Dynamic stereo microscopy for studying particle sedimentation.

We demonstrate a new method for measuring the sedimentation of a single colloidal bead by using a combination of optical tweezers and a stereo microscope based on a spatial light modulator. We use optical tweezers to raise a micron-sized silica bead to a fixed height and then release it to observe its 3D motion while it sediments under gravity. This experimental procedure provides two independent measurements of bead diameter and a measure of Faxén's correction, where the motion changes due to presence of the boundary.

Optical shield: measuring viscosity of turbid fluids using optical tweezers.

The viscosity of a fluid can be measured by tracking the motion of a suspended micron-sized particle trapped by optical tweezers. However, when the particle density is high, additional particles entering the trap compromise the tracking procedure and degrade the accuracy of the measurement. In this work we introduce an additional Laguerre-Gaussian, i.e. annular, beam surrounding the trap, acting as an optical shield to exclude contaminating particles.

Peptide gels of fully-defined composition and mechanics for probing cell-cell and cell-matrix interactions in vitro.

Current materials used for in vitro 3D cell culture are often limited by their poor similarity to human tissue, batch-to-batch variability and complexity of composition and manufacture. Here, we present a "blank slate" culture environment based on a self-assembling peptide gel free from matrix motifs. The gel can be customised by incorporating matrix components selected to match the target tissue, with independent control of mechanical properties. Therefore the matrix components are restricted to those specifically added, or those synthesised by encapsulated cells. The flexible 3D culture platform provides full control over biochemical and physical properties, allowing the impact of biochemical composition and tissue mechanics to be separately evaluated in vitro. Here, we demonstrate that the peptide gels support the growth of a range of cells including human induced pluripotent stem cells and human cancer cell lines. Furthermore, we present proof-of-concept that the peptide gels can be used to build disease-relevant models. Controlling the peptide gelator concentration allows peptide gel stiffness to be matched to normal breast (<1 kPa) or breast tumour tissue (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, the peptide gels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions affect the size, shape and organisation of breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and other diseases.

The self-assembly, elasticity, and dynamics of cardiac thin filaments.

Solutions of intact cardiac thin filaments were examined with transmission electron microscopy, dynamic light scattering (DLS), and particle-tracking microrheology. The filaments self-assembled in solution with a bell-shaped distribution of contour lengths that contained a population of filaments of much greater length than the in vivo sarcomere size ( approximately 1 mum) due to a one-dimensional annealing process. Dynamic semiflexible modes were found in DLS measurements at fast timescales (12.5 ns-0.0001 s). The bending modulus of the fibers is found to be in the range 4.5-16 x 10(-27) Jm and is weakly dependent on calcium concentration (with Ca2+ > or = without Ca2+). Good quantitative agreement was found for the values of the fiber diameter calculated from transmission electron microscopy and from the initial decay of DLS correlation functions: 9.9 nm and 9.7 nm with and without Ca2+, respectively. In contrast, at slower timescales and high polymer concentrations, microrheology indicates that the cardiac filaments act as short rods in solution according to the predictions of the Doi-Edwards chopsticks model (viscosity, eta approximately c(3), where c is the polymer concentration). This differs from the semiflexible behavior of long synthetic actin filaments at comparable polymer concentrations and timescales (elastic shear modulus, G' approximately c(1.4), tightly entangled) and is due to the relative ratio of the contour lengths ( approximately 30). The scaling dependence of the elastic shear modulus on the frequency (omega) for cardiac thin filaments is G' approximately omega(3/4 +/- 0.03), which is thought to arise from flexural modes of the filaments.

Effect of sol-gel transition on shear-induced drop deformation in aqueous mixtures of gellan and kappa-carrageenan.

In this work, we present an experimental methodology to investigate the dynamics under shear flow of a drop that is gelling as a consequence of a temperature quench. The experiments were carried out on the system water/gellan/kappa-carrageenan in the biphasic region of the phase diagram, the gellan-rich phase being used as the dispersed phase. Gelation was brought about by lowering the temperature during flow after steady state drop deformation had been reached. Simple shear flow was applied by using a parallel plate apparatus equipped with optical microscopy and image analysis, which made it possible to monitor drop shape evolution before, during, and after gelation. The onset of gelation trapped drop deformation, thus producing anisotropic particles. The fingerprint of gelation was the simultaneous tumbling of the drops, which rotated as rigid ellipsoids under the action of shear flow. Interfacial tension between the two equilibrium phases was determined at different times during the temperature quench by analyzing drop retraction upon cessation of flow. Up to gelation, no significant change was observed in the measured values.

Integrating microfluidic generation, handling and analysis of biomimetic giant unilamellar vesicles.

The key roles played by phospholipids in many cellular processes, has led to the development of model systems, to explore both lipid-lipid and lipid-peptide interactions. Biomimetic giant unilamellar vesicles represent close facsimiles of in vivo cellular membranes, although currently their widespread use in research is hindered by difficulties involving their integration into high-throughput techniques, for exploring membrane biology intensively in situ. This paper presents an integrated microfluidic device for the production, manipulation and high-throughput analysis of giant unilamellar vesicles. Its utility is demonstrated by exploring the lipid interaction dynamics of the pore-forming antimicrobial peptide melittin, assessed through the release of fluorescent dyes from within biomimetic vesicles, with membrane compositions similar to mammalian plasma membranes.

The internal dynamic modes of charged self-assembled peptide fibrils.

Photon correlation spectroscopy is used to study the internal dynamics of self-assembled charged peptide fibrils. Short neutral and charged polymeric aggregates have diffusive modes due to whole macromolecular motion. For long semiflexible fibrils the logarithm of the intermediate scattering function follows a q(2)t(3/4) scaling at long times consistent with a Kratky-Porod free energy and preaveraged Oseen hydrodynamics. Persistence lengths on the order of micrometers are calculated for the peptide fibrils consistent with estimates from the liquid-crystalline phase behavior. Fibril diameters (5-35 nm) calculated from the initial decay of the correlation functions are in agreement with transmission electron microscopy measurements.