Interplay between flow and diffusion in capillary alginate hydrogels.

Alginate gels with naturally occurring macroscopic capillaries have been used as a model system to study the interplay between laminar flow and diffusion of nanometer-sized solutes in real time. Calcium alginate gels that contain homogeneously distributed parallel-aligned capillary structures were formed by external addition of crosslinking ions to an alginate sol. The effects of different flow rates (0, 1, 10, 50 and 100 µl min(-1)) and three different probes (fluorescein, 10 kDa and 500 kDa fluorescein isothiocyanate-dextran) on the diffusion rates of the solutes across the capillary wall and in the bulk gel in between the capillaries were investigated using confocal laser scanning microscopy. The flow in the capillaries was produced using a syringe pump that was connected to the capillaries via a tube. Transmission electron microscopy revealed an open aggregated structure close to the capillary wall, followed by an aligned network layer and the isotropic network of the bulk gel. The most pronounced effect was observed for the 1 nm-diameter fluorescein probe, for which an increase in flow rate increased the mobility of the probe in the gel. Fluorescence recovery after photobleaching confirmed increased mobility close to the channel, with increasing flow rate. Mobility maps derived using raster image correlation spectroscopy showed that the layer with the lowest mobility corresponded to the anisotropic layer of ordered network chains. The combination of microscopy techniques used in the present study elucidates the flow and diffusion behaviors visually, qualitatively and quantitatively, and represents a promising tool for future studies of mass transport in non-equilibrium systems.

µPIV methodology using model systems for flow studies in heterogeneous biopolymer gel microstructures.

A methodology for studying flow in heterogeneous soft microstructures has been developed. The methodology includes: (1) model fractal or random heterogeneous microstructures fabricated in PDMS and characterised using CLSM; (2) µPIV measurements; (3) Lattice-Boltzmann simulations of flow. It has been found that the flow behaviour in these model materials is highly dependent on pore size as well as on the connectivity and occurrence of dead ends. The experimental flow results show good agreement with predictions from the Lattice-Boltzmann modelling. These simulations were performed in geometries constructed from 3D CLSM images of the actual PDMS structures. Given these results, mass transport behaviour may be predicted for even more complex structures, like gels or composite material in, e.g., food or biomaterials. This is a step in the direction towards predictive science with regards to tailoring soft biomaterials for specific mass transport properties.

A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae.

Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.

A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning.

Cells naturally exist in a dynamic chemical environment, and therefore it is necessary to study cell behaviour under dynamic stimulation conditions in order to understand the signalling transduction pathways regulating the cellular response. However, until recently, experiments looking at the cellular response to chemical stimuli have mainly been performed by adding a stress substance to a population of cells and thus only varying the magnitude of the stress. In this paper we demonstrate an experimental method enabling acquisition of data on the behaviour of single cells upon reversible environmental perturbations, where microfluidics is combined with optical tweezers and fluorescence microscopy. The cells are individually selected and positioned in the measurement region on the bottom surface of the microfluidic device using optical tweezers. The optical tweezers thus enable precise control of the cell density as well as the total number of cells within the measurement region. Consequently, the number of cells in each experiment can be optimized while clusters of cells, that render subsequent image analysis more difficult, can be avoided. The microfluidic device is modelled and demonstrated to enable reliable changes between two different media in less than 2 s. The experimental method is tested by following the cycling of GFP-tagged proteins (Mig1 and Msn2, respectively) between the cytosol and the nucleus in Saccharomyces cerevisiae upon changes in glucose availability.

Optical systems for single cell analyses.

BACKGROUND: Data extracted from a population of cells represent the average response from all cells within the population. Even when the cells are genetically identical, cell-to-cell variations and genetic noise can make the cells respond in completely different ways. To understand the mechanisms behind the behaviour of a population, the cells must also be analysed on an individual basis. OBJECTIVE: This review highlights the use of optical manipulation, microfluidics and advanced fluorescence imaging techniques for the acquisition of single cell data. CONCLUSION: By implementation of these three techniques, it is possible to achieve a deeper insight into the principles underlying cellular functioning and a more thorough understanding of the phenomena often observed in cell populations, thus facilitating research in drug discovery.

Controlling enzymatic reactions by geometry in a biomimetic nanoscale network.

We demonstrate that a transition from a compact geometry (sphere) to a structured geometry (several spheres connected by nanoconduits) in nanotube-vesicle networks (NVNs) induces an ordinary enzyme-catalyzed reaction to display wavelike properties. The reaction dynamics can be controlled directly by the geometry of the network, and such networks can be used to generate wavelike patterns in product formation. The results have bearing for understanding catalytic reactions in biological systems as well as for designing emerging wet chemical nanotechnological devices.

Controlled initiation of enzymatic reactions in micrometer-sized biomimetic compartments.

We present a technique to initiate chemical reactions involving few reactants inside micrometer-scale biomimetic vesicles (10(-12) to 10(-15) L) integral to three-dimensional surfactant networks. The shape of these networks is under dynamic control, allowing for transfer and mixing of two or several reactants at will. Specifically, two nanotube-connected vesicles were filled with reactants (substrate and enzyme, respectively) by microinjection. Initially, the vesicles are far apart and any diffusive mixing (on relevant experimental time scales) between the contents of the separated vesicles is hindered because of the narrow diameter and long axial extension of the nanotube. To initiate a reaction, the vesicles were brought close together, the nanotube was consumed by the vesicles and at a critical distance, the nanotube-vesicle junctions were dilated leading to formation of one spherical reactor, and hence mixing of the contents. We demonstrate the concept using a model enzymatic reaction, which yields a fluorescent product (two-step hydrolysis of fluorescein diphosphate by alkaline phosphatase), where product formation was measured as a function of time using a FRAP fluorescence microscopy protocol. By comparing the enzymatic activity with bulk measurements, the enzyme concentration inside the vesicle could be determined. Reactions could be followed for systems having as few as approximately 15 enzyme molecules confined to a reactor vesicle. To describe the experiments we use a simple diffusion-controlled reaction model and solve it using a survival probability approach. The agreement with experiment is qualitative, but the model describes the trends well. It is shown that the model correctly predicts (i) single-exponential decay after a few seconds, and (ii) that the substrate decay constant depends on the number of enzymes and geometry of reaction container. The numerical correction factor Lambda is introduced in order to ensure semiquantitative agreement between experiment and theory. It was shown that this numerical factor depends weakly on vesicle radius and number of enzymes, thus it is sufficient to determine this factor only once in a single calibration measurement.

Nanofluidic networks based on surfactant membrane technology.

We explore possibilities to construct nanoscale analytical devices based on lipid membrane technology. As a step toward this goal, we present nanotube-vesicle networks with fluidic control, where the nanotube segments reside at, or very close (<2 microm) to optically transparent surfaces. These nanofluidic systems allow controlled transport as well as LIF detection of single nanoparticles. In the weak-adhesion regime, immobilized vesicles can be approximated as perfect spheres with nanotubes attached at half the height of the vesicle in the axial (z) dimension. In the strong-adhesion regime (relative contact area, Sr* approximately 0.3), nanotubes can be adsorbed to the surface with a distance to the interior of the nanotubes defined by the membrane thickness of approximately 5 nm. Strong surface adsorption restricts nanotube self-organization, enabling networks of nanotubes with arbitrary geometries. We demonstrate LIF detection of single nanoparticles (30-nm-diameter fluorescent beads) inside single nanotubes. Transport of nanoparticles was induced by a surface tension differential applied across nanotubes using a hydrodynamic injection protocol. Controlled transport in nanotubes together with LIF detection enables construction of nanoscale fluidic devices with potential to operate with single molecules. This opens up possibilities to construct analytical platforms with characteristic length scales and volume orders of magnitudes smaller than employed in traditional microfluidic devices.

Nanotube-vesicle networks with functionalized membranes and interiors.

We describe nanotube-vesicle networks with reconstituted membrane protein from cells and with interior activity defined by an injection of microparticles or molecular probes. The functionality of a membrane protein after reconstitution was verified by single-channel ion conductance measurements in excised inside-out patches from the vesicle membranes. The distribution of protein, determined by fluorescence detection, in the network membrane was homogeneous and could diffuse via a nanotube connecting two vesicles. We also show how injecting small unilamellar protein-containing vesicles can differentiate the contents of individual containers in a network. The combination of membrane activity and interior activity was demonstrated by ionophore-assisted accumulation, and internal Calcium Green-mediated detection, of Ca2+ within a single network container. This system can model a variety of biological functions and complex biological multicompartment structures and might serve as a platform for constructing complex sensor and computational devices.

Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies.

We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120 degrees between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks.