Nam T. Vu

Integrated microfluidic and imaging platform for a kinase activity radioassay to analyze minute patient cancer samples

Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric (32)P-ATP-labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL-positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples.

A β-Camera Integrated with a Microfluidic Chip for Radioassays Based on Real-Time Imaging of Glycolysis in Small Cell Populations

An integrated β-camera and microfluidic chip was developed that is capable of quantitative imaging of glycolysis radioassays using 18F-FDG in small cell populations down to a single cell. This paper demonstrates that the integrated system enables digital control and quantitative measurements of glycolysis in B-RafV600E–mutated melanoma cell lines in response to specific B-Raf inhibition. Methods: The β-camera uses a position-sensitive avalanche photodiode to detect charged particle–emitting probes within a microfluidic chip. The integrated β-camera and microfluidic chip system was calibrated, and the linearity was measured using 4 different melanoma cell lines (M257, M202, M233, and M229). Microfluidic radioassays were performed with cell populations ranging from hundreds of cells down to a single cell. The M229 cell line has a homozygous B-RafV600E mutation and is highly sensitive to a B-Raf inhibitor, PLX4032. A microfluidic radioassay was performed over the course of 3 days to assess the cytotoxicity of PLX4032 on cellular 18F-FDG uptake. Results: The β-camera is capable of imaging radioactive uptake of 18F-FDG in microfluidic chips. 18F-FDG uptake for a single cell was measured using a radioactivity concentration of 37 MBq/mL during the radiotracer incubation period. For in vitro cytotoxicity monitoring, the β-camera showed that exposure to 1 μM PLX4032 for 3 days decreased the 18F-FDG uptake per cell in highly sensitive M229 cells, compared with vehicle controls. Conclusion: The integrated β-camera and microfluidic chip can provide digital control of live cell cultures and allow in vitro quantitative radioassays for multiple samples simultaneously.

Performance Characteristics of BGO Detectors for a Low Cost Preclinical PET Scanner

PETbox is a low-cost benchtop PET scanner dedicated to high throughput preclinical imaging that is currently under development at our institute. This paper presents the design and characterization of the detectors that are used in the PETbox system. In this work, bismuth germanate scintillator was used for the detector, taking advantage of its high stopping power, high photoelectric event fraction, lack of intrinsic background radiation and low cost. The detector block was segmented into a pixelated array consisting of 20 × 44 elements, with a crystal pitch of 2.2 mm and a crystal cross section of 2 mm × 2 mm. The effective area of the array was 44 mm × 96.8 mm. The array was coupled to two Hamamatsu H8500 position sensitive photomultiplier tubes, forming a flat-panel type detector head with a sensitive area large enough to cover the whole body of a typical laboratory mouse. Two such detector heads were constructed and their performance was characterized. For one detector head, the energy resolution ranged from 16.1% to 38.5% full width at half maximum (FWHM), with a mean of 20.1%; for the other detector head, the energy resolution ranged from 15.5% to 42.7% FWHM, with a mean of 19.6 %. The intrinsic spatial resolution was measured to range from 1.55 mm to 2.39 mm FWHM along the detector short axis and from 1.48 mm to 2.33 mm FWHM along the detector long axis, with an average of 1.78 mm. Coincidence timing resolution for the detector pair was measured to be 4.1 ns FWHM. These measurement results show that the detectors are suitable for our specific application.

Performance evaluation of PETbox: A low cost bench top PET scanner dedicated to high throughput preclinical imaging

PETbox is designed to be a low cost and easy to use bench top small animal PET scanner dedicated for high throughput quantitative pharmacokinetic and pharmacodynamic studies. To achieve this goal, the scanner is integrated with a complete animal management system that provides life support including reproducible positioning, temperature control, anesthesia, real-time monitoring of animal respiration and a pathogen barrier. This approach minimizes the overall cost and complexity of preclinical PET imaging and should enable non-imaging scientists to embrace the technology. The system uses two opposing detector heads, each one consisting of a pixilated BGO array coupled to two H8500 multi-channel photomultiplier tubes. The BGO crystals were segmented into 20 ? 44 arrays with a pixel pitch of 2.2 mm and a total active area of 44 mm ? 96.8 mm. Position and timing signals from the photomultiplier tube readout circuitry were connected to a field programmable gate array (FPGA) board with eight ADC channels, each running at 100 MHz. Signal processing algorithms were developed for the FPGA to process received PET events and raw list-mode data were generated by the FPGA board and transferred to a host PC for storage. Basic system performance parameters were measured. The system has an average intrinsic spatial resolution of 1.72 mm FWHM along detector long axis and 1.84 mm FWHM along detector short axis. The coincidence timing resolution was measured to be 4.1 ns FWHM. The average energy resolution of the crystals was 19.8% and the absolute sensitivity of the system was measured to be 3.8% at the center of the gantry. Initial imaging studies were also performed with live mice. A mouse tumor xenograft was imaged 1 hour after a 32uCi [18F]FDG injection for 20 minutes. 3D images were generated using a ML-EM method. Results demonstrate the capability and potential of the PETbox system for dedicated high throughput mouse studies such as biodistribution and organ uptake quantification.

Performance of an integrated microfluidic chip and position sensitive APD for the detection of beta emitting probes in cell cultures

A new device has been developed that allows imaging of charged particle emitting probes in a microfluidic circuit using a position sensitive avalanche photodiode (PSAPD). Microfluidic chips are an emerging technology that have been used in applications such as the synthesis of new molecular probes and incubation of live cell cultures in microfluidic chambers. This new device works by direct contact of the PSAPD detector with the substrate layer of a microfluidic chip and provides 2- dimensional images of the distribution of beta emitting probes over time. The spatial resolution of the PSAPD detector for beta particle imaging was evaluated with 18F line sources. The FWHM was measured to be 0.4 mm at the center of the field of view. The sensitivity of the device for 18F beta particle detection was evaluated as a function of substrate thickness between the source and detector surface. One of the applications of this new detector system is for the imaging of live cell cultures in a microfluidic environment. Preliminary images have also been acquired showing the uptake of [18F]fluorodeoxyglucose ([18F]FDG) probes in live cells incubated in a microfluidic chamber. Images of [18F]FDG uptake were acquired with less than 200 incubated cells over a period of 3 days. An increase in cellular [18F]FDG uptake was also observed by increasing the time period between cell feeding and [18F]FDG incubation in the cell chamber.

Direct Detection of Beta Particles on a Microfluidic Chip using Position Sensitive APDs

A new device is being developed that will allow imaging of the distribution of charged particles in microfluidic circuits, using position sensitive avalanche photodiodes (PSAPD). Microfluidic chips are an emerging technology that will facilitate the synthesis and study of new molecular imaging probes. These poly-dimethylsiloxane (PDMS) chips are low in cost to custom design and produce, and their development/prototyping cycles are relatively short. They can contain a variety of microcircuitry and microwells for manipulating nanoliter volumes of solutions. The new imaging device will be created by direct contact of a PSAPD detector with the fluidic layer of a PDMS chip and will provide 2-dimensional images of the distribution of beta emitting radiolabeled probes on the chip. Furthermore, the new device will also be capable of quantifying small amounts of these radiolabeled probes over time. The PSAPD will be used for the direct detection of positrons and other charged particles. Preliminary studies were performed in our laboratory to test the feasibility of detecting small amounts of radioactive positron emitting fluorodeoxyglucose (FDG) in a prototype device. Results have shown that sensitivities as low as 56 plusmn 0.1 pCi in a 4 mm2 ROI (14 pCi/mm2) and line pair spatial resolution of 0.5 mm can be achieved.

MARS: a mouse atlas registration system based on a planar x-ray projector and an optical camera

This paper introduces a mouse atlas registration system (MARS), composed of a stationary top-view x-ray projector and a side-view optical camera, coupled to a mouse atlas registration algorithm. This system uses the x-ray and optical images to guide a fully automatic co-registration of a mouse atlas with each subject, in order to provide anatomical reference for small animal molecular imaging systems such as positron emission tomography (PET). To facilitate the registration, a statistical atlas that accounts for inter-subject anatomical variations was constructed based on 83 organ-labeled mouse micro-computed tomography (CT) images. The statistical shape model and conditional Gaussian model techniques were used to register the atlas with the x-ray image and optical photo. The accuracy of the atlas registration was evaluated by comparing the registered atlas with the organ-labeled micro-CT images of the test subjects. The results showed excellent registration accuracy of the whole-body region, and good accuracy for the brain, liver, heart, lungs and kidneys. In its implementation, the MARS was integrated with a preclinical PET scanner to deliver combined PET/MARS imaging, and to facilitate atlas-assisted analysis of the preclinical PET images.

Design and initial performance of PETbox4, a high sensitivity preclinical imaging tomograph

PETBox4, currently under development at the Crump Institute is a new tomograph dedicated to preclinical imaging of mice. This system presents a significant improvement on sensitivity and spatial resolution compared to the first generation PETBox. We report here on its design and initial performance characteristics.

Detection of Beta Particles in a Microfluidic Chip Using a Scintillator and CCD

A new device is being developed that will allow imaging of the distribution of charged particles in microfluidic circuits, using a scintillator, a lens and a charge coupled device (CCD). Microfluidic chips are an emerging technology that will facilitate the study of molecular processes in pico-liter levels in a finely controlled manner. The ability to quantify low amounts of radioactivity in a microfluidic chip will provide researchers with a platform to investigate molecular processes in a controlled in-vitro environment. The new detector system consists of a plastic scintillator, a C-mount compact lens, and a Sony ICX285AL interline-transfer CCD cooled with a Peltier device. A microfluidic chip filled with fluoro-deoxy-glucose (FDG) solution was coupled to a plastic scintillator and the set up was placed inside a light-tight box for imaging. Preliminary studies were performed to test the feasibility of using the scintillator-based CCD detector system for this application. The CCD performance parameters were characterized by the photon transfer curve. The camera gain constant and read-out noise were measured to be 0.746 e-/ADU (analog-to-digital unit) and 8 e-, respectively. The dark current was also investigated with different temperatures and binning factors. The spatial resolution was measured and line pairs of FDG in a microfluidic chip were discernable down to a 0.5 mm separation. The system was able to quantify the activity level reliably down to 5 nCi/mm2.

Design and characterization of a biomedical device capable of pico-CI level beta detection for the study of cell metabolism

A new system is being developed to allow imaging of charged particles in microfluidic chip using position sensitive avalanche photodiodes (PSAPD). The new imaging system was created by placing the PSAPD detector with close proximity to the PDMS microchip. The new system therefore is capable of quantifying tiny amounts of these radiolabeled probes over time. The PSAPD can be used for the direct detection of positrons and other charged particles. In our studies, the performance of the system has been carried out. A gradient of solution containing radioactive positron emitting fluorodeoxyglucose (FDG) was imaged from a microchip. Results have shown that sensitivities as low as 56 plusmn 0.1 pCi in a 4 mm2 region of interest (ROI) (14 pCi/mm2) and line pair spatial resolution of 0.5 mm can be achieved. Moreover, the application to cell biology was successfully performed in which currently radioactive signal from less than one hundred cells can be detected and resolved clearly in the system.