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

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°

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

Micromachined pipettes integrated in a flow channel for single DNA molecule study by optical trapping

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 set-up (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, and the powder blasting of glass to create the fluid connections. The volume of the flow cell is 2 µl. The pipettes have a length of 130 µm, a width of 5-10 µm, a round opening of 1 micron 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

Biophysical Characterization of CD6—TCR/CD3 Interplay in T Cells

Activation of the T cell receptor (TCR) on the T cell through ligation with antigen-MHC complex of an antigen-presenting cell (APC) is an essential process in the activation of T cells and induction of the subsequent adaptive immune response. Upon activation, the TCR, together with its associated co-receptor CD3 complex, assembles in signaling microclusters that are transported to the center of the organizational structure at the T cell-APC interface termed the immunological synapse (IS). During IS formation, local cell surface receptors and associated intracellular molecules are reorganized, ultimately creating the typical bull's eye-shaped pattern of the IS. CD6 is a surface glycoprotein receptor, which has been previously shown to associate with CD3 and co-localize to the center of the IS in static conditions or stable T cell-APC contacts. In this study, we report the use of different experimental set-ups analyzed with microscopy techniques to study the dynamics and stability of CD6-TCR/CD3 interaction dynamics and stability during IS formation in more detail. We exploited antibody spots, created with microcontact printing, and antibody-coated beads, and could demonstrate that CD6 and the TCR/CD3 complex co-localize and are recruited into a stimulatory cluster on the cell surface of T cells. Furthermore, we demonstrate, for the first time, that CD6 forms microclusters co-localizing with TCR/CD3 microclusters during IS formation on supported lipid bilayers. These co-localizing CD6 and TCR/CD3 microclusters are both radially transported toward the center of the IS formed in T cells, in an actin polymerization-dependent manner. Overall, our findings further substantiate the role of CD6 during IS formation and provide novel insight into the dynamic properties of this CD6-TCR/CD3 complex interplay. From a methodological point of view, the biophysical approaches used to characterize these receptors are complementary and amenable for investigation of the dynamic interactions of other membrane receptors