a 4.7 power dependence on pCO). These results and the interdependent progress of oxygen saturation, partial pressure, fracture rate and remaining polymer that we model bear on pathogenesis and particularly on vaso-occlusive crises, which result from red cell rigidification and from cellular adhesion due to polymer-dependent cellular damage. Under these mechanisms, the lungs may play an important role in initiating pathology; and remediation of dysfunction by breathing assists is potentially prophylactic.

We report femtosecond Laser-induced Breakdown Spectroscopy (fs-LIBS) measurements on several amino acids (Serine, Glutamine, and Cysteine) and Albumin protein solutions mixed with ficoll at different concentrations. The goal is to assess the effects of a host matrix on the identification and spectral characterization of amino acids by fs-LIBS. fs-LIBS utilizes an intense short laser pulse to obliterate a sample into basic constituents and to record the emission spectrum of atoms, ions, and molecules in the cooling down of the plasma plume. Several spectral peaks associated primarily with elemental composition of a sample were observed in the fs-LIBS spectra in a range from 200 to 950 nm. In addition, some molecular information associated with diatomic vibrational modes in certain molecules such as C-C and C-N were also obtained. The presence of ficoll affects the relative intensity and broadening of the C-N band, which is a signature of the amino acids. The influence of ambient gas on the fs-LIBS measurements was also analyzed under helium and argon environments, and a different behavior of peaks associated with He and Ar as a function of amino acids and ficoll mixture composition was observed. The fs-LIBS data and their analysis compare favorably with those derived from Fourier Transform Infrared Spectroscopy (FTIR). Interpretation of the spectral information enclosed in the emission of the diatomic molecules during laser ablation may lead to a better understanding of plume chemistry with a direct consequence on chemical analysis of complex samples such as amino acids. Altogether, the results demonstrate the potential of fs-LIBS technique as a detection method of biomolecules and for probing interactions of these biomolecules with a host matrix.

We investigate the effect of dehydration on the diffusion of fluorescent nanoprobes embedded in aqueous solutions and gels of Poly(Vinyl Alcohol) (PVA) polymers. using a custom-made optical chamber and applying fluorescence correlation spectroscopy (FCS), we measured changes in fluorescence intensity and, simultaneously time-correlation functions at various stages during the dehydration of samples of the solutions or gels. The samples were prepared at different concentrations and cross-link densities, with glutaraldehyde being the cross-linker. We have analyzed the data and determined the changes of the concentrations of the nanoprobes and PVA, as well as the apparent diffusion times of the nanoprobes during the dehydration. For rhodamine fluorophores (TAMRA, MW =430 Da), we find that diffusion slows down in both shrinking samples. The drying rates of samples of the solution and gel are different, though both samples were prepared with the same PVA concentrations and subjected to the same conditions. Moreover, the diffusion time and its changes in the drying sample of gel is larger than those in the corresponding drying sample of the solution. We also measured changes of the elastic modulus of samples of dehydrating gels prepared at different concentrations and cross-link density, the goal being to establish whether there is a correlation between the changes of the elastic modulus and those of the nanoprobe diffusion.

We apply time-resolved fluorescence confocal microscopy and fluorescence correlation spectroscopy to examine the movements of fluorescently-labeled HIV-1 virions (∼120 nm) embedded in crude human cervical mucus. Particle-tracking analysis indicates that the motion of most virions is decreased 200-fold compared to that in water and is not driven by typical diffusion. Rather, the time-dependence of their ensemble-averaged mean-square displacements is proportional to τα + v2τ2, describing a combination of anomalous diffusion at short time scales (α ∼ 0.3) and flow-like behavior at longer times, τ being the lag time. We attribute the flow-like behavior to slowly-relaxing mucus matrix that follows mechanical perturbations such as stretching and twisting of the sample. Further analysis of the tracks and displacements of individual virions indicates differences in the local movements among the virions, including constrained motion and infrequent jumps, perhaps due to abrupt changes in matrix structure. We surmise that these differences are related not only to possible variations in the local microenvironments experienced by each individual virion but, perhaps, also to variations in the surface structure of the virions themselves. Possible changes in the microenvironments due to slow structural changes may provide a means for some virions to move and reach the port of entry, the underlying cervical mucosa.