Realization and Characterization of a Four-Channel Integrated Optical Young Interferometer

In this paper, we report the realization and characterization of a four-channel integrated optical Young interferometer (YI), which enables simultaneous and independent monitoring of three binding processes. The simultaneous and independent measurement of three different glucose concentrations shows the multi-purpose feature of such device. The phase resolution for different pairs of channels was /spl sim/1/spl times/10/sup -4/ fringes, which corresponds to a refractive index resolution of /spl sim/8.5/spl times/10/sup -8/ . The observed errors, which are caused due to mismatching of spatial frequencies of individual interference patterns with those determined from the CCD camera, have been reduced by using different reduction schemes. In addition, we have investigated a novel method for discrimination of the refractive index change from the thickness of a bound layer during an immunoreaction, as well as measuring the temperature change the takes place during such a process

Using Magnetic Probes to Study Receptor Clustering in Live Cells

During pathogen recognition T-Cell Receptors form microclusters which are believed to be the central signalling units. These structures could hold the secret behind the exceptional sensitivity of T-Cells in distinguishing single triggering ‘agonist’ peptides against a background of thousands. We have developed a biophysical approach based on magnetic tweezers that allows us to study the players involved in these receptor clusters and their dynamics. We use antibody functionalized magnetic beads to target CD3, a subunit of the TCR Complex to induce TCR clustering. Using magnetic tweezers, we move the beads along the cell membrane and simultaneously measure trafficking of co-receptors and proteins involved in the complex using confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). We study co-receptor CD6, which is considered a co-stimulator for cell activation during cluster formation. Our findings suggest that while CD6 is not physically associated with TCR complex, it gets recruited into the TCR clusters. There it is partially immobilized and moves along as clusters are displaced. The diffusion coefficient of CD6 is higher in bead-stimulated cells, whereas CD6 outside clusters diffuse faster than those within clusters. We are also downscaling this method to induce formation of receptor nanoclusters, in order to explore the effects of physical receptor oligomerization on the activity of TCR and Epidermal Growth Factor Receptors

Plasticity of the MAPK signaling network in response to mechanical stress

Cells display versatile responses to mechanical inputs and recent studies have identified the mitogen-activated protein kinase (MAPK) cascades mediating the biological effects observed upon mechanical stimulation. Although, MAPK pathways can act insulated from each other, several mechanisms facilitate the crosstalk between the components of these cascades. Yet, the combinatorial complexity of potential molecular interactions between these elements have prevented the understanding of their concerted functions. To analyze the plasticity of the MAPK signaling network in response to mechanical stress we performed a non-saturating epistatic screen in resting and stretched conditions employing as readout a JNK responsive dJun-FRET biosensor. By knocking down MAPKs, and JNK pathway regulators, singly or in pairs in Drosophila S2R+ cells, we have uncovered unexpected regulatory links between JNK cascade kinases, Rho GTPases, MAPKs and the JNK phosphatase Puc. These relationships have been integrated in a system network model at equilibrium accounting for all experimentally validated interactions. This model allows predicting the global reaction of the network to its modulation in response to mechanical stress. It also highlights its context-dependent sensitivity

Direct Integration of Micromachined Pipettes in a Flow Channel for Single DNA Molecule Study by Optical Tweezers

We have developed a micromachined flow cell consisting of a flow channel integrated with micropipettes. The flow cell is used in combination with an optical trap setup (optical tweezers) to study mechanical and structural properties of λ-DNA molecules. The flow cell was realized using silicon micromachining including the so-called buried channel technology to fabricate the micropipettes, the wet etching of glass to create the flow channel,\ud and the powder blasting of glass to make the fluid connections. The volume of the flow cell is 2 µl. The pipettes have a length of 130 m, a width of 5–10 µm, a round opening of 1 um and can be processed with different shapes. Using this flow cell we stretched single molecules (λ-DNA) showing typical force-extension curves also found with conventional techniques. These pipettes can be\ud also used for drug delivery, for injection of small gas bubbles into a liquid flow to monitor the streamlines, and for the mixing of liquids to study diffusion effects. The paper describes the design, the fabrication and testing of the flow cell

Waveguide-coupled micro-ball lens array suitable for mass fabrication

We demonstrate a fabrication procedure for the direct integration of micro-ball lenses on planar integrated optical channel waveguide chips with the aim to reduce the divergence of light that arises from the waveguide in both horizontal and vertical directions. Fabrication of the lenses is based on photoresist reflow which is a procedure that allows for the use of photolithography for careful alignment of the lenses with respect to the waveguides and enables mass production. We present in detail the design and fabrication procedures. Optical characterization of the fabricated micro-ball lenses demonstrates a good performance in terms of beam-size reduction and beam shape. The beam half divergence angle of 1544 nm light is reduced from 12.4° to 1.85°