Paul G. Charette

Biosensing based on surface plasmon resonance and living cells

We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents.

Fabrication of silicon nitride waveguides for visible-light using PECVD: a study of the effect of plasma frequency on optical properties

This paper presents work aimed at optimizing the fabrication of silicon nitride Si(x)N(y) thin-film visible-light planar waveguides using plasma-enhanced chemical vapour deposition (PECVD). The effects of plasma frequency, precursor gas ratio, and thermal annealing in relation to waveguide optical properties (refractive index, propagation losses) are studied. Experimental results over a wide range of precursor gas ratios show convincingly that waveguides fabricated using low-frequency PECVD have lower propagation losses in the visible range compared to waveguides of equal refractive index fabricated with high-frequency PECVD.

Fabrication of high resistivity cold-implanted InGaAsP photoconductors for efficient pulsed terahertz devices

A multiple-energy, high fluence, MeV Fe ion implantation process was applied at 83 K to heavily damage a low band gap (0.79 eV) epitaxial InGaAsP layer. Optimal rapid thermal annealing conditions were found and produced a fast photoconductor with high resistivity (up to 2500 Ωcm) and Hall mobility around 400 cm2V−1s−1. Short photocarrier trapping times (0.3 ps – 3 ps) were observed via transient differential reflectivity measurements. Furthermore, photoconductive terahertz devices with coplanar electrodes were fabricated and validated. Under pulsed excitation with a 1550 nm femtosecond fiber laser source, antennas based on Fe-implanted InGaAsP are able to emit broadband radiation exceeding 2 THz. Given such specifications, this new material qualifies as a worthy candidate for an integration into optical terahertz spectrometer designs.

Numerical method for high accuracy index of refraction estimation for spectro-angular surface plasmon resonance systems.

An eigenvector analysis based algorithm is presented for estimating refractive index changes from 2-D reflectance/dispersion images obtained with spectro-angular surface plasmon resonance systems. High resolution over a large dynamic range can be achieved simultaneously. The method performs well in simulations with noisy data maintaining an error of less than 10(-8) refractive index units with up to six bits of noise on 16 bit quantized image data. Experimental measurements show that the method results in a much higher signal to noise ratio than the standard 1-D weighted centroid dip finding algorithm.

In vivo intravital endoscopic confocal fluorescence microscopy of normal and acutely injured rat lungs

We present a new lung imaging technique based on endoscopic confocal fluorescence microscopy (ECFM), which is a new method that is able to provide cellular and structural assessment of living tissue using a small confocal probe in direct contact with the visceral pleura. To observe distal airspace structure and cellular condition in normal and injured lungs (hyperoxic and bleomycin challenged), we used fluorescent-specific marker contrast and ECFM. Alveolar space ECFM with spectral analyses were performed at 488-nm excitation using FITC-labeled markers or naturally fluorescent dyes. The normal lung was compared with the sick lung, where our in vivo imaging experiments correlated well with results obtained with corresponding ex vivo conventional assays. Four main elements pertaining to the acute lung injury/acute respiratory distress syndrome (ALI/ARDS) pathophysiology and established early key events were specifically studied: alveolar epithelial membrane phenotype, lung cell apoptosis, neutrophil recruitment, and edema. ECFM allowed visualization of (i) fine-tuned ultrastructural lectin (RCA-1) and sialoglycoprotein (RTI40) epithelial cell membrane expression, (ii) YO-PRO-1-related DNA linking of lung cell apoptosis, (iii) PKH2 green fluorescent cell linker-labeled neutrophil tracking in lung microcirculatory network and airspaces, (iv) FITC-dextran plasma contrast and extravasation with edema formation. ECFM provides reliable results to corresponding ex vivo fluorescent methods. ECFM, using the minimally invasive Five-1® optical instrument and specific fluorescent markers, is able to provide real-time potentially useful imaging of live unfixed normal and injured lung tissue with promising developments for improving bedside diagnostic and decision-making therapeutic strategy in patients with ALI.

Implementation Study of Single Photon Avalanche Diodes (SPAD) in

Single Photon Avalanche Diodes (SPAD) are known for their excellent timing performance which enables Time of Flight capabilities in positron emission tomography (PET). However, current array architectures juxtapose the SPAD with its ancillary electronics at the expense of a poor fill factor of the SPAD array. The 3D vertical integration of SPADs and readout electronics represents a solution to the aforementioned problem. Compared to systems with external electronics readout, 3D vertical integration reduces the SPAD interconnect parasitic capacitance while greatly increasing the photosensitive area and improving overall performances. This paper presents the implementation of two SPAD structures designed for PET. The SPAD structures are designed using Teledyne DALSA high voltage (HV) CMOS technology targeted for a 3-dimensional single photon counting module (3DSPCM). SPAD with two types of guard ring (diffusion-based and virtual guard ring) are designed, fabricated and characterized. All structures are based on a p + anode in an n-well cathode and are implemented along with active quenching circuits for proper characterization. The results show that the contact distribution and the anode-cathode spacing impact the dark count rate (DCR). The design of SPADs with a diffusion guard ring have a DCR down to 3 s- 1μm-2 at room temperature, afterpulsing probability of , timing resolution of 27 ps FWHM and PDE of 49% at 480 nm.

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Surface plasmon resonance (SPR) has developed into a powerful approach for label-free monitoring of cellular behavior. Most cellular responses, however, involve a complex cascade of molecular events which makes identifying the specific components of cellular behavior dynamics contributing to the aggregate SPR signal problematic. Recently, a number of groups have used surface plasmon-enhanced fluorescence (SPEF) microscopy on living cells. In this work, we show that SPEF microscopy can be used to identify the molecular mechanisms responsible for SPR detection of cellular processes. By specifically labeling the actin cytoskeleton in human epithelial kidney cells (HEK 293) and rat vascular smooth muscle cells (A7r5), we correlate cell reorganization observed in SPEF with SPR signal variations reflecting aggregate cellular changes. HEK 293 cells stimulated with angiotensin-II exhibited transient contraction, appearing as an SPR signal decrease with a subsequent increase above the initial baseline. SPEF micrographs showed a decrease in cellular area followed by actin densification and cell spreading. A7r5 stimulated with Latrunculin A showed actin cytoskeleton depolymerization, generating a steady SPR signal decrease, with SPEF micrographs showing extensive collapse of cell actin structures. We observed that SPR monitoring of cellular response is strongly dependent on minute variations in cellular footprint on the substrate as well as changes in the molecular density in the basal portions of the cells. Therefore, combining SPR with imaging of selective fluorescent markers by SPEF allows a more comprehensive deconvolution of the cellular signal in relation to molecular events within the cells.

HV CMOS Technology

This paper presents a buried quad p-n junction (BQJ) photodetector fabricated with a HV (high-voltage) CMOS process. Multiple buried junction photodetectors are wavelength-sensitive devices developed for spectral analysis applications where a compact integrated solution is preferred over systems involving bulk optics or a spectrometer due to physical size limitations. The BQJ device presented here is designed for chip-based biochemical analyses using simultaneous fluorescence labeling of multiple analytes such as with advanced labs-on-chip or miniaturized photonics-based biosensors. Modeling and experimental measurements of the spectral response of the device are presented. A matrix-based method for estimating individual spectral components in a compound spectrum is described. The device and analysis method are validated via a test setup using individually modulated LEDs to simulate light from 4-component fluorescence emission.

Identification of the molecular mechanisms in cellular processes that elicit a surface plasmon resonance (SPR) response using simultaneous surface plasmon-enhanced fluorescence (SPEF) microscopy

New radiotracer developments for nuclear medicine imaging require the analysis of blood as a function of time in small animal models. A microfluidic device was developed to monitor the radioactivity concentration in the blood of rats and mice in real time. The microfluidic technology enables a large capture solid angle and a reduction in the separation distance between the sample and detector, thus increasing the detection efficiency. This in turn allows a reduction of the required detection volume without compromising sensitivity, an important advantage with rodent models having a small total blood volume (a few ml). A robust fabrication process was developed to manufacture the microchannels on top of unpackaged p-i-n photodiodes without altering detector performance. The microchannels were fabricated with KMPR, an epoxy-based photoresist similar to SU-8 but with improved resistance to stress-induced fissuring. Surface passivation of the KMPR enables non-diluted whole blood to flow through the channel for up to 20 min at low speed without clotting. The microfluidic device was embedded in a portable blood counter with dedicated electronics, pumping unit and computer control software for utilisation next to a small animal nuclear imaging scanner. Experimental measurements confirmed model predictions and showed a 4- to 19-fold improvement in detection efficiency over existing catheter-based devices, enabling a commensurate reduction in sampled blood volume. A linear dose-response relationship was demonstrated for radioactivity concentrations typical of experiments with rodents. The system was successfully used to measure the blood input function of rats in real time after radiotracer injection.

CMOS buried Quad p-n junction photodetector for multi-wavelength analysis

During the last years native chemical ligation (NCL) gained in popularity as a method allowing the chemical synthesis of large peptides and entire proteins. NCL is particularly well‐suited for chemoselective and nondenaturing attachment of biomolecules on solid substrates. In the present work, we show the feasibility of monitoring of peptide synthesis, NCL and its catalysis on silicon oxide modified gold surfaces by surface plasmon resonance (SPR). NCL of a model peptide—bradykinin thioester—was carried out and monitored with a custom‐built SPR apparatus. Solid‐phase produced bradykinin thioester was ligated to the surface in the presence of variable concentrations of 4‐mercaptophenylacetic acid as transthioesterification catalyst. At catalyst concentration of 48 mM and above, the NCL reaction was maximal and identical to the reaction of the purified peptide‐mercaptophenylacetic acid thioester. SPR curves indicate typical first‐order kinetics with t1/2 of 81 s for this aryl thioester, but of 104 min for the primary alkyl thioester.