Heat and water transfer in a rotating drum containing solid substrate particles

In previous work we reported on the simulation of mixing behavior of a slowly rotating drum for solid-state fermentation (SSF) using a discrete particle model. In this investigation the discrete particle model is extended with heat and moisture transfer. Heat transfer is implemented in the model via interparticle contacts and the interparticle heat transfer coefficient is determined experimentally. The model is shown to accurately predict heat transfer and resulting temperature gradients in a mixed wheat grain bed. In addition to heat transfer, the addition and subsequent distribution of water in the substrate bed is also studied. The water is added to the bed via spray nozzles to overcome desiccation of the bed during evaporative cooling. The development of moisture profiles in the bed during spraying and mixing are studied experimentally with a water-soluble fluorescent tracer. Two processes that affect the water distribution are considered in the model: the intraparticle absorption process, and the interparticle transfer of free water. It is found that optimum distribution can be achieved when the free water present at the surface of the grains is quickly distributed in the bed, for example, by fast mixing. Alternatively, a short spraying period, followed by a period of mixing without water addition, can be applied. The discrete particle model developed is used successfully to examine the influence of process operation on the moisture distribution (e.g., fill level and rotation rate). It is concluded that the extended discrete particle model can be used as a powerful predictive tool to derive operating strategies and criteria for design and scale-up for mixed SSF and other processes with granular media.     

Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation.

Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by monitoring spontaneous equilibrium fluctuations in the local concentration of a fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, submicromolar samples by directly "counting particles". We introduce the application of two-photon excitation to scanning FCS and discuss its important advantages for this technique. We demonstrate the capability of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, and then with B phycoerythrin. The detection of multiple species in a single sample is shown, using mixtures containing both sphere sizes. The method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/ dissociation equilibrium for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good agreement with previously published values. In addition, oligomer dissociation induced by pH titration from pH 8 to pH 5.0 is detectable for the enyme phosphofructokinase. The possibility of measuring dissociation kinetics by scanning two-photon FCS is also demonstrated using phosphofructokinase.     

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.     

Light scattering and fluorescence by small particles having internal structure.

We consider two related, yet distinct queries: 1. How does the internal morphology of a small particle affect the elastic light scattering signals? We have devised an algorithm, presently accurate for particles comparable only to small biological spheres (diameter less than 1 micron), which suggests that light scattering is sensitive to internal morphology only in the backward directions. Accordingly, observations should be obtained in these directions when probing for internal morphology. 2. How are fluorescent signals affected when the active molecules are variously distributed within small particles? One cannot assume that the fluorescent signals are simply proportional to the number of active molecules contained in the particle because there may also be a dependence upon the geometrical and optical properties of the particle and upon the particular spatial distribution of these molecules within the particle. Indeed, even the measured emission spectrum may be affected by such morphological features. Here, too, these calculations are mainly restricted to small particles (diameter less than 1 micron) in which the fluorescent molecules are isotropic and immobile. Under these conditions the effects are quite dramatic. These effects should be considered in quantitative procedures which utilize fluorescence for determining the concentration of specific molecules in small particles such as biological cells. They may provide a clue for discriminating among cells which differ morphologically or in which the spatial distribution of the fluorescent moiety differs. These effects may be minimized by utilizing a light source which is polarized perpendicularly to the scattering plane.     

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay. For this purpose a theoretical model of the initial agglutination kinetics, based on particle diffusion combined with a steric factor determined by the level of specific and nonspecific agglutination, was developed. The theoretical model combined with the experimental data can be used to optimize an agglutination-based assay with regard to sensitivity and dynamic range and to estimate the affinity, receptor surface density, molecular and binding site sizes, and level of nonspecific binding that is present in the assay. The experimental results are in good agreement with the theoretical model, indicating the usefulness of the model for immunoagglutination assay optimization.     

Electrical Sizing of Particles in Suspensions: III. Rigid Spheroids and Red Blood Cells

The processes involved during the passage of a suspended particle through a small cylindrical orifice across which exists an electric field are investigated experimentally for an approximate prolate spheroid in the form of two tangent, rigid spheres (ragweed pollen particles) and for fresh, human red blood cells. Oscillograms of current pulses produced by both types of particles are presented and discussed in terms of particle shape and orientation and the effects of the hydrodynamic field. It is concluded that all the particles enter the orifice with their major axes aligned parallel to the orifice axis (electric field), but that during their passage some are rotated by the hydrodynamic field. Cells with their equatorial plane perpendicular to a radius of the orifice change their orientation with respect to the electric field as they are rotated, the others do not; only in the former case is there any deformation. It is shown that the bimodal or skewed size distributions can be explained on this basis, and that size (shape factor × volume) is actually a normally distributed variable (P > 95%). The average size of samples from 10 healthy adults was found to be 102.7 μ³ with a coefficient of variation of 1.8%. For a volume of 87 μ³, this corresponds to a shape factor of 1.18, an axial ratio (assuming a perfect oblate spheroid) of 0.26, and an equivalent major axis of 8.6 μ. The effect of high electric fields on red cell size distributions is mentioned.     

Selective identification of the paternal mitochondrion in living sea urchin eggs and embryos by chlorotetracycline

When sea urchin eggs are pretreated with fluorescent chelate probe chlorotetracycline (CTC) and then fertilized with unlabeled sperm, a small, brightly fluorescent particle resembling the mitochondrion of free-swimming sperm both in size and fluorescent staining characteristics appears in the egg cytoplasm. This particle first appears near the base of the insemination cone and, like the paternal mitochondrion identified in previous ultrastructural studies, remains closely associated with the male pronucleus during its microtubule-dependent migration toward the egg center. These similarities strongly suggest that the fluorescent particle observed in the cytoplasm of living, CTC-pretreated sea urchin eggs is, in fact, the mitochondrion of the fertilizing sperm.     

Micronization of Organic Materials by Crystallization with Compressed Gases

A pilot plant is presented, which has been built to prepare fine particles by the P recipitation with a C ompressed Fluid A ntisolvent (PCA process). This technique offers interesting applications for products, which are produced in small amounts, as certain pharmaceuticals or energetic materials. In this contribution the micronization of paracetamol and tartaric acid is presented. Liquid solutions of tartaric acid in acetone, ethanol and methanol/ethanol mixtures have been sprayed into supercritical carbon dioxide used as antisolvent. The mean particle size of the precipitated powder can be manipulated by changing the precipitation pressure and solvent type however the precipitation temperature has no significant influence on the particle size. Paracetamol is micronized from acetone, methanol and DMF and morphologies from needles to spheres were found depending on the solvent. The particle size was in the submicron range.     

The effect of non-aggregating proteins upon the gelation of sickle cell hemoglobin: model calculations and data analysis.

The scaled particle theory for mixtures of hard spheres is used to calculate the effect of added proteins of varying size upon the solubility of sickle cell hemoglobin. For a given added weight, smaller macromolecules are more effective in lowering the solubility of sickle cell hemoglobin. Calculations based upon this model agree with many recently reported observations. The observed effect of the addition of myoglobin or hemoglobin α-chains on the minimum gelling concentration of sickle cell hemoglobin (Benesch $\text{et al.}$), however, is smaller than predicted. We suggest that this difference may arise from self-association of the added species.     

Rotational diffusion coefficients of a small,spherical subunit flexibly tethered to a larger sphere

The rotational diffusion coefficients of a small spherical particle, which is flexibly anchored to the surface of a much larger sphere, are calculated using the hydrodynamic theory of segmentally flexible particles. The model is intended for representing the rotational mobility of a small residue or chromophore in the surface of a globular macromolecule. The coefficients are found to be essentially independent, or to vary slowly with the relative dispositions of the spheres. They are also insensitive to the size ratio when this ratio is high enough. These findings support the use of an approximative treatment proposed by Wegener in which the small conformation dependence is averaged out. The resulting averages are tentatively used in the Lipari-Szabo model for restricted rotational diffusion in a cone. It is concluded that the rotational relaxation of the small sphere has three components: (i) a torsional rotation with the same diffusion coefficient as the free sphere; (ii) a perpendicular wobbling with a diffusion coefficient several (five in a typical case) times smaller; and (iii) an overall rotation of the whole macromolecule, that will appear in a much longer time scale if the two spheres have quite distinct sizes.     

Spatial pattern characterization of linear polarization-sensitive backscattering Mueller matrix elements of human serum albumin sphere suspension

Human serum albumin (HSA) nanometer or micron particles represent promising drug-carrier systems. The azimuthal and radial variations of a linear polarization-sensitive backscattering Mueller matrix were experimentally studied in two cases: the scattering particle was smaller or larger in size to the probing wavelength of 780 nm. The results show that the twofold and fourfold structures are characteristic of small particle size suspension, whereas the eightfold structure is characteristic of large particle size suspension. Moreover, for both particle size suspensions, the element patterns have strong radial dependence when the suspension concentration and the incident power of laser change. In addition, for both particle size suspensions, the rotational symmetry of each element is lost in the case of oblique incidence but the multifold structure is maintained. Some suggestions for applications of Mueller matrix imaging in biomedical optics are provided.     

Interaction forces between red cells agglutinated by antibody. I. Theoretical.

A general method of calculating forces, torques, and translational and rotational velocities of rigid, neutrally buoyant spheres suspended in viscous liquids undergoing a uniform shear flow has been given by Arp and Mason (1977). The method is based on the matrix formulation of hydrodynamic resistances in creeping flow by Brenner and O'Neill (1972). We describe the solution of the Brenner-O'Neill force-torque vector equation in terms of the particle and external flow field coordinates and derive expressions for the normal force acting along, and the shear force acting perpendicular to, the axis of the doublet of spheres, the latter explicitly given for the first time. The equations consist of a term comprising force and torque coefficients obtained from the matrices of the hydrodynamic resistances (functions of the distance h between sphere surfaces which have been computed), and terms comprising the orientation of the doublet axis relative to the coordinates of the external flow field and the shear stress (which can be experimentally determined). We have applied the theory to a system of doublets of sphered, hardened human red cells of group A or B antigenic type cross-linked by the corresponding antibody at a fixed interparticle distance. Working from studies of the breakup of doublets of red cells in an accelerating Poiseuille flow, given in the succeeding paper, we are able to compute the hydrodynamic force required to separate the two spheres. Previous work has shown that the theory can be applied to doublets in a variable shear, Poiseuille flow, provided the ratio of particle to tube diameter is small. In calculating the force-torque coefficients it was assumed that the cells are crosslinked by antibody with h = 20 nm.     

In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction

Recently, it has become possible to directly detect changes in neuropeptide vesicle dynamics in nerve terminals in vivo and to measure the release of neuropeptides induced experimentally or evoked by normal behavior. These results were obtained with the use of transgenic fruit flies that express a neuropeptide tagged with green fluorescent protein. Here, we describe how vesicle movement and neuropeptide release can be studied in the larval Drosophila neuromuscular junction using fluorescence microscopy. Analysis methods are described for quantifying movement based on time lapse and fluorescence recovery after photobleaching data. Specific approaches that can be applied to nerve terminals include single particle tracking, correlation and Fourier analysis. Utilization of these methods led to the first detection of vesicle mobilization in nerve terminals and the discoveries of activity-dependent capture of transiting vesicles and post-tetanic potentiation of neuropeptide release. Overall, this protocol can be carried out in an hour with ready Drosophila.     

Mapping Diffusion in a Living Cell via the Phasor Approach

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created.     

Aggregation and disaggregation kinetics of human blood platelets: Part I. Development and validation of a population balance method.

Hydrodynamic shear stress of sufficient intensity is known to cause platelet activation and aggregation and to alter the effects of biochemical platelet agonists and antagonists. In this work, a population balance equation (PBE) model is developed for analysis of platelet aggregation and disaggregation kinetics under the influence of a shear field. The model incorporates both aggregation and disaggregation by splitting and/or erosion mechanisms. This paper, the first of a series of three, deals with the formulation, simplification, and validation of the PBE and with the estimation of parameters involved in the PBE. These population parameters include collision efficiency, void fraction (related to the particle collision diameter), and the breakage rate coefficient. The platelet particle size distribution is determined experimentally, both initially and at some later times. The PBE can then be used to match satisfactorily the observed particle histograms, by appropriate choice of parameters of the model as functions of time, platelet size, and magnitude of physical or chemical stimuli. Besides providing information on adhesive forces and on the rates of aggregation and disaggregation, these parameters infer the physical properties of platelets and platelet aggregates. These properties are of potential value in increasing our understanding of the processes involved in thrombotic disease and/or therapy. A numerical procedure for solving the PBE is validated by application to simple cases for which analytical solutions are available. The model is applied to analysis of experiments, and parameter sensitivity studies are used to order the importance of the parameters and to reduce the complexity of the model. The simplified model is shown to give good agreement with experimental observations.     

Pair distribution functions in small systems: implications for protein structure analysis.

A general formula is derived for the relation between the pair correlation function and the histogram of interparticle distances in small nonuniform systems. The formula is applied to random packings of spheres in a spherical container, which are generated by a Monte Carlo method. When measured properly, the resultant correlation functions are very similar to ones in bulk systems with the same volume fraction of particles. In contrast, the density is very nonuniform as a function of distance from the center of the container. The variation is an order of magnitude for the number density of particle centers, or severalfold for the occupied volume fraction. It is described how these results can be used to analyze the forces that determine protein structure.     

Dependence of Protein Folding Stability and Dynamics on the Density and Composition of Macromolecular Crowders

We investigate the effect of macromolecular crowding on protein folding, using purely repulsive crowding particles and a self-organizing polymer model of protein folding. We find that the variation in folding stability with crowder size for typical α-, β-, and α/β-proteins is well described by an adaptation of the scaled particle theory. The native state, the transition state, and the unfolded protein are treated as effective hard spheres, with the folded and transition state radii independent of the size and concentration of the crowders. Remarkably, we find that, as the effective unfolded state radius is very weakly dependent on the crowder concentration, it can also be approximated by a single size. The same model predicts the effect of crowding on the folding barrier and therefore refolding rates with no adjustable parameters. A simple extension of the scaled-particle theory model, assuming additivity, can also describe the behavior of mixtures of crowding particles.     

Measurement of biological aerosol with a fluorescent aerodynamic particle sizer (FLAPS): correlation of optical data with biological data

Biological aerosol measurement in real time is anurgent military requirement that also has manypotential non-military applications. Such detectioncapabilities will be useful in environmentalmonitoring, for example, in gathering information inperceived hazardous areas like housing developmentsdownwind of sewage treatment plants.Experience gained from measuring fluorescence signalsof single bacterial spores under flow cytometry usingUV excitation at 340--360 nm, was applied to concepttesting of a prototype instrument, built to do thesame for aerosols. This machine was capable ofresolving particle size as well as fluorescenceintensity of each particle under laboratory and fieldconditions; it was called the fluorescent aerodynamicparticle sizer (FLAPS). A second generation FLAPS(FLAPS2) was designed to be smaller, power efficientand field portable. FLAPS2 was challenged underrefereed conditions in blind trials to determine if itcould detect biological aerosols in natural fieldenvironment. This paper describes practical aspects ofmeasuring biological aerosols when the results must becompared to reference samplers that provide culturableor ``live' data. Treatment of particle size andfluorescence information is discussed with respect toFLAPS and reference data fidelity. Finally, anobjective method is introduced to evaluate FLAPS datacorrelation to reference data. The measurementssuggest that there is positive correlation betweenFLAPS measurements and live biological aerosolparticles.     

Modeling the correlation functions of conformational motions in proteins

A first principles calculation of the correlation function for conformational motion (CM) in proteins is carried out within the framework of a microscopic model of a protein as a heterogeneous system. The fragments of the protein are assumed to be identical hard spheres undergoing the CM within their conformational potentials about some mean equilibrium positions assigned by the tertiary structure. The memory friction function (MFF) for the generalized Langevin equation describing the CM of the particle is obtained on the basis of the direct calculation which is feasible for the present model of the protein due to the existence of a natural large parameter, viz. the ratio of the minimal distance between the mean equilibrium positions of the particles (approximately 7A) to the amplitude of their CM (<1A). A relationship between the MFF and the correlation functions of the CM of the particles is derived which makes their calculation to be a self-consistent mathematical problem. The general analysis of the MFF is exemplified by a simple model case in which the mean equilibrium positions of the particles form a regular lattice so that the correlation functions for all particles are the same. In this case the MFF is shown to be an infinite series of the powers of the auto-correlation function whose coefficients are independent on temperature. The latter is a result of the abstraction of the interaction potential by that of hard spheres which actually corresponds to the high temperature limit. On the examples of cubic and triangular lattices the coefficients are shown to be non-negative values which increase with the increase of the packing density of the particles and quickly tend to zero with the increase of their index. Thus the MFF can be approximated by a polynomial of the correlation function and the resulting mathematical equation is analogous to the one from the dynamic theory of liquids. The correlation function of the CM is obtained by numerical solution of the equation. At realistic packing densities for proteins it exhibits transparent non-exponential decay and includes two relaxation processes: the first one on the intermediate timescale (tens of picoseconds) and the second on the long timescale (its characteristic time is about tens of nanoseconds at small values of the friction coefficient and increases by orders of the magnitude with the increase of the latter). Thus the present approach provides the microscopic basis for previous phenomenological models of cooperative dynamics in proteins.     

Application of europium(III) chelate-dyed nanoparticle labels in a competitive atrazine fluoroimmunoassay on an ITO waveguide

We have demonstrated the use of an optical indium tin oxide (ITO) (quartz) waveguide as a new platform for immunosensors with fluorescent europium(III) chelate nanoparticle labels (Seradyn) in a competitive atrazine immunoassay. ITO as a solid surface facilitated the successful use of particulate labels in a competitive assay format. The limit of detection in the new nanoparticle assay was similar to a conventional ELISA. The effect of particle size on bioconjugate binding kinetics was studied using three sizes of bioconjugated particle labels (107, 304, and 396nm) and a rabbit IgG/anti-IgG system in a 96-well plate. A decrease in particle size resulted in faster binding but did not increase the assay sensitivity. Flux calculations based on the particle diffusivity prove that faster binding of the small particles in this study was primarily due to diffusion kinetics and not necessarily to a higher density of antibodies on the particle surface. The results suggest that ITO could make a good platform for an optical immunosensor using fluorescent nanoparticle labels in a competitive assay format for small molecule detection. However, when used in combination with fluorescent particulate labels, a highly sensitive excitation/detection system needs to be developed to fully utilize the kinetic advantage from small particle size. Different regeneration methods tested in this study showed that repeated washings with 0.1 M glycine-HCl facilitated the reuse of the ITO waveguide.