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Current Research |
Individual Projects and Group Projects are often connected to the AAA Problems in Science, problems with solutions that promise to have a far-reaching impact on science as a whole.
Process Analytical Technologies
Sara J. Hamilton, Amanda E. Lowell
Process analytical technologies (PAT) represent the future of pharmaceutical analysis. U.S. drug products are of generally high quality, but there is an increasing trend toward manufacturing-related problems that lead to recalls, shortages of essential drugs, disruption of manufacturing operations, and interference with new drug approvals. PATs offer means of reducing manufacturing problems. Modern spectrometric technology is capable of accomplishing much more in pharmaceutical analysis than it currently does. Hyperspectral UV-visible, near-IR, and IR imagers in combination with tunable lasers are now being applied in Dr. Lodder's labs to remote sensing of small samples. Improving the pharmaceutical industry to reduce manufacturing problems depends on such rapid online sample analysis replacing old QA methods.
In Dr. Lodder's lab, NIR imaging
of tablets inside blister packs has been shown to analyze over
1000 samples simultaneously in seconds. Remote sensing of lysine
crosslinks in single gelatin capsules at large distances (e.g.,
one mile) demonstrates that useful spectra can be obtained remotely
with good signal-to-noise ratio. These studies, in conjunction
with successful experiments demonstrating remote sensing of bacterial
biofilms, suggest that hyperspectral imaging will be profitably
applied to in situ cleaning validation and sample analysis in
a production environment.
Acoustic-Resonance Spectrometry as a
Process Analytical Technology for Rapid and Accurate Tablet Identification
Joseph Medendorp
The US FDA frequently orders recalls of tablets because of labeling problems, e.g., the wrong tablet appears in a bottle. A rapid, nondestructive method of online analysis and label comparison before shipping could obviate the need for recall or disposal of a batch of mislabeled drugs, thus saving a company considerable expense. ARS is accurate and precise as well as inexpensive and nondestructive, and the sensor is constructed from readily available parts, suggesting utility as a PAT. Click here for an animation demonstrating ARS of tablets.
Multispectral Imaging of Intact Pharmaceutical
Products
Imran Malik, Mela Poonacha, Jennifer
Moses
Near-IR spectrometry and multivariate analysis has been used for analysis of intact tablets, and to discriminate between different tablet formulations inside blister packages. Current experiments test the hypothesis that visible / near-IR and IR imaging spectrometry permits the identity and composition of multiple individual tablets to be determined simultaneously through packaging. For example, near-IR cameras collect thousands of spectra simultaneously from a field of blister-packaged tablets. The BEST chemometric-imaging algorithm is used to draw probability-density contour plots that reveal tablet composition. The number of tablets that a typical near-IR camera can currently analyze simultaneously is approximately 1300.
Astrobiology
and Spectroscopy
Kah-Siew Ho, Imran Malik, and Mela Poonacha
The ASRG has joined Project Argus, a worldwide microwave sky survey coordinated by the SETI League, a privately funded research foundation. The hydrogen line telescope is based on a parabolic reflector dish and a cylindrical feed-horn cavity and low-noise amplifier (LNA), and can be adjusted in azimuth and elevation to view the full sky. The telescope can monitor over 10,000 channels simultaneously for narrowband microwave signals - signals assumed to be of intelligent origin. Both human-assisted and completely automated signal screening methods are being employed to achieve faster data screening rates. Data are also screened for outbursts from Mira variable stars, pulsars, and comets, which also have signals in the same microwave region.
A near-infrared optical telescope with an aperture of one-meter has been constructed to detect interstellar laser transmissions. The near-IR telescope (4760 to 25000 cm-1) is being operated at the University of Kentucky. The telescope comprises a one meter visible / near-IR Fresnel lens, an aluminum compound parabolic concentrator, tilting interference filters, a robot for detector translation and star tracking, a liquid nitrogen Dewar and detectors (PbS, InSb, and InGaAs) and a 50 MHz preamplifier with 26 dB gain (max. data rate approximately 3 Gb per minute). Autocorrelation and cross-correlation are applied in screening the data collected.
Sensing in
Astrobiology Using Laser Arrays
J. Clay Harris
Our NSF-funded research deals with computational mathematics and a new paradigm called dynamic data-driven application simulations (DDDAS). We are working on a programmable, networked portable low cost mil-spec sensor-based system using DDDAS in extreme aqueous environments. DDDAS algorithms interact with three data collection components to provide early contaminant detection methods that can be used in remediation efforts to protect coastal facilities and nearby ecosystems from chemical spills. The research in this project is extendable to other environments, including remote planets and moons.
Near-Field Near-Infrared and Microwave
Microscopy
Charlie Symons, Joseph Medendorp
Transmission-mode near-field scanning microscopes (NSM) in the near-infrared and microwave spectral regions are being modeled to provide understanding of near-field optics and imaging. Near-field spectrometric imaging involves the electromagnetic scattering characteristics of objects illuminated by the near field of a sub-wavelength-sized aperture. In this project some unusual phenomena are being investigated, including a near-field focusing effect, as well as an impedance-based image-shape effect. Two computational models have been used to characterize NSMs, moment-method and finite-difference time-domain models. These two models are being applied to the analysis of the NSM for various configurations, and experimental near-field imaging measurements are compared with numerical predictions.
Nanotechnology
and Sensing of Vulnerable Atherosclerotic Plaque
Clay Harris and Rob Dolan
This research is developing an MFC-NSOM, and combining it with the concept of sensing arrays utilizing ISP methodology capable of ICI and HICI. In this way, sensors will be created that not only obtain large quantities of data, but also analyze that data immediately. The development of a nanowire "sniffer" capable of measuring CRP in vulnerable plaque will provide a method for the identification of patients susceptible to sudden cardiac death. Further research and development of these analytical tools and methods may reduce the number of deaths due to vulnerable plaque.
Virtual
Mouse Project
Clay Harris, Jay Hatcher
How do you simulate an entire creature on the metabolic scale? Being able to predict how mice will react will be a novel and groundbreaking new method for drug discovery. The calculations made in virtual cells and virtual mice can be transferred to synthetic biologists who are working to build a cell and eventually a mouse "from the ground up."
Imaging algorithms for massively
parallel supercomputers and intraarterial fiber-optic "cameras"
are used in this project. The nondestructive chemical analysis
of single lesions over time gives a new understanding of the mechanisms
of lesion formation and growth. With other traditional techniques,
vascular tissue must be removed before cholesterol and ox-LDL
can be assayed. The advantage of the near-IR technique is that
repeated analyses are possible of the same lesion during lesion
formation and/or treatment. The technique will make possible tests
of theories of lesion formation that are not possible with traditional
techniques.
Near-Infrared and Infrared Imaging
Analysis of Lipid Metabolism and Energy Expenditure
Lisa Cassis
Near-infrared (1000 - 3000 nm) spectrometry, which employs an external light source for determination of chemical composition, has been previously utilized for industrial determination of the fat content of commercial meat products, for in vivo determination of body fat, and in our laboratories for determination of lipoprotein composition in carotid artery atherosclerotic plaques. Infrared (3000 - 5000 nm) imaging, which measures the surface temperature of objects, was previously examined for quantitation of energy expenditure. However, no previous studies have examined combined near-IR/IR imaging as a tool for the determination of energy expenditure. The purpose of this study was to utilize near-IR and IR spectrometry under a variety of experimental conditions for determination of superficial lipid composition and surface heat emission in rats. Results demonstrate that near-IR spectrometry was capable of predicting the genotype of Zucker obese rats before the development of obesity (3 and 17 days of age). Pharmacologic analysis of the dose-dependent effects of norepinephrine (NE) and angiotensin II (AII) administration on superficial interscapular lipid composition by near-IR spectrometry demonstrated that both agents mobilized lipids albeit with different lipid spectral profiles. Using a tunable-range video camera with different band pass filters, near-IR imaging of adult obese rats demonstrated increases in superficial lipid composition. Moreover, IR imaging with the tunable-range video camera allowed for regional-specific measurements of surface heat emission in conscious, freely moving rats. Following treatment with isoproterenol (ISO) and AII, tail heat emission increased, but dorsal body heat emission differed between the two pharmacologic agents. Chronic administration of low doses of AII resulted in an increase in dorsal surface heat emission. Collectively, these results demonstrate that with a single-instrument, indexes of superficial lipid composition and surface heat emission can be obtained quickly and noninvasively for the analysis of energy expenditure.
Spectrophotometric and Electrophoretic Analysis of Carotid Plaque Lipoproteins
Amanda R. Tatro
Determining the number of proteins present in an atherosclerotic plaque and the roles played by these proteins is essential in the future to diagnosis and treatment of atherosclerosis. In vitro plaque protein electrophoresis and in vivo near-IR spectrometry are used to provide qualitative and quantitative information on the proteins involved in carotid atherosclerotic lesions. The information garnered from electrophoresis is used to test the hypothesis that particular proteins in combination show significant correlation to patient medical history and lesion pathology. For example, bruit, from medical history, and fibrous cap, from pathology reports, are now known to involve similar concentrations of the same proteins. Near-IR spectra of these proteins indicate that all but one are probable lipoproteins. In addition, the concentration of the 18 kD protein is related to lesion size, which in turn has been related to severity of disease. Duplex ultrasound measurements such as shown below are used to determine lesion size and are compared to protein concentrations determined by electrophoresis to demonstrate a relationship between the concentration of the 18 kD protein and the the size of the lesion.
Lipoprotein Determination in Single Cells by Near-Infrared Spectromicrography
Jennifer L. Moses
A near-infrared InSb focal plane
array camera is being used to collect images of human carotid
plaques during carotid endarterectomies. The excised plaques are
later analyzed for lipoprotein content by ultracentrifugation
and gel electrophoresis. The results from the electrophoresis
indicate a correlation between the size of atherosclerotic plaque
and the concentration of a 93 kD protein in the plaque, and plaque
size is correlated to incidence of stroke. Experiments now underway
are designed to test two hypotheses:
(1) that a near- infrared PtSi CCD camera provides better in
vivo spatial resolution and more functional pixels on a plaque
image than the InSb focal plane array camera, with sufficient
signal-to-noise ratio for macroscopic lipoprotein determination
in individual plaque cells.
(2) that the oxLDL 93kD protein is transported into plaque cells
from serum by infiltrating macrophages.
The InSb camera typically has more failed pixels and has a lower
spatial resolution than the PtSi camera. The SEE and SEP for analytes
using the InSb camera and PtSi camera have been compared in the
laboratory using prepared samples to create a calibration curve.
The increased spatial resolution of the PtSi camera makes it easier
to select spectra from microscopic sections made from the excised
plaque. The spectra collected by attaching the PtSi camera to
a microscope are also compared to a visual picture of a stained
slide from the same plaque for reference. The near-IR microscope
shown below uses an InSb focal plane array camera and can be used
with either blackbody light sources with interference filters
or a Nd:YAG-pumped KTP/OPO tunable near-IR laser system.
Computerized Assignment of Near-IR Absorbances to Molecular Motions of Proteins and Peptides
Elizabeth G. Kraemer
Factor analysis of near-IR spectra of proteins and peptides in samples often reveals 20 or more possible proteins above the spectral noise level, even in in vivo near-IR spectrometry. One approach to understanding changes in near-IR spectra of biological systems is to propose a model on a molecular scale and calculate spectra for the molecular changes to see if they are consistent with any portion of the spectra observed in actual samples. This approach has been traditionally a difficult one to follow in the near-infrared because the signals are combinations and overtones of the molecular motions involved. However, continuing advances in instrument and computer technology are making the modeling approach attractive again.
Using a parallel supercomputer, semiempirical and ab initio molecular orbital calculations can be made from a proposed reaction model. Correlation energy calculations using Moller-Plesset perturbation theory and configuration interaction (CI) are typically required to achieve acceptable energy accuracy for fundamental molecular motions. Computation of force constants and harmonic vibrational analysis and determination of intensities for vibrational transitions must be done at the MP2 and CI levels. The calculated spectral model is reconciled to the spectral factor analysis through constrained optimization that uses linear programming to solve the assignment problem, and QQ analysis of residuals to detect model and/or assignment failures. This form of near-IR spectral modeling is being applied to polymers for controlled drug release microspheres, hard gelatin capsules, and lipoproteins in human carotid artery plaque. The figure below shows an intermediate (from lysine side chains) in the crosslinking process of proteins in gelatin for capsules. Over a dozen such intermediates, reactants, and products have been used in the modeling process described above to calculate near-IR spectra for comparison with collected spectra and reconstructed spectra from factor analysis.
A new near-IR InSb focal plane array video camera is being used to image carotid plaques during carotid endarterectomy. We have just completed a new program that manipulates frames from the camera for analysis was just completed. This new program is entirely menu/mouse driven, and enables users to grab a frame collected at any wavelength just by pointing at it. The mouse can be used to zoom in and out on any selected portion of the image, and to rescale the colors and change color weighting schemes on any area of the image. More importantly, the mouse can be used to select any feature in the image at one wavelength, and the complete spectra (absorbances from all frames) are automatically selected simultaneously and stored in a 2-D variable that can be passed to all of the 90+ programs that we have developed over the past ten years. The completion of this new imaging program will accelerate greatly our analysis of spectra obtained with the near-IR imaging system.
Figure 1 is a near-IR image of a carotid endarterectomy taken at 2312 nm, where -CH3 groups in lipids have an absorbance. The carotid plaque is the most dense red area toward the center of the image. The white circle on left is a white marble placed as a reference in the field of the camera (the black marble also used as an intensity reference is not visible at this wavelength, except for a small highlight to the right of the white marble). The long white features of the image are the tissue retractors used to hold the incision open. The marble and stainless steel instruments appear white in the image because they reflect the most near-IR light. The purple area to the far right is the surgical drape, which reflects most of the near-IR light falling upon it. The color scheme, from lowest light absorbance to highest, is white, violet, blue, green, yellow, orange, and red.
Figure 2 is a zoom-in image of the carotid from Figure 1, shown at the protein amide absorbance wavelength at 2180 nm. The carotid artery appears orange and a red plaque appears at the carotid bifurcation.
Near-IR spectra obtained at d wavelengths are represented as single points in a d-dimensional hyperspace. The HP Superdome calculates the probability that certain regions of the carotid plaques are normal tissue based on their spatially resolved near-IR spectra. If the tissue is abnormal, the direction of the spectral vector of the abnormal tissue in wavelength hyperspace identifies the constituents that make that region abnormal, and the length of the vector (scaled by the probability of a normal spectrum lying in that direction) gives the amount of constituents present. The probabilities calculated on the HP are converted to images on a workstation.
The development of a magnetohydrodynamic acoustic-resonance near-infrared (MAReNIR) spectrometer is currently underway. The MAReNIR spectrometer is a novel device for noninvasive chemical analysis. A major application for the device is near-infrared detection and quantification of cholesterol and lipoproteins simultaneously in serum samples and perhaps even in vivo. Near-IR spectrometry has been shown to be an effective method of determining cholesterol and lipoproteins in a human blood matrix, but variations in sodium ion concentrations constitute a significant interference in results. Knowledge of the ion concentration enables one to overcome the interference to cholesterol, but such knowledge is difficult to obtain noninvasively. The MAReNIR spectrometer overcomes the sodium ion interference by inducing ion motion in a magnetic field with a tunable acoustic wave (see Figure 3). The moving ions create an electrical current that is picked up by electrodes in a cuvette or on the surface of the skin. Measuring the current continuously in a computerized pattern recognition algorithm reveals the ion concentration, and permits accurate analyses of cholesterol. The acoustic wave itself is used to improve identification and quantification of similar apolipoproteins (such as apoA-I and apoA-II) in solution by modulating their conformations and hence their near-IR spectra (through hydrogen bonding). In addition, the acoustic waves help to set the near-IR spectral baseline by establishing the bulk density of tissue samples in vivo.
The MAReNIR spectrometer will be used to create a desktop clinical instrument for analyzing blood for lipoproteins and cholesterol simultaneously and without reagents in physician's offices. Present analytical techniques are error-prone, slow, and expensive. An analytical method that minimizes sample handling and time of analysis will make lipoprotein measurements more accurate by reducing degradation of the sample during the analysis. The spectrometric nature of the instrument may enable it to determine accurately cholesterol and lipoprotein levels directly through the skin as well.
A unique center for near-infrared imaging was constructed around a tunable KTP/OPO laser system granted by the National Institutes of Health to a group of medical researchers at the University of Kentucky. The laser system consists of a MIRAGE 3000B Mid-Infrared Optical Parametric Generator and a Continuum NY81-10 Nd:YAG Pump Laser (see Figure 4). The system provides tunable near-IR light with a wavelength from 1.4 to 4.1 micrometers with an effective power of 3.3 million watts. Figure 5 shows the mirrors in the laser OPO: (a) shows the laser off and (b) shows the laser on. From left to right, the three mirrors are for the 532 nm OPO beam, the 1064 nm OPA beam, and the output coupler, which reflects the Nd:YAG fundamental into a beam dump. Figure 6 shows the KTP optical parametric amplifier crystals that produce output near-IR light (signal and idler beams). Additional hardware and software were consturcted at the University of Kentucky to make the laser more useful; this equiptment is described in the Appendix. A laser control program was constructed and is detailed in a flow chart. The laser detector unit that was constructed is shown in Figure 7. The light is used in medical imaging experiments. The laser system is the first of its kind to be used in near-IR medical research, and allows the production of video images to monitor the progression of disease. These video images contain more specific chemical information on lipids and proteins than is available through CAT or MRI imaging.
The equipment is employed in the collection of data for several multidisciplinary projects aimed at improving human health through state-of-the-art near-infrared/supercomputer techniques. Each project involves the use of near-infrared spectrometry to image noninvasively chemical and structural changes associated with disease. Traditional methods of analysis typically require the removal of tissue samples, comparison between different subjects, or other indirect means of determining progression or effectiveness of treatment of disease. Because the new system permits nondestructive imaging of disease in living subjects, it enables the researchers to monitor disease directly, within individual subjects, which is a superior method of evaluating experimental treatments.
The system is used primarily in projects that are based in three broad components:
The Stroke Program, which will employ the equipment in the imaging of patient volunteers with carotid atherosclerosis. The laser will be used as a bright, tunable light source to obtain transcutaneous 3-D spatial resolution of plaques, with chemical composition profiles collected at various stages in lesion development. These data will be correlated with results from duplex ultrasound and lipoprotein electrophoresis from Stroke Program projects. This project seeks to investigate factors that contribute to stroke and treatments that can modulate degree of damage that results from stroke.
The in vivo chemical analysis and high-resolution imaging of the structure of atherosclerotic plaques using a near-IR fiber-optic catheter. New imaging algorithms for massively parallel supercomputers and intraarterial fiber-optic cameras are the most recent technological discoveries in this project. The nondestructive chemical analysis of single lesions over time virtually guarantees a new understanding of the mechanisms of lesion formation and growth.
1) Near-IR Spectrometric Imaging Design
The laser system has been used to test experimental catheters that are being developed for use in several projects investigating atherosclerosis. The catheters are now being used to monitor disease progression in living animals and their results will eventually be extended to human studies. The catheter's distal reflector tips are constructed of gold plated steel aand are only 450 micrometers wide. Confirmation that individual tips, imported from Germany, meet the necessary focusing and reflectance specifications can be achieved only by testing with the new laser. Use of a distal reflector catheter tip that is out of specifications would move the focal point and cause light loss, which would waste time, research funds, and subjects. Some projects are supported by the National Sciences Foundation and the Kentucky Affiliate of the American Heart Association.
2) Catheter Studies
The laser has also been used as the light source in clinical tests of functioning catheters (described above). In these studies, rabbits are maintained on a high-cholesterol diet to create fatty streaks and lesions in their arteries. The catheters are inserted into the femoral artery and advanced to the aortic arch. As the catheters are slowly withdrawn, near-IR chemical analyses of the lesion can be performed. With all other traditional techniques, vascular tissue must be removed from the animal before cholesterol and ox-LDL can be assayed. The advantage of the near-IR technique is that repeated analyses are possible of the same lesion in the same animal during lesion development.
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