Rohit Bhargava, Ph.D. - US grants

This award from the Instrumentation for Materials Research program supports instrument acquisition of a Fourier Transform Infrared Imaging System for Multicomponent Polymer Dynamics Research and Student Education at the University of Akron. The investigators will assemble one of the world's most powerful FTIR imaging systems that will have unprecedented sensitivity and speed for spatially resolved spectroscopic measurements and be a uniquely powerful technique for studying all types of polymer diffusion in various materials including nano-networks and nanocomposites and for studying phase separations and confinement effects. The acquisition of the proposed instrument will provide the timely training of a future generation of scientists skillful in vibrational spectroscopic imaging that will soon replace the existing 5,000 infrared microscopes in the world. The research will provide a novel learning experience in polymer science and engineering, enabling the largest interdisciplinary polymer program at University of Akron to be the first to possess this emerging technology for research and education.

This award from the Instrumentation for Materials Research program supports instrument acquisition of a Fourier Transform Infrared Imaging System for Multicomponent Polymer Dynamics Research and Student Education at the University of Akron. The investigators will assemble one of the world's most powerful FTIR imaging systems that will have unprecedented sensitivity and speed for spatially resolved spectroscopic measurements and be a uniquely powerful technique for studying all types of polymer diffusion in various materials including nano-networks and nanocomposites and for studying phase separations and confinement effects. The acquisition of the proposed instrument will provide the timely training of a future generation of scientists skillful in vibrational spectroscopic imaging that will soon replace the existing 5,000 infrared microscopes in the world. The research will provide a novel learning experience in polymer science and engineering, enabling the largest interdisciplinary polymer program at University of Akron to be the first to possess this emerging technology for research and education.

Technical Summary: Non-destructive and non-invasive imaging in three dimensions allows for the analysis of intact samples of a diverse nature. Several techniques are available for 3D imaging from the atomic to macroscopic scales, such as x-ray diffraction, micro and nano x-ray computed tomography, positron emission tomography, and fluorescence confocal microscopy. In some specimens, the limitations of these techniques require more advanced instrumentation to produce high-contrast, high-resolution images. Some samples do not diffract x-rays consistently, are sensitive to visible light, display dynamic behavior within the sampling time, diffract light, are non-fluorescent, or do not include heavy atom labels. Confocal Raman microscopy provides a non-invasive and non-destructive method for 3D imaging of these and other challenging samples. The new instrument will allow for collection of Raman spectra at multiple positions within a sample can be used to generate images containing an abundance of chemical information that includes time-resolved investigations of fast dynamic processes. Confocal Raman spectroscopy will be used to analyze polymers, semiconductors, solid-state materials, live cells, and nanomaterials. This instrument will be the only multi-user-accessible multi-wavelength (532, 633, and 785 nm laser sources) confocal Raman microscope on campus. The instrument has relevance in a diverse array of research at Illinois and will be housed at the Microscopy Suite of the Imaging Technology Group at the Beckman Institute for broad access across campus. The Imaging Technology group and collaborating faculty will continue to enhance science education and training through a variety of outreach projects for a diverse group of students and staff.

Layman Summary:
Non-destructive and non-invasive imaging in three dimensions allows for the analysis of intact samples of a diverse nature. Several techniques are available for 3D imaging from the atomic to macroscopic scales but are limited in their ability to probe complex materials. Confocal Raman microscopy provides a non-invasive and non-destructive method for 3D imaging of challenging samples and is required for the advancement of several projects, providing a platform for optical and spectroscopic analysis in materials characterization. Configured to allow for diversity in sample handling, the instrument will support a broad range of ongoing Illinois research programs in more than 18 different laboratories. Key research areas have been identified that would greatly benefit from acquisition of this advanced instrument including self-healing systems, multi-functional polymers, electronic materials, and cells & tissues. The instrument has relevance in a diverse array of research at Illinois and will be housed at the Microscopy Suite of the Imaging Technology Group at the Beckman Institute for broad access across campus. This instrument will be the only multi-user-accessible multi-wavelength confocal Raman microscope on campus. The Imaging Technology group and collaborating faculty will continue to enhance science education and training through a variety of outreach projects for a diverse group of students and staff.

ABSTRACT Nearly 240,000 men are diagnosed with prostate cancer (PCa) every year in the United States but there is no clinical test that can effectively determine whether their tumors will progress to life-threatening disease or remain indolent. Consequently, a majority of men with low-risk PCa who should simply undergo active surveillance (AS) instead receive costly treatments with major long-term adverse effects. A multidisciplinary team of investigators comprising clinicians, biologists, and bioengineers, who individually have developed key technologies, now seek to combine resources to directly address this long-standing clinical problem. The proposed project focuses on validating a novel technology platform that combines label-free and quantum dot-labeled spectral imaging to predict PCa progression. Illustrating the need and utility of our technology is the specific choice of assays we are utilizing here. The Mayo team has shown that rearrangements and/or copy number variant levels of five genomic regions in tumor cells in formalin fixed and paraffin embedded (FFPE) biopsy specimens can be useful in determining risk of PCa aggressiveness. However, these markers cannot be developed into a robust, clinical assay due to the current limitations of technology: Needle biopsy specimens often contain only a small amount of cancer, and even when cut into thin sections, the employment of a 5-probe assay is often simply not possible because of the limited capacity to multiplex conventional FISH probes. Furthermore, it is not possible to simultaneously identify cell types in sections labeled for fluorescence, so it is not clear whether cells that stain positive or negative for the FISH probes comprise cancer or stromal cells. This is the general problem our technology will address ? lack of multiplexed molecular quantitation and identification of (non)responsive cells. The Illinois team has shown that using infrared (IR) spectral imaging, the tumor microenvironment can be profiled and new predictive information can be obtained. However, this approach needs to be validated in a larger trial. Our project addresses the technology and validation needs by combining (a) FISH-probes based on quantum dots to identify specific molecular alterations with (b) cell/tissue identification using label-free infrared spectroscopic imaging. While both technologies has been independently developed and demonstrated to be effective, they have not been integrated in a complementary manner nor together used to address PCa needs. Here, we propose a test and validation of this combination technology via a cohort of PCa specimens that have already been genomically profiled. The combined technology's validation will also test its effectiveness in providing a practical test for PCa prognosis with statistical models and measures that will be compared to the current gold standards. Success in this proposal will enable the production of a robust assay strategy to determine which men are best candidates for AS or for more aggressive treatment ? which would be transformative for prostate healthcare. Validation of this technology also paves the way for combined molecular and cellular analysis in all tissues and for all types of molecular targets, which can vastly expand our capacity to provide accurate and specific diagnoses that can guide therapy in a wide range of pathologies.

This Major Research Instrumentation (MRI) grant awarded to the University of Illinois at Urbana-Champaign provides funding for the acquisition of a 3D-bioprinting instrument. Efforts at the University of Illinois are seeking to develop the next generation of tissue engineering solutions for critical healthcare needs such as musculoskeletal regeneration, models of cancer, cardiovascular repair, and controlling stem cell behavior. The EnvisionTEC 3D-Bioplotter will provide critical capabilities to fabricate large, geometrically complex, and cell-laden tissue biomaterials that mimic features of native tissues. This bioplotter will be operated as a shared-use bioprinting facility housed within the Carl R. Woese Institute for Genomic Biology (IGB) at the University of Illinois and will provide a critical resource to a broad user-base with diverse backgrounds to meet the needs of the tissue engineering community on our campus. The bioplotter will enhance the interdisciplinary training environment at the University of Illinois for trainees to gain the skills and confidence to address emergent challenges at the intersection of the biological, physical, and quantitative sciences. The bioplotter will also be integrated into innovative outreach and education programs at the IGB designed to expose students and the public to the transformative research programs within our institute.



Advances in tissue engineering rely on successful integration of biomaterials, cells, and biologics such as growth factors and small molecules to address significant health care challenges. The EnvisionTEC 3D-Bioplotter acquired through this MRI program will be housed within the Carl R. Woese Institute for Genomic Biology (IGB) at the University of Illinois. This bioplotter system will enable transformative developing research efforts aimed at developing novel technologies and fundamental insight regarding biological phenomena required to promote the regeneration of human tissues and organs. The 3D-Bioplotter system will facilitate critical areas of biomaterial research across the University of Illinois community due to its capacity to generate large, geometrically complex, multiphase, and cell-laden tissue constructs. The bioplotter will be used to print diverse libraries of cell-infused hydrogel, ceramic, and polymeric bioinks, as well as composites thereof at the resolution required to mimic features of native tissue and organs in the body. This system provides critical operational capabilities for a biomaterials fabrication user-facility that serves the University of Illinois research community, specifically sterile operation, flexibility for bioink customization, the capacity for clinical-scale print volumes, and integrated feedback control essential for reproducibility. The bioplotter will be localized within the convergence-based research ecosystem at the IGB, which integrates disciplines of biology with technologies and approaches from engineering, computer science, physics, chemistry, mathematics, and the social sciences. The bioplotter will be managed as an open access facility to serve the needs of researchers from multiple departments and colleges across our campus, making it a central hub for tissue engineering efforts at the University of Illinois at Urbana-Champaign.

Abstract Molecular chirality is at the heart of many chemical processes that determine life and drives significant research in development and disease. All life has chiral asymmetry with naturally occurring molecules and long-range assemblies being of distinct handedness. Many exogenous molecules, for example those useful as drugs, also have a distinct enantiomeric dependence for their efficacy in benefiting human health. Thus, measurement of molecular chirality is of critical importance across the medical sciences. Vibrational Circular Dichroism (VCD) spectroscopy has emerged as a powerful platform for quantifying chirality and molecular structure. However, imaging has not been demonstrated due to technological challenges. VCD measurements are largely of homogeneous materials, neat or in solution and probed with sensitive Fourier transform infrared (FT-IR) spectrometers. Microscopy would require ~105 reduction of the typical sensing volume and increase in speed that would make imaging feasible. Instead of utilizing FT-IR spectroscopy, we built a custom quantum cascade laser (QCL) microscope to demonstrate feasibility of a point scanning VCD instrument capable of acquiring spectra rapidly across all fingerprint region wavelengths in both transflection and transmission configurations. Moreover, for the first time, we also demonstrate the VCD imaging performance of our instrument for site-specific chirality mapping of biological tissue samples. However, the feasibility data also point to several technological and conceptual challenges that this project seeks to address in developing a practical prototype. The prototype to be developed here, termed vibrational circular dichroism imaging microscope or VIM, aims to record chirality from microscopically heterogeneous biomedical samples. We propose a design for VIM using a laser scanning approach to minimize artifacts and maximize signal. Starting from a de novo design, we will use commercial and custom optics, custom electronics for control and data management, and in-house software to develop the prototype. Next, we model the VCD image formation process and develop the analytical methods for VIM. The theoretical model developed here builds on our models of IR microscopy and will guide prototype development while ultimately provide greater accuracy, precision and assurance to data recorded. Finally, we validate the performance and broad utility of VIM using well-characterized samples. Together, the work will develop new VCD imaging technology that opens capability to measure and research a wide variety of biological problems.

Abstract Colorectal cancer (CRC) is one of the leading causes of death in the US. Active screening and early intervention in risky cancers can lead to good outcomes; however, a bottleneck in rapidly delivering appropriate patient care is the long time period for histologic assessment and lack of precision in predicting disease severity. Morphological assessments prevalent in histology are useful but resource intensive and not predictive enough. Molecular techniques to complement traditional pathology are emerging but often require much more effort and time, without being especially compatible with histologic assessments. Here, we seek to develop a technology that measures the chemical content of tissues, does not require reagents, is entirely compatible with clinical workflows and leverages modern artificial intelligence (AI) techniques to provide real-time histologic assessment. The foundation of our approach is a new design for an infrared spectroscopic imaging system that is faster than any reported, offers a higher spatial and spectral quality and uses a solid immersion lens with a fixed focus at the sealed surface of the lens to enable use by a minimally trained person. In conjunction with the instrument, we develop AI algorithms that measure the chemical content of tissue and use it to provide (a) conventional pathology images without the use of dyes (?stainless staining?), and (b) histologic assessment based on molecular data, which can provide complementary composition, disease and risk of lethal cancer images akin to conventional pathology. The instrument will be usable by laboratory technicians, without the need to prepare thin sections from excised tissue and will provide information in minutes. Using preliminary data from human patients on over 850 tissue microarray (TMA) samples from 8 TMAs and 30 surgical resections, we validate the use of technology in providing complete histologic and disease grade assessment. Statistical methods will be used to assess the results rigorously and quantitative milestones guide the entire approach. We then translate the results to fresh tissue chunks, providing histology minutes after tissue is extracted from the body. Finally, we use the detailed tumor and microenvironment information available from the tissue to segment patients into a ?high risk? and ?low risk? group. The availability of rapid histologic assessment can help prevent delays in providing care, provide intraoperative assessment, and add more information to morphologic assessments following screening, enabling a wide use in CRC and other cancer pathologies.

Abstract Molecular understanding of tumors relies greatly on appropriate samples to be prepared from epithelial cells in tissues. Epithelial cells, however, are often surrounded by other cell types and extracting pure populations of these cells is crucial for correct biospecimen preparation and resulting accuracy of molecular assays. Laser microdissection (LM) has contributed immensely in this effort due to its high spatial specificity in the extraction of defined cell populations and ease of use. While LM has enhanced the precision of biochemical analysis, several drawbacks remain. The necessity of staining and human supervision limits throughput, molecular yield and purity of samples. There is little explicit control or confidence in the purity of extracted cell populations while it is difficult to extract multiple cells from the same sample. Combining the morphologic specificity of microscopy and molecular sensitivity of spectroscopy, infrared (IR) spectroscopic imaging been employed to automate histopathologic recognition in complex tissues using artificial intelligence algorithms applied of spectral data. This project will demonstrate a completely automated instrument by coupling LM with IR microscopy. Termed spectroscopy-assisted laser microdissection (SLaM), the developed prototype will be validated using state of the art IR imaging systems and commercial LCM in terms of accuracy, speed and type fidelity. Last, the approach will be applied to extract cells of different types from the same prostate sample to demonstrate the capability to multiplex LM (muxLM) from the same tissue. The project directly addresses the need to reduce the time- and labor-intensive nature of LM. SLaM can maximize the quality and utility of biological samples used for downstream analyses by automation, high throughput and precision while enabling a comprehensive acquisition of cells without user fatigue or error, thereby providing a sample of higher integrity and quality for cancer molecular analysis.