Design and Construction of a Multi-Tiered Minimal Actin Cortex for Structural Support in Lipid Bilayer Applications.

Artificial lipid bilayers have revolutionized biochemical and biophysical research by providing a versatile interface to study aspects of cell membranes and membrane-bound processes in a controlled environment. Artificial bilayers also play a central role in numerous biosensing applications, form the foundational interface for liposomal drug delivery, and provide a vital structure for the development of synthetic cells. But unlike the envelope in many living cells, artificial bilayers can be mechanically fragile. Here, we develop prototype scaffolds for artificial bilayers made from multiple chemically linked tiers of actin filaments that can be bonded to lipid headgroups. We call the interlinked and layered assembly a multiple minimal actin cortex (multi-MAC). Construction of multi-MACs has the potential to significantly increase the bilayer's resistance to applied stress while retaining many desirable physical and chemical properties that are characteristic of lipid bilayers. Furthermore, the linking chemistry of multi-MACs is generalizable and can be applied almost anywhere lipid bilayers are important. This work describes a filament-by-filament approach to multi-MAC assembly that produces distinct 2D and 3D architectures. The nature of the structure depends on a combination of the underlying chemical conditions. Using fluorescence imaging techniques in model planar bilayers, we explore how multi-MACs vary with electrostatic charge, assembly time, ionic strength, and type of chemical linker. We also assess how the presence of a multi-MAC alters the underlying lateral diffusion of lipids and investigate the ability of multi-MACs to withstand exposure to shear stress.

Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as "silent translocation." By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

Cerebral Blood Flow Response to Simulated Hypovolemia in Essential Hypertension: A Magnetic Resonance Imaging Study.

Hypertension is associated with raised cerebral vascular resistance and cerebrovascular remodeling. It is currently unclear whether the cerebral circulation can maintain cerebral blood flow (CBF) during reductions in cardiac output (CO) in hypertensive patients thereby avoiding hypoperfusion of the brain. We hypothesized that hypertension would impair the ability to effectively regulate CBF during simulated hypovolemia. In the present study, 39 participants (13 normotensive, 13 controlled, and 13 uncontrolled hypertensives; mean age±SD, 55±10 years) underwent lower body negative pressure (LBNP) at -20, -40, and -50 mmHg to decrease central blood volume. Phase-contrast MR angiography was used to measure flow in the basilar and internal carotid arteries, as well as the ascending aorta. CBF and CO decreased during LBNP (P<0.0001). Heart rate increased during LBNP, reaching significance at -50 mmHg (P<0.0001). There was no change in mean arterial pressure during LBNP (P=0.3). All participants showed similar reductions in CBF (P=0.3, between groups) and CO (P=0.7, between groups) during LBNP. There was no difference in resting CBF between the groups (P=0.36). In summary, during reductions in CO induced by hypovolemic stress, mean arterial pressure is maintained but CBF declines indicating that CBF is dependent on CO in middle-aged normotensive and hypertensive volunteers. Hypertension is not associated with impairments in the CBF response to reduced CO.

Mechanically Enhancing Planar Lipid Bilayers with a Minimal Actin Cortex.

All cells in all domains of life possess a cytoskeleton that provides mechanical resistance to deformation and general stability to the plasma membrane. Here, we utilize a two-dimensional scaffolding created by actin filaments to convey mechanical support upon relatively fragile planar bilayer membranes (black lipid membranes, BLMs). Robust biomembranes play a critical role in the development of protein nanopore sensor applications and might also prove helpful in ion-channel research. Our investigation utilizes a minimal actin cortex (MAC) that is formed by anchoring actin filaments to lipid membranes via a biotin-streptavidin-biotin bridge. We characterize the joined structure using various modes of optical microscopy, electrophysiology, and applied mechanical stress (including measurements of elastic modulus). Our findings show the resulting structure includes a thin supporting layer of actin. Electrical studies indicate that the integrity of the MAC-bilayer composite remains unchanged over the limits of our tests (i.e., hours to days). The actin filament structure can remain intact for months. Minimalistic layering of the actin support network produces an increase in the apparent elastic modulus of the MAC-derivatized bilayer by >100×, compared to unmodified BLMs. Furthermore, the resistance to applied stress improves with the number of actin layers, which can be cross-linked to arbitrary thicknesses, in principle. The weblike support structure retains the lateral fluidity of the BLM, maintains the high electrical resistance typical of traditional BLMs, enables relatively uninhibited molecular access to the lipid surface from bulk solution, and permits nanopore self-assembly and insertion in the bilayer. These interfacial features are highly desirable for ion-channel and nanopore sensing applications.

Imaging Active Surface Processes in Barnacle Adhesive Interfaces.

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.

Proximal Capture Dynamics for a Single Biological Nanopore Sensor.

Single nanopore sensors enable capture and analysis of molecules that are driven to the pore entry from bulk solution. However, the distance between an analyte and the nanopore opening limits the detection efficiency. A theoretical basis for predicting particle capture rate is important for designing modified nanopore sensors, especially for those with covalently tethered reaction sites. Using the finite element method, we develop a soft-walled electrostatic block (SWEB) model for the alpha-hemolysin channel that produces a vector map of drift-producing forces on particles diffusing near the pore entrance. The maps are then coupled to a single-particle diffusion simulation to probe capture statistics and to track the trajectories of individual particles on the µs to ms time scales. The investigation enables evaluation of the interplay among the electrophoretic, electroosmotic, and thermal driving forces as a function of applied potential. The findings demonstrate how the complex drift-producing forces compete with diffusion over the nanoscale dimensions of the pore. The results also demonstrate the spatial and temporal limitations associated with nanopore detection and offer a basic theoretical framework to guide both the placement and kinetics of reaction sites located on, or near, the nanopore cap.

Shell Structure and Growth in the Base Plate of the Barnacle Amphibalanus amphitrite.

The base plate of the acorn barnacle Amphibalanus amphitrite (equivalent to Balanus amphitrite) is composed of hierarchically scaled, mutually aligned calcite grains, adhered to the substratum via layered cuticular tissue and protein. Acorn barnacles grow by expanding and lengthening their side plates, under which the cuticle is stretched, and adhesive proteins are secreted. In barnacles with mineralized base plates, such as A. amphitrite, a mineralization front follows behind, radially expanding the base plate at the periphery. In this study, we show that the new mineralization develops above the adhesion layers in a unique trilayered structure. Calcite crystallites in each of the layers have distinct sizes, varying from coarse-grained (>1 µm across) in the upper layer, to fine-grained (∼1 µm) in the middle layer, to nanoparticulate (∼40 nm) in the basal layer. The fine-grained crystallites dominate the growth front, comprising the bulk of the shell at the periphery, with later coarse grain development on the top of the base plate (toward the barnacle interior) and nanocrystalline calcite templating underneath in contact with the cuticle/protein layer. While the coarse-grained calcite on the upper surface contains a range of crystal orientations, the underlying fine-grained and nanocrystalline calcite are mutually oriented to within a few degrees of each other. Electron diffraction and X-ray absorption spectroscopy confirm that all of the crystallites are calcite, and metastable aragonite or amorphous calcium carbonate (ACC) phases are not observed. The complex morphology of the leading edge of the base plate suggests that crystallization initiates with the emplacement of mutually aligned fine-grained calcite, followed by the accumulation of coarser grains above and nucleation of highly oriented nanocrystalline grains below.

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate.

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.

The entry of HCl through soluble surfactants on sulfuric acid: effects of chain branching.

Gas-liquid scattering experiments are used to determine how a soluble, branched surfactant (2-ethylbutanol) controls the entry of gaseous HCl molecules into 60 and 68 wt % D2SO4 at 213 K. Short-chain alcohols spontaneously segregate to the surfaces of these sulfuric acid solutions, which are representative of aerosol droplets in the lower stratosphere. We find that 2-ethylbutanol enhances HCl entry at low surface coverages, most likely because it provides extra interfacial OH groups that aid HCl dissociation. This enhancement disappears at higher coverages as the alkyl chains crowd each other and block access to the acid. The branched alcohol impedes HCl entry more effectively than its unbranched isomer 1-hexanol, implying that the larger 2-ethybutanol footprint on the surface blocks more HCl molecules from reaching the alcohol-acid interface. This behavior contrasts sharply with gas transport through long-chain monolayers, where branching introduces gaps that allow more facile passage. The experiments suggest that short-chain surfactants with extended footprints may impede transport more effectively than their unbranched isomers.

Temperature sculpting in yoctoliter volumes.

The ability to perturb large ensembles of molecules from equilibrium led to major advances in understanding reaction mechanisms in chemistry and biology. Here, we demonstrate the ability to control, measure, and make use of rapid temperature changes in fluid volumes that are commensurate with the size of single molecules. The method is based on attaching gold nanoparticles to a single nanometer-scale pore formed by a protein ion channel. Visible laser light incident on the nanoparticles causes a rapid and large increase of the adjacent solution temperature, which is estimated from the change in the nanopore ionic conductance. The temperature shift also affects the ability of individual molecules to enter into and interact with the nanopore. This technique could significantly improve sensor systems and force measurements based on single nanopores, thereby enabling a method for single molecule thermodynamics and kinetics.

The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine.

Over 15 years ago, the ability to electrically detect and characterize individual polynucleotides as they are driven through a single protein ion channel was suggested as a potential method for rapidly sequencing DNA, base-by-base, in a ticker tape-like fashion. More recently, a variation of this method was proposed in which a nanopore would instead detect single nucleotides cleaved sequentially by an exonuclease enzyme in close proximity to one pore entrance. We analyze the exonuclease/nanopore-based DNA sequencing engine using analytical theory and computer simulations that describe nucleotide transport. The available data and analytical results suggest that the proposed method will be limited to reading <80 bases, imposed, in part, by the short lifetime each nucleotide spends in the vicinity of the detection element within the pore and the ability to accurately discriminate between the four mononucleotides.

Barnacle Balanus amphitrite adheres by a stepwise cementing process.

Barnacles adhere permanently to surfaces by secreting and curing a thin interfacial adhesive underwater. Here, we show that the acorn barnacle Balanus amphitrite adheres by a two-step fluid secretion process, both contributing to adhesion. We found that, as barnacles grow, the first barnacle cement secretion (BCS1) is released at the periphery of the expanding base plate. Subsequently, a second, autofluorescent fluid (BCS2) is released. We show that secretion of BCS2 into the interface results, on average, in a 2-fold increase in adhesive strength over adhesion by BCS1 alone. The two secretions are distinguishable both spatially and temporally, and differ in morphology, protein conformation, and chemical functionality. The short time window for BCS2 secretion relative to the overall area increase demonstrates that it has a disproportionate, surprisingly powerful, impact on adhesion. The dramatic change in adhesion occurs without measurable changes in interface thickness and total protein content. A fracture mechanics analysis suggests the interfacial material's modulus or work of adhesion, or both, were substantially increased after BCS2 secretion. Addition of BCS2 into the interface generates highly networked amyloid-like fibrils and enhanced phenolic content. Both intertwined fibers and phenolic chemistries may contribute to mechanical stability of the interface through physically or chemically anchoring interface proteins to the substrate and intermolecular interactions. Our experiments point to the need to reexamine the role of phenolic components in barnacle adhesion, long discounted despite their prevalence in structural membranes of arthropods and crustaceans, as they may contribute to chemical processes that strengthen adhesion through intermolecular cross-linking.

Information content in fluorescence correlation spectroscopy: binary mixtures and detection volume distortion.

When properly implemented, fluorescence correlation spectroscopy (FCS) reveals numerous static and dynamic properties of molecules in solution. However, complications arise whenever the measurement scenario is complex. Specific limitations occur when the detection region does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory, or when properties of multiple fluorescent species are assessed simultaneously. A simple binary solution of diffusers, where both mole fraction and diffusion constants are sought, can face interpretive difficulty. In order to better understand the limits of FCS, this study systematically explores the relationship between detection-volume distortion, diffusion constants, species mole fraction, and fitting methodology in analyses that utilize a two-component autocorrelation model. FCS measurements from solution mixtures of dye-labeled protein and free dye are compared to simulations, which predict the performance of FCS under a variety of experimental circumstances. The results reveal a range of conditions necessary for performing accurate measurements and describe experimental scenarios that should be avoided. The findings also provide guidelines for obtaining meaningful measurements when grossly distorted detection volumes are utilized and generally assess the latent information contained in FCS datasets.

Accurate optical analysis of single-molecule entrapment in nanoscale vesicles.

We present a nondestructive method to accurately characterize low analyte concentrations (0-10 molecules) in nanometer-scale lipid vesicles. Our approach is based on the application of fluorescence fluctuation analysis (FFA) and multiangle laser light scattering (MALLS) in conjunction with asymmetric field flow fractionation (AFFF) to measure the entrapment efficiency (the ratio of the concentration of encapsulated dye to the initial bulk concentration) of an ensemble of liposomes with an average diameter less than 100 nm. Water-soluble sulforhodamine B (SRB) was loaded into the aqueous interior of nanoscale liposomes synthesized in a microfluidic device. A confocal microscope was used to detect a laser-induced fluorescence signal resulting from both encapsulated and unencapsulated SRB molecules. The first two cumulants of this signal along with the autocorrelation function (ACF) were used to quantify liposome entrapment efficiency. Our analysis moves beyond typical, nonphysical assumptions of equal liposome size and brightness. These advances are essential for characterizing liposomes in the single-molecule encapsulation regime. Our work has further analytical impact because it could increase the interrogation time of free-solution molecular analysis by an order of magnitude and form the basis for the development of liposome standard reference materials.

HCl uptake through films of pentanoic acid and pentanoic acid/hexanol mixtures at the surface of sulfuric acid.

Molecular beam scattering experiments are used to investigate collisions and reactions of HCl with deuterated sulfuric acid containing 0-0.2 M pentanoic acid (PA) and mixtures of PA and hexanol. Surface tension measurements at 296 K indicate that PA segregates to the surface of the acid, reaching coverages of 58% and 52% of maximum packing on 60 and 68 wt % D(2)SO(4), respectively. We find that these films increase HCl entry into the acid at low PA surface coverage at 213 K. This enhancement is attributed to the dissociation of HCl molecules that come into contact with surface COOH groups and protonate them. At higher coverages, the PA film becomes more compact and impedes HCl uptake. Comparisons with films of pure hexanol and pentanoic acid/hexanol mixtures indicate that surface OH groups are more effective than COOH groups in catalyzing HCl entry. They also suggest that the PA films consist of patchy regions of tightly packed molecules, which are pushed away from the surface upon addition of the more surface active hexanol. HCl entry into the pure and mixed films can be analyzed quantitatively using a two-step model in which adsorbed HCl molecules penetrate between the alkyl chains and then dissociate at the surfactant-acid interface.

Heterogeneous translational dynamics of rhodamine B in polyelectrolyte multilayer thin films observed by single molecule microscopy.

The lateral diffusion dynamics of rhodamine B (RB) in polyelectrolyte multilayer (PEM) thin films has been studied with single-molecule confocal fluorescence microscopy. The films were made with sodium poly(sodium 4-styrenesulfonate) (PSS) and poly(diallydimethlyammonium chloride) (PDDA). Analysis of the real-time emission intensity traces reveals three diverse components of translational motion: (1) fast diffusion of RB through the confocal detection volume; (2) reversible tracer adsorption processes; and (3) nanoconfined diffusion. These processes cover a wide range of time scales. Analysis via fluorescence correlation spectroscopy (FCS) involves multicomponent fitting of the autocorrelated emission data. The model includes a free Brownian diffusion parameter, D, and two rate constants of desorption, k(-1) and k(-2). For RB in a PSS/PDDA thin film made with 0.01 M NaCl in the polyelectrolyte buildup solutions, D = 1.7 x 10(-7) cm(2)/s, k(-1) = 30 s(-1), and k(-2) = 0.1 s(-1). FCS was also performed on RB/PEM samples made with NaCl concentrations of the buildup solutions ranging from 0.01 to 0.7 M. A weak dependence of D and k(-1) on NaCl concentration was observed while k(-2) increased linearly with [NaCl].

Surfactant control of gas transport and reactions at the surface of sulfuric acid.

Aerosol particles in the atmosphere are tiny chemical reactors that catalyze numerous reactions, including the conversion of benign gases into ozone-destroying ones. In the lower stratosphere, these particles are often supercooled mixtures of water and sulfuric acid. The different species present at the surface of these droplets (H(2)O, H(3)O(+), HSO(4)(-), H(2)SO(4), and SO(4)(2-)) stand at the "gas-liquid frontier"; as the first to be struck by impinging molecules, these species provide the initial environment for solvation and reaction. Furthermore, aerosol particles may contain a wide range of organic molecules, some of which migrate to the surface and coat the droplet. How do ambient gases dissolve in the droplet if it is coated with an organic layer? At one extreme, monolayer films of insoluble, long-chain alcohols can dramatically reduce gas transport, packing so tightly at the surface of water that they impede water evaporation by factors of 10,000 or more. Shorter chain surfactants are expected to pack less tightly, but we wondered whether these incomplete monolayers also block gas transport and whether this system could serve as a model for understanding the surfaces of atmospheric aerosol particles. To address these questions, our research focuses on small, soluble surfactants such as butanol and hexanol dissolved in supercooled sulfuric acid. These amphiphilic molecules spontaneously segregate to the surface and coat the acid but only to a degree. Gas-liquid scattering experiments reveal that these porous films behave in surprisingly diverse ways: they can impose a barrier (to N(2)O(5) hydrolysis), be "invisible" (to water evaporation), or even enhance gas uptake (of HCl). The transition from obstacle to catalyst can be traced to specific interactions between the surfactant and each gas. For example, the hydrolysis of N(2)O(5) may be impeded because of its large size and because alcohol molecules that straddle the interface limit contact between N(2)O(5) and its H(3)O(+) and H(2)O reaction partners. However, these same alcohol molecules assist HCl dissociation because the alcohol OH groups provide extra interfacial protonation sites. Interestingly, butanol does not impede water evaporation, in part because the butyl chains pack much more loosely than insoluble, long-chain surfactants. Through these investigations, we hope to gain insight into the mechanisms by which surfactants on sulfuric acid and other aqueous solutions affect transport and reactivity at the gas-liquid interface.

Solvation of nitrophenol isomers: consequences for solute electronic structure and alkane/water partitioning.

Solute partitioning across a variety of alkane/aqueous interfaces was examined as a function of solute and alkane solvent structure. Solutes include p-nitrophenol (PNP), 3,5-dimethyl-p-nitrophenol (3,5-DMPNP), and 2,6-dimethyl-p-nitrophenol (2,6-DMPNP), the latter two being isomers distinguished solely by the location of methyl substituents on the aromatic ring. The alkane solvents included cylohexane, methylcyclohexane, octane, and iso-octane (2,2,4-trimethylpentane). PNP partitioned preferentially into the water by factors as high as 160:1. The dimethyl isomers partitioned more equally between water and the different alkanes. 2,6-DMPNP showed a 3-fold greater affinity for the alkane phase than 3,5-DMPNP. Ab initio calculations were used to characterize the molecular and electronic structure of the three solutes and to quantify individual contributions to each solute's solvation energy in model aqueous and alkane phases. Differences between 2,6-DMPNP and 3,5-DMPNP partitioning are interpreted based on the ability of the methyl groups in 2,6-DMPNP to weaken hydrogen bonding between the phenol group and adjacent water molecules. This diminished solvation interaction reduces the barrier to solute migration into the nonpolar organic phase despite the fact that 2,6-DMPNP has a larger (calculated) permanent, ground-state dipole than 3,5-DMPNP.

A distributed algorithm for multi-tau autocorrelation.

Network data-transfer times in distributed simulation environments can be reduced by performing data analysis at the remote source, if the analytical technique does not require the entire set of data at once. This novel multi-tau autocorrelation algorithm allows time-domain data records to be processed in discrete, distributed segments and combined at a later point in time. The new approach agrees with autocorrelation results performed by concatenating the discrete segments before correlation, but it operates with significantly shortened processing times. The multi-tau algorithm also benefits from reduced memory requirements since it does not require access to the entire data record at once, and from improved scalability since the multi-tau algorithm has order O(N), while fast Fourier transform autocorrelation algorithms have order O(N log N). This distributed algorithm has particular utility in simulations of fluorescence correlation spectroscopy or photon correlation spectroscopy.

Numerical fluorescence correlation spectroscopy for the analysis of molecular dynamics under nonstandard conditions.

The suitability of mathematical models used to extract kinetic information from correlated data constitutes a significant issue in fluorescence correlation spectroscopy (FCS). Standard FCS equations are derived from a simple Gaussian approximation of the optical detection volume, but some investigations have suggested this traditional practice can lead to inaccurate and misleading conclusions under many experimental circumstances, particularly those encountered in one-photon confocal measurements. Furthermore, analytical models cannot be derived for all measurement scenarios. We describe a novel numerical approach to FCS that circumvents conventional analytical models, enabling meaningful analyses even under extraordinarily unusual measurement conditions. Numerical fluorescence correlation spectroscopy (NFCS) involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation based on an experimentally determined 3D map of the detection volume. Model parameters are adjusted iteratively to minimize the residual differences between synthetic and experimental correlation curves. In order to reduce analysis time, we distribute calculations across a network of processors. As an example of this new approach, we demonstrate that synthetic autocorrelation curves correspond well with experimental data and that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume.