Andrew J. Berger - US grants

0086797
Berger
Vibrational Raman spectroscopy is a precise tool for the identification and quantification of molecular species. Because the spectral bands are narrow, signals from many compo-nents can be resolved and analyzed simultaneously. Consequently, Raman spectroscopy provides a method for analyzing multiple components' concentrations in complex sam-ples accurately, non-invasively, and non-destructively. These attributes are valuable in the medical field for both in vivo and in vitro measurements.

Acquisition of Raman spectra is limited by the effects of intrinsic sample autofluor-escence. For most biological samples, the spectral background from fluorescence will typically be much (greater than100 times) larger than the Raman signal, and fluorescence shot noise can be the limiting source of noise. Consequently, fluorescence limits Raman spec-troscopy from being a more widely applicable technique for biomedical purposes.

The investigators propose to develop a novel Raman spectroscopy system based upon wavelength shifting to suppress these fluorescence effects. Such a system would be able to acquire fluorescence-corrected Raman spectra with unprecedented speed and accuracy. The technique uses a laser whose wavelength is varied over a range of < 1 nm at kHz fre-quencies. Because Raman spectra shift with the laser wavelength while fluorescence re-mains stable, the fluorescence background can be efficiently rejected; at the same time, detection in the kHz regime reduces the fluoresence shot noise significantly relative to DC detection. To date, the published implementations of wavelength-shifted Raman spectroscopy have used either steady-state detection, in which the noise-suppression advantage is lost, or single-wavelength scanning, which makes them too slow to com-pete with present multipixel systems. The proposed system would combine all of the ad-vantages and should significantly improve concentration predictions. In addition, sup-pression of fluorescence signal and noise could open new avenues for biomedical Ra-man spectroscopy, such as visible-excitation experiments (avoided because of extremely high fluorescence) and higher-contrast Raman imaging.

This proposal is broken into four Specific Aims, which are (1) to assemble the above Raman system and acquire a first round of spectra; (2) to optimize the signal-to-back-ground and signal-to-noise of the system and compare values to other Raman modali-ties; (3) to increase the optical throughput of the spectrometer via spatial Fourier-trans-form techniques; and (4) to conduct experiments to detect clinically relevant analytes in biological samples and phantoms.

This proposal combines basic spectroscopy with advanced instrumentation, provid-ing a solid platform for training graduate and undergraduate students in biomedical optics. Through Raman spectroscopy, discussion of related topics in absorption, fluo-rescence, and diffuse photon migration will arise. Conference attendance and presen-tations by graduate students (for which funding is explicitly requested) will further in-crease exposure to the field. Within the laboratory, students will gain direct experience assembling lasers, optics, spectrographs, and detectors into a functioning system and also programming computers to control the system. This will nurture broadly applica-ble skills for future use in both academic and industrial laboratory settings.

[unreadable] DESCRIPTION: A novel approach for measuring the bacterial composition of oral plaques, using optical scattering ("Raman") spectroscopy, is proposed. Raman spectroscopy measures the characteristic vibrational frequencies of a molecule and is, therefore, well-suited for discriminating between similar bacterial species. The proposed method has the potential to be faster, simpler, and more accurate in analyzing the major components of microbial communities than plating and immunofluorescence techniques. The ability to measure bacterial concentrations within minutes by placing samples directly in a laser beam, without preparation or labeling, would be attractive in many laboratory settings. [unreadable] [unreadable] Experiments will be performed on a series of increasingly complex bacterial samples, from titrated mixtures of two streptococci to bacterial ensembles harvested from the oral plaque of human volunteers. In each phase of the study, spectra of at least twenty samples will be obtained using state-of-the-art Raman spectroscopy. Due to the use of a confocal microscope, only very small sample volumes (0.1 microliters) will be required, and spectral scans will last on the scale of minutes. Spectral differences from sample to sample, caused by variations in chemical content, will be detected and exploited to speciate and enumerate bacterial samples. The target of each experiment will be Raman-based prediction of bacterial concentrations, compared to reference values. Accuracy of prediction will be characterized in each step, providing benchmarks for subsequent studies on more complex samples. [unreadable] [unreadable] At the conclusion of these studies, the potential for Raman spectroscopy to analyze bacterial content in oral plaques will be thoroughly explored, and the feasibility of developing practical techniques for widespread usage in oral biology research will be clarified. In addition, the methodologies developed during this investigation will be broadly applicable to other problems requiring quantitative discrimination between two or more similar organisms in biological mixtures. [unreadable] [unreadable]

CBET-0754698
Andrew Berger, University of Rochester

An optical probing and imaging technique called Integrated Raman and Angular scattering Microscopy (IRAM) will be developed for the purpose of simultaneously studying the chemical and structural composition of single cells. The engineering challenge of unifying Raman and elastic angular-scattering measurements on a high-resolution microscopy platform will first be addressed through basic studies on microbeads and immune cells. The development of dendritic immune cells from immature to mature phase will then be studied using the IRAM technique. Specifically, chemical and structural changes will be sought at various timepoints to shed light on the signal pathways that steer the differentiation process.

0931687
Berger

This project aims to improve the use of near-infrared light for studying infants' brain activity. Although current optical methods of monitoring brain activity in infants show promise, many poorly-corrected variables (physical, physiological, and anatomical) still corrupt the data from too many infant participants. Through the research team's combination of expertise in biophotonics, brain and cognitive sciences, and pediatrics, the sources of noise will be reduced to levels where meaningful scans can be performed on babies routinely. The method used will be near-infrared spectroscopy (NIRS), which measures changes in the absorption of light by blood in the scalp and brain. The instrumentation will include dedicated "scalp-only" channels whose source-detector separations (~5 mm) are too short to be sensitive to brain signals. When one of these channels' readout is subtracted from standard channels (with separations of 20-35 mm), much physiological noise is cancelled, and stimulus related activations become easier to observe. The researchers' home-built corrected-near infrared-spectroscopy (C-NIRS) system for adults will be restructured for infant studies and enhanced with more sources and detectors, enabling measurements from a stimulated region and concurrent monitoring of a "control" region. An instrument of this scale designed with dedicated near-channel noise-suppression will be the first of its kind. In parallel, a more flexible silicone-based headband will be developed to fit a range of head shapes, be comfortable and safe for infants, and reduce the amplitude of motion artifacts in the data. The new instrument will be tested on a cohort of 60 healthy, alert infant participants (age 6-9 mos.), along with a commercial near-infrared instrument that lacks correction channels.
Measurement protocols will focus on well-studied visual and auditory stimulation paradigms. The reductions in physiological noise and motion artifacts should yield a higher "hit rate" of meaningful data runs using the C-NIRS instrument than for traditional NIRS, thereby establishing C-NIRS as a valuable tool in the arsenal of the cognitive scientist studying infant development.

DESCRIPTION (provided by applicant): Significance: Glucocorticoid (GC) treatment, a common anti-inflammatory prescription for rheumatoid arthritis, has a dangerous side effect: it elevates risk for osteoporosis and bone fracture. To reduce this effect, other drugs are being tested in combination with GC. The efficacy of these cocktails is often studied in a transgenic mouse model of rheumatoid arthritis, using X-ray imaging and mechanical tests to assess bone quality. Unfortunately, radiographic density correlates only weakly with early stages of bone fragility, and mechanical tests require sacrifice of the animal and cannot be translated to human patients. New methods are needed to study the progression of bone deterioration in animals (and humans) over time, particularly to detect alterations in the early stages where intervention is most valuable. Innovation: We propose the use of Raman spectroscopy (RS), a chemically-sensitive optical scattering technique, to monitor bone quality through the intact limbs of living, GC-treated mice. In preliminary tests, Raman spectra of excised bones from such mice exhibit lower mineral-to-matrix ratios than from control animals, and the ratio correlates strongly with mechanical tests of bone strength. Separate tests have verified that Raman signatures from mouse bones can be gathered through the soft tissue of intact limbs. These results motivate the hypothesis that RS can monitor individual mice noninvasively at multiple time points and detect early signs of bone alteration under various GC treatment regimens. As an added benefit, no ionization radiation is used, unlike the common microCT and DXA (dual X-ray absorptiometry) modalities. Specific aims: The project's first specific aim is to identify the Raman spectral features from ex vivo cortical bone that correlate most strongly with gold-standard assays of bone quality. Groups of mice will be paired to study three variables: wild-type vs. transgenic, GC-treated vs. placebo, and GC-alone vs. GC-plus- drug. The second specific aim is to obtain this same Raman information transcutaneously in living animals, using optimized probe geometry and data analysis to reduce interference from overlying soft tissue. Achieving this aim will enable robust monitoring of bone quality in individual animals over several weeks, producing histories of bone fragility onset in individual mice with unprecedented detail. Such histories should be rich sources of information about how GC treatment compromises bone quality and about the preventive capabilities of complementary drugs, providing feedback for disease management and treatment. Relevance: The proposed work directly addresses NIBIB's mission of "developing technologies for early disease detection and assessment of health status," as well as NIAMS's mission to "support research into the causes, treatment, and prevention of arthritis and musculoskeletal and skin diseases." PUBLIC HEALTH RELEVANCE: People with rheumatoid arthritis are often given medications to reduce the inflammation. Unfortunately, these medications themselves have side effects that elevate risk for bone fracture. This study will develop a noninvasive optical method of measuring bone fragility in arthritic mice as they receive both anti-inflammatory medication and complementary drugs that try to preserve bone health. By providing a better way of tracking bone fragility in living animal models, this work will generate new understanding of how bone disorders develop and how medicines can treat them more effectively in both animals and humans.

Illumination with beams of light in unconventional polarization states departs from the usual, textbook description. Two popular examples of unconventional polarization states are radial and azimuthal polarizations (sometimes called Cylindrical Vector Beams). A wide variety of unconventional polarizations exist -- some have been well studied, while many remain unexplored. This research activity brings together three investigators that have been studying unconventional polarization states and their mathematical representation, the propagation and focusing of polarized light, and light scattering from small particles such as those found within biological cells.

This investigation makes use of a novel mathematical representation (complex-focus basis) for modeling optical focusing and scattering, the study of radial, azimuthal, and Full Poincare fields as representatives of a complex-focus basis, and the scattering of unconventional polarization states from mesoscopic particles, including cell organelles. In the process, we also address the formulation and testing of a theory of partially coherent unconventional polarization states, the coupling of unconventional polarization states to nanostructures, and develop numerically efficient theoretical models for coherent and partially coherent light propagation. We make use of the recently-introduced concept of stress- engineered optical elements to adapt an existing light scattering microscope to carry out polarization- sensitive scattering experiments. In the process, we are advancing optical physics by introducing new analytic tools for scattering analysis, a new experimental tool for rapid-acquisition pupil polarimetry, and find better ways to use polarization as a tool for improving projection imaging systems such as LCD projectors and semiconductor lithography systems.

Polarization--the vector nature of light--influences the scattering of light in profound ways, and is therefore fundamental to how we gain information about a scatterer from the scattered light. This has been used to good result in advancing cell identification for immune cell research, for example. Scattering is also important in the inspection processes that are used to guarantee high quality semiconductor circuits for computers and electronic devices. A better understanding of the physics of new polarization states will therefore have a broad impact on fields such as optical engineering and biomedical optics. The educational impact is seen annually through involvement by both undergraduates and graduate students, not merely as research assistants, but as students in training who are encouraged toward independent initiatives. Our role as educators at the Institute of Optics offers us a unique platform from which to take the results of the work, bring them into the classroom at the MS and BS levels, and to take the exciting features of polarized light with us on educational activities in area schools and science museums. Our presence at the Institute of Optics offers technology transfer to over 30 companies who are members of our Industrial Associates Program.

This engineering education research initiation grant seeks to engage an engineering faculty member with physics education researchers to understand the alignment between students' mathematical and conceptual understanding in optics. The research will use an innovative interview method to elicit both conceptual and mathematical representations of optical polarization and plane waves.

The broader significance and importance of this project arises from its ability to inform others how students learn to integrate conceptual and mathematical representations of abstract ideas. The investigator will also engage undergraduate students in the planned research, thus contributing to their professional development and mentoring. This project overlaps with NSF's strategic goals of transforming the frontiers through preparation of an engineering workforce with new capabilities and expertise. Additionally NSF's goal of innovating for society is enabled by creating results and research that are useful for society by informing educational policy and practices.

1402345
Berger

Significance:
The familiar saying "every journey begins with a single step" has a special meaning in the human body: many large-scale changes are triggered by a few "early responder" cells. Examples include allergic reactions, wound healing, and cancerous transformations. Even when many "identical" cells are exposed to the same conditions in a laboratory, some respond faster; such differences are clues to understanding how an entire process unfolds, and perhaps how medicine can influence it. Unfortunately, most methods of measuring cellular responses have one of the following limitations: (a) they only calculate averages over many cells, (b) they measure each cell only once, or (c) they inject chemicals that alter the cells' function. In this project, a new microscope system will be built without these limitations. Using only low levels of light, many individual cells will be monitored for extended periods of time, producing chemical and structural "histories" of the cells as they respond to stimuli. This measurement technique will be useful for identifying early-responding cells and understanding how they lead to large-scale behavior in the human body. In a particular case of interest, a cellular transformation called "platelet degranulation" will be studied because it is associated with increased levels of neurological illness in HIV-positive individuals.


Technical description:
This project applies a new biophotonic measurement technique, holographic angular-domain elastic scattering (HADES), to studies of single cells responding to stimuli. Angular scattering is highly sensitive to the average size of organelles, and therefore to changes in cellular contents. Holography will enable first-principles analysis of single cells? angular scattering for the first time. The HADES technique will be combined in a microscope with Raman spectroscopy, a complementary technique that provides chemical specificity. This instrument will acquire morphological and chemical "histories" of single cells as they undergo changes, for example (a) endothelial cells receiving damage to their mitochondrial membranes, (b) immune cells being stimulated, and (b) platelet cells undergoing degranulation, a process of interest to research on HIV-associated neurocognitive disorder (HAND). Such reactions can vary greatly from one cell to the next, and these differences can be important parameters to study. Yet many analytical methods do not provide single-cell data, and many that do so require exogenous labels that alter the very process being studied. There is therefore a need for non-labeling, non-destructive methods that can obtain detailed information repeatedly from single cells, without affecting cellular function or viability. The HADES microscopy platform will provide rich information for characterizing cellular transformations and their variability on a cell-by-cell basis.

This award is being made jointly by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Biophotonics, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate).

Project Summary Bone fragility fractures that occur in the absence of significant trauma are often associated with primary or secondary osteoporosis, and can result in serious patient morbidity and increased mortality rates. Prediction of bone fracture risk primarily relies on measures of bone mineral density (BMD), which is strongly correlated with bone strength, but not with fracture risk. Alternatively, Raman spectroscopy (RS), an optical technique that can provide information on mineral crystallinity, composition, and relative degree of mineralization (mineral/matrix ratio), as well as collagen composition and cross-linking, has emerged as a promising technique for assessment of bone strength and fracture risk. We have recently shown that RS can detect biochemical changes that occur in mouse models of rheumatoid arthritis (RA), glucocorticoid (GC)-induced osteoporosis (GIOP), and osteogenesis imperfecta, which correlated with independent measures of biomechanical strength and fracture toughness. We have also developed instrumentation to enable the first diagnostically-sensitive transcutaneous Raman measurements of murine bone on intact limbs, along with sophisticated algorithms to reduce optical contributions from overlying soft tissue, but these measurements were only made ex vivo on tissue specimens. In this application, we will develop new instrumentation and algorithms to adapt our transcutaneous RS measurements on live animals. More importantly, based on unpublished data demonstrating that bone ends (epiphyses) exhibit more discriminate RS differences than mid-shaft (diaphysis) regions, we will redesign the excitation/collection optics in our RS platform to provide a larger range of source- detector separation, greater variety in sampling depth, and ultimately improve the ability to resolve the spectral contributions from interfering soft tissues in the more anatomically and biochemically complex epiphyses regions (Aim 1). We will then validate and correlate regional (epiphysis versus diaphysis) transcutaneous Raman spectroscopy measurements with regional and whole bone mechanics (measures of bone quality) in juvenile, skeletally mature, and aged mice (Aim 2). Finally, the studies will demonstrate that our transcutaneous RS platform has the sensitivity to detect longitudinal reductions in bone quality in mouse models of RA and GIOP over time, and improvements in bone quality in response to anti-resorptive and anabolic treatments (Aim 3). Upon completion, the proposed studies will have validated a disruptive technology for pre-clinical, non-contact optical assessment of bone fragility and fracture risk, which are undetectable by standard metrics such as BMD. While successful completion of this work will yield a new instrument in our research toolbox to advance our understanding of mechanisms of osteoporosis and to evaluate efficacy of new drugs in preclinical models, we hope that the progress we make here will allow this non-invasive technology to be scaled-up and translated in the not too distant future as an experimental diagnostic tool in the clinic.

It has been widely recognized that understanding and controlling the most elemental unit of light - the photon - is essential to innovating light-based technology as well as improving critical elements of our nation's defense and security. The students in this REU Site program, Nano-, Bio-, and Quantum Photonics at the University of Rochester, Institute of Optics, will participate in investigations at the frontiers of photonic research contributing to discoveries in nanoscience, bioscience and quantum science. The primary objective of the REU is an immersive experience in science and engineering at the forefront of photonics research. A diverse cohort of students from underrepresented groups and institutions with limited research opportunities, will participate in interdisciplinary projects that touch on topics in the life sciences, quantum sciences and nanotechnology. Ultimately this REU program will enhance our nation's capabilities by training top-quality students to become key contributors in the commercial photonics sector, leaders on the frontiers of fundamental photonic science and engineering, and the very best educators in colleges and universities across the nation.

Over 9 summer weeks, the University of Rochester will host 12 undergraduate students who will participate in interdisciplinary research on exemplary projects such as photonic spectroscopy for bone quality monitoring and novel device fabrication for generating quantum states of light. Professional development activities will also expose the REU students to the excellent career opportunities, both academic and industry based, available in photonics. The Institute of Optics will provide a world-class research environment in photonics for the participants, let them participate in its long-running Industrial Associates program for networking and interviews, and expose them to the dynamic Photonics community through interactions with the Rochester Regional Photonics Cluster. A product of this program will be to create a well-defined pathway to a career in photonics for the REU participants' with a particular focus on underrepresented demographics, undergraduates from partner universities without strong photonics research programs, and community college students' who might not otherwise recognize such a career path is possible. It is anticipated that many of the projects will lead to original and publishable research results in the photonics sciences. In addition to the research opportunities, existing resources at the Institute of Optics - the Institute Summer School and Industrial Associates Program - will provide research specific photonics training and a mechanism for continued contact with the REU cohort. Within the greater Rochester community, the REU program will leverage both AIM Photonics and the Rochester Regional Photonics Cluster to inform the participants of all the potential career possibilities that exist in the photonics sector.