Size-selective analyte detection with a Young interferometer sensor using multiple wavelengths.

We present a method to discriminate between analytes based on their size using multiple wavelengths in a Young interferometer. We measured the response of two wavelengths when adding 85 nm beads (representing specific binding), protein A (representing non-specific binding) and D-glucose (inducing a bulk change) to our sensor. Next, the measurements are analysed using a approach based on theoretical analysis, and a ratio-based analysis approach to discriminate between bulk changes and the binding of the different sized substances. Moreover, we were able to discriminate binding of 85 nm beads from binding of protein A (~2 nm) in a blind experiment using the ratio-based approach. This can for example be used to discriminate specific analyte binding of larger particles from non-specific binding of smaller particles. Therefore, we believe that by adding size-selectivity we can strongly improve the performance of the Young interferometer sensor and integrated optical interferometric sensors in general.

Chip based common-path optical coherence tomography system with an on-chip microlens and multi-reference suppression algorithm.

We demonstrate an integrated optical probe including an on-chip microlens for a common-path swept-source optical coherence tomography system. This common-path design uses the end facet of the silicon oxynitride waveguide as the reference plane, thus eliminating the need of a space-consuming and dispersive on-chip loop reference arm, thereby obviating the need for dispersion compensation. The on-chip micro-ball lens eliminates the need of external optical elements for coupling the light between the chip and the sample. The use of this lens leads to a signal enhancement up to 37 dB compared to the chip without a lens. The light source, the common-path arm and the detector are connected by a symmetric Y junction having a wavelength independent splitting ratio (50/50) over a much larger bandwidth than can be obtained with a directional coupler. The signal-to-noise ratio of the system was measured to be 71 dB with 2.6 mW of power on a mirror sample at a distance of 0.3 mm from the waveguide end facet. Cross-sectional OCT images of a layered optical phantom sample are demonstrated with our system. A method, based on an extended Fourier-domain OCT model, for suppressing ghost images caused by additional parasitic reference planes is experimentally demonstrated.

Syntenin-1 and ezrin proteins link activated leukocyte cell adhesion molecule to the actin cytoskeleton.

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.

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 °.

Classification of dynamical diffusion states in single molecule tracking microscopy.

Single molecule tracking of membrane proteins by fluorescence microscopy is a promising method to investigate dynamic processes in live cells. Translating the trajectories of proteins to biological implications, such as protein interactions, requires the classification of protein motion within the trajectories. Spatial information of protein motion may reveal where the protein interacts with cellular structures, because binding of proteins to such structures often alters their diffusion speed. For dynamic diffusion systems, we provide an analytical framework to determine in which diffusion state a molecule is residing during the course of its trajectory. We compare different methods for the quantification of motion to utilize this framework for the classification of two diffusion states (two populations with different diffusion speed). We found that a gyration quantification method and a Bayesian statistics-based method are the most accurate in diffusion-state classification for realistic experimentally obtained datasets, of which the gyration method is much less computationally demanding. After classification of the diffusion, the lifetime of the states can be determined, and images of the diffusion states can be reconstructed at high resolution. Simulations validate these applications. We apply the classification and its applications to experimental data to demonstrate the potential of this approach to obtain further insights into the dynamics of cell membrane proteins.

Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.

Size-selective detection in integrated optical interferometric biosensors.

We present a new size-selective detection method for integrated optical interferometric biosensors that can strongly enhance their performance. We demonstrate that by launching multiple wavelengths into a Young interferometer waveguide sensor it is feasible to derive refractive index changes from different regions above the waveguide surface, enabling one to distinguish between bound particles (e.g. proteins, viruses, bacteria) based on their differences in size and simultaneously eliminating interference from bulk refractive index changes. Therefore it is anticipated that this new method will be ideally suited for the detection of viruses in complex media. Numerical calculations are used to optimize sensor design and the detection method. Furthermore the specific case of virus detection is analyzed theoretically showing a minimum detectable virus mass coverage of 4 × 10(2) fg/mm(2) < (typically corresponding to 5 × 10(1) particles/ml).

Simultaneous time-resolved measurement of the reaction rates and the refractive index of photopolymerization processes.

We explore the use of imaging surface plasmon resonance (iSPR) to simultaneously measure the refractive index and reaction rates of the commercially available Ormocore photosensitive resist during photopolymerization. To this end, we adapted a commercially available iSPR device. We demonstrate good accuracy in the measurement of the refractive index determined independently of the thickness of the polymerized film. Furthermore, we demonstrate that the refractive index is proportional to the degree of cure (double bond conversion) of the resist. This allows the determination of the reaction rates of the polymerization processes, which show reasonable agreement with photodifferential scanning calorimetry measurements.

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.

An ultrasensitive young interferometer handheld sensor for rapid virus detection.

Future viral outbreaks are a major threat to societal and economic development throughout the world. A rapid, sensitive and easy-to-use test for viral infections is essential to prevent and control such viral pandemics. Furthermore, a compact, portable device is potentially very useful in remote or developing regions without easy access to sophisticated laboratory facilities. In this report we discuss the application of a Young interferometer sensor device for ultrasensitive and real-time detection of viruses. The essential innovation in this technique is the combination of an integrated optical interferometric sensor with antibody-antigen recognition approaches to yield very sensitive, rapid virus detection. The technology is amenable to miniaturization and mass production and, thus, has significant potential to be developed into a handheld, point-of-care device.

Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers.

Phagocytosis is an important process of the immune system by which pathogens are internalized and eliminated by phagocytic cells. Upon internalization, the phagosome matures and acidifies while being transported in a centripetal fashion. In this chapter, we describe protocols for simultaneous imaging of phagosomal acidification as well as their spatial manipulation by magnetic tweezers. First, we describe the protocols for functionalization of magnetic microbeads with pH-sensitive dyes and pH calibration of these particles. We also describe the preparation of magnetic tweezers and the calibration of forces that can be generated by these tweezers. We provide details of the design of the custom electrical and optical setup used for simultaneous imaging of phagosomal pH and phagosome's location. Finally, we provide a detailed description of the data analysis methodology.

Temperature modulation of the visible and near infrared absorption and scattering coefficients of human skin.

We determine temperature effect on the absorption and reduced scattering coefficients (mu(a) and mu(s)(')) of human forearm skin. Optical and thermal simulation data suggest that mu( a) and mu(s)(') are determined within a temperature-controlled depth of approximately 2 mm. Cutaneous mu(s)(') change linearly with temperature. Change in mu(a) was complex and irreversible above body normal temperatures. Light penetration depth (delta) in skin increased on cooling, with considerable person-to-person variations. We attribute the effect of temperature on mu(s)(') to change in refractive index mismatch, and its effect on mu(a) to perfusion changes. The reversible temperature effect on mu (s)(' ) was maintained during more than 90 min. contact between skin and the measuring probe, where temperature was modulated between 38 and 22 degrees C for multiple cycles While temperature modulated mu(s)(' ) instantaneously and reversibly, mu(a) exhibited slower response time and consistent drift. There was a statistically significant upward drift in mu(a) and a mostly downward drift in mu( s)(') over the contact period. The drift in temperature-induced fractional change in mu(s)(') was less statistically significant than the drift in mu(s)('). Deltamu( s)(') values determined under temperature modulation conditions may have less nonspecific drift than mu(s)(') which may have significance for noninvasive determination of analytes in human tissue.

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.

Magnetic nanoparticles as mediators of ligand-free activation of EGFR signaling.

BACKGROUND: Magnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells. METHODOLOGY/PRINCIPAL FINDINGS: The EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength. CONCLUSIONS/SIGNIFICANCE: We demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Integrin-dependent activation of the JNK signaling pathway by mechanical stress.

Mechanical force is known to modulate the activity of the Jun N-terminal kinase (JNK) signaling cascade. However, the effect of mechanical stresses on JNK signaling activation has previously only been analyzed by in vitro detection methods. It still remains unknown how living cells activate the JNK signaling cascade in response to mechanical stress and what its functions are in stretched cells.We assessed in real-time the activity of the JNK pathway in Drosophila cells by Fluorescence Lifetime Imaging Microscopy (FLIM), using an intramolecular phosphorylation-dependent dJun-FRET (Fluorescence Resonance Energy Transfer) biosensor. We found that quantitative FRET-FLIM analysis and confocal microscopy revealed sustained dJun-FRET biosensor activation and stable morphology changes in response to mechanical stretch for Drosophila S2R+ cells. Further, these cells plated on different substrates showed distinct levels of JNK activity that associate with differences in cell morphology, integrin expression and focal adhesion organization.These data imply that alterations in the cytoskeleton and matrix attachments may act as regulators of JNK signaling, and that JNK activity might feed back to modulate the cytoskeleton and cell adhesion. We found that this dynamic system is highly plastic; at rest, integrins at focal adhesions and talin are key factors suppressing JNK activity, while multidirectional static stretch leads to integrin-dependent, and probably talin-independent, Jun sensor activation. Further, our data suggest that JNK activity has to coordinate with other signaling elements for the regulation of the cytoskeleton and cell shape remodeling associated with stretch.

Intracellular manipulation of chromatin using magnetic nanoparticles.

Magnetic tweezers are widely used for manipulating small magnetic beads inside the cell cytoplasm in order to gain insight into the structural and mechanical properties of the cytoskeleton. Here we discuss the use of magnetic tweezers for the study of nuclear architecture and the mechanical properties of chromatin in living cells. A custom-built, dedicated micro magnetic tweezer set-up is described. We review progress that has been made in applying this technology for the study of chromatin structure and discuss its prospects for the in situ analysis of nuclear architecture and chromatin function.

Biofunctionalized lipid-polymer hybrid nanocontainers with controlled permeability.

We have successfully developed, for the first time, a novel polymer-lipid hybrid nanocontainer with controlled permeability functionality. The nanocontainer is made by nanofabricating holes with desired dimensions in an impermeable polymer scaffold by focused ion beam drilling and sealing them with lipid bilayers containing remote-controlled pore-forming channel proteins. This system allows exchange of solutions only after channel activation at will to form temporary pores in the container. Potential applications are foreseen in bionanosensors, nanoreactors, nanomedicine, and triggered delivery.

Direct observation of nanomechanical properties of chromatin in living cells.

Precise manipulation of nanometer-sized magnetic particles using magnetic tweezers has yielded insights into the rheology of the cell cytoplasm. We present first results using this approach to study the nanomechanics of the cell nucleus. Using a custom-designed micro-magnetic-tweezers instrument, we can achieve sufficiently high magnetic forces enabling the application and measurement of controlled distortion of the internal nuclear structure on the nanometer scale. We precisely measure the elasticity and viscosity inside the nucleus of living HeLa cells. The high value of the Young's modulus (Y = 2.5 x 10(2) Pa) measured relative to the cytoplasm is explained by a large-scale model for in vivo chromatin structure using a polymer network model.

Fast, ultrasensitive virus detection using a Young interferometer sensor.

We report the application of an integrated optical Young interferometer sensor for ultrasensitive, real-time, direct detection of viruses. We have validated the sensor by detecting herpes simplex virus type 1 (HSV-1), but the principle is generally applicable. Detection of HSV-1 virus particles was performed by applying the virus sample onto a sensor surface coated with a specific antibody against HSV-1. The performance of the sensor was tested by monitoring virus samples at clinically relevant concentrations. We show that the Young interferometer sensor can specifically and sensitively detect HSV-1 at very low concentrations (850 particles/mL). We have further demonstrated that the sensor can specifically detect HSV-1 suspended in serum. Extrapolation of the results indicates that the sensitivity of the sensor approaches the detection of a single virus particle binding, yielding a sensor of unprecedented sensitivity with wide applications for viral diagnostics.

Drift correction in a multichannel integrated optical young interferometer.

We demonstrate that in a sensor based on a multichannel Young interferometer, the phase information obtained for different pairs of channels can be used to correct the long-term instability (drift) due to temperature differences between measuring and reference channels, the drift in the alignment of the setup, etc. Experiments show that the nature of a major part of the drift is such that the drift present in one of the channels can be determined by interpolation of the drift measured in the two adjacent channels. It is shown that a drift reduction of 10 times can be achieved as compared with the situation in which no correction is applied. We anticipate that these findings will permit the exploitation of the extreme sensitivity of interference-based sensors to a much greater extent.