Yu-Hwa Lo - US grants

9410905 Lo When communication and computing systems merge in the coming information age, schemes of interfacing photonics and electronics become increasingly important. One ideal scenario will be integrating photonics and electronics together to form optoelectronic integrated circuits (OEIC's). Such OEIC's will not only provide efficient O/E and E/O interfaces but also have applications in massively parallel interconnect and signal processing. Since transistors and optical devices (laser and detectors), the workhorses for electronics and photonics, are mostly made of silicon and III-V compound semi-conductors respectively, the most sensible OEIC's should combine III-V optoelectronics with Si electronics. If the technology for such III-V/Si OEIC's becomes available, exciting new areas such as the integration of optoelectronics with nanoelectronics and micromachining can be explored. The future impact of these new areas might be very significant. One fundamental problem for Si-based OEIC's is that high quality III-V compound epitaxy can not be formed reliably on Si substrates. Besides, the integration process of forming III-V/Si OEIC's has not been developed, hindered by the process incompatibilities between III-V and Si materials. To solve these technical bottlenecks, this research will focus on three subjects: (1) to develop viable technology to integrate III-V epitaxial layers on silicon substrates, (2) to develop processes to integrate III-V optical components with silicon electronic circuits, and (3) to demonstrate functional III-V/Si OEIC's. ***

This proposal has two components, a research proposal and an education plan. The objective of the research program is to develop innovative concepts and technologies to fabricate long wavelength (1.3/1.55 micron) vertical-cavity surface- emitting lasers (VC-SELs) and WDM surface emitting laser arrays for communication, interconnect, and signal processing. The VC-SELs developed in this program are expected to outperform the existing devices in terms of threshold current and spectral purity. Above all, the VC-SELs should have a substantial cost advantage over conventional single mode DFB and DBR lasers. The goal of the proposers education plan is to educate future university teachers, to educate capable and conscientious engineers who can contribute to the advance of sciences and technologies, and to cultivate engineers who can educate themselves to fit in the ever changing, increasingly challenging work environment. Two other important aspects in his education plan are in the improvement of minority and pre-college science education.

The objectives of the proposed research and education program are: (1) to develop a viable technology for compliant substrates which can be used to grow defect-free heteroepitaxial materials of large (>15%) lattice mismatches and different lattice structures, and (2) to understand the underlining physics of compliant substrates and to establish a physical model to quantitatively describe the phenomenon of substrate compliance. In brief, this program aims to establish the theory and technology of compliant universal substrates. As a result of this research, one or two types of substrates, perhaps GaAs or Si, will be sufficient to accommodate a large variety of compound semiconductor epitaxial materials, including those having no proper conventional substrates on which to grow. The proposed compliant universal substrate is essentially made of an ultra thin (10 A to 100A) single crystal (e.g. GaAs or Si) bonded to a GaAs or Si bulk crystal with a large (> 10 degrees) twist angle. It is believed that the microstructure of the twist boundary and the extremely thin bonded layer play a critical role in making the structure compliant to the heteroepitaxial overlayer. We conceive that the twist bonded thin portion of the bicrystal behaves somewhat similar to a free standing thin template to achieve its compliance. Our preliminary experimental results show that InGaP, GaSb, and InSb, with 1%, 8%, and 15% mismatch to GaAs, respectively, can be grown defect-free on the compliant universal substrates. This proposal brings together the expertise of Lo, who has developed the concept of the compliant universal substrate, and of Sass, who has carried out extensive studies of the structure of grain boundaries contained in bicrystals with the same geometry as in the compliant universal substrate. The research team made of Lo, Sass, 3 Ph.D. students and at least 4 undergraduates will perform three tasks: fundamental materials research, compliant substrate technology development, and material and device demonstrations. Through these efforts, the physical mechanisms, technologies, and applications of compliant universal substrates are anticipated to be established. Compliant universal substrates have been a dream for many decades for people working on semiconductor materials and devices. The realization of compliant universal substrates will remove one of the most serious road blocks in semiconductor research and significantly advance the technologies of microelectronics, optoelectronics, and the integration of both. The proposed research will contribute substantially to achieving this goal.

[unreadable] DESCRIPTION (provided by applicant): There are approximately 100 trillion microorganisms inhabited in a human body. The intimate interactions between these microorganisms and the host have a profound impact of the physiology of the human body. The majority of these microorganisms have yet to be fully characterized, mainly due to the difficulties in growing them in laboratory conditions. Here we propose the development of an efficient and scalable method to obtain genome sequences from single microbial cells. This will eliminate the need to obtain pure laboratory culture, thus allow us to systematically characterize the genome structure of microbial communities that resides in different parts of the human body. The proposed project contains three major components. 1. Development of low-cost and disposable cell sorting devices that integrate microfluidics with micro- scale optical components. Such a lab-on-chip cell sorter will be able to identify and isolate single microbial cells from samples that contain hundreds to thousands of different species and often are contaminated with free DNAs from the host and other sources. 2. Development of a micro-well based polymerase cloning device for simultaneous amplification of genomes from hundreds to thousands of single microbial cells in parallel. Using such a device, we will prepare sufficient amount of DNAs from single cells for whole genome shotgun sequencing, a critical step to obtain genome sequences from single cells. 3. Development of an integrate pipeline for dissecting the genome composition of human microbiome at the single cell resolution. We will test this pipeline by isolating and sequencing genome from approximately 35 single microbial cells at different levels of relative abundance from the mouse distal intestine. The propose method will provide the research community a new tool to identify unknown microbial species, to study their metabolic functions and to better understand the host-microbe interactions under various physiological and disease conditions. PUBLIC HEALTH RELEVANCE: The distribution and activities of microorganisms in the human body have a profound impact on the health of the host. In this proposed project we will develop an efficient and scalable method to obtain genome sequences from single cells in complex microbial communities. Characterizing the genomic composition and structures of these microorganisms is a necessary step before their metabolic functions and the interactions with the host can be study in great detailed. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Flow cytometers are an indispensable biomedical tool that measures the angular scattering of light from a cell/sample and/or the fluorescence the cell/sample emits, creating the basis for routine blood counts, cancer screenings, and a wide range of other diagnostics and assays. Flow cytometers employ high-quality optical systems and complex electrical or mechanical cell sorting devices to enable sensitive, reproducible detection and sorting of cells, but these same systems make the instrument expensive to fabricate and maintain. The aim of the project is to create a low-cost, compact flow cytometer that can be widely available to clinicians and researchers. The system will also possess unique functionality unavailable by today's system. Plastic photonic circuits will be integrated with microfluidics and microacoustically actuated cell sorters on a microchip platform. This design readily allows the creation of a sorting scheme for the ordered removal of a small number of cells of interest, allowing researchers to remove the specific cells they are interested in for additional individual studies, such as the genotyping of individual cancer cells. Device design will be carried out based on combined results from optical and fluidic simulations performed for polymer-based structures. Over the course of three years, several device test and redesign cycles will be made possible by the rapid prototyping allowed by polymer replica molding and the use of off-the-shelf piezoelectric actuators, LED/diode laser sources, and Si photodetectors. Successful demonstration of the feasibility of this device will be measured by (1) the realization of a cell detection module capable of simultaneously detecting forward scatter, side scatter, and fluorescence from standard cytometry calibration polymer beads with sufficient sensitivity to yield a coefficient of variation with 11% of that measured by a commercial benchtop cytometer. (2) the development of a system prototype with capabilities sufficient for removal of a single cell from a high-throughput flow channel (i.e. up to 100,000 cells/min per channel, multiplied by the number of channels). (3) the ability to choose and remove individual cells in flow to a spatially-indexed sorting channel for further study (e.g. genotyping of single cancer cells). [unreadable] [unreadable] The development of an inexpensive, disposable chip-based flow cytometer would make the device more widely available from a financial standpoint to both clinicians and researchers alike, helping to lower net testing costs, contain biohazard materials, and provide a more rapid turnaround on results from the wide range of tests currently run on flow cytometers, such as routine clinical bloodwork, HIV and cancer progression monitoring, or research-based cell studies. In addition, the development of our proposed single-cell sorting capabilities would automate the isolation of individual cells of interest, making it an invaluable tool for facilitating biomedical breakthroughs in areas such as our understanding of cancer, where the ability to perform single-cell genotyping for studies of the evolution of cancers will be critical. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Fluorescence-activated-cell-sorting (FACS) or flow cytometry enables clinicians and researchers to quantitatively characterize the physical (cell size, shape, granularity) and biochemical (DNA content, cell cycle distribution, cell surface markers, and viability) properties of cells. With the capability of high-throughput sorting to enrih biospecimens and extract rare cell types, a state-of-the-art flow cytometer makes it possible to conduct rare-event studies such as the identification or isolation of bacterial cells, stem cells, r tumor cells. However, current flow cytometers that detect multiple colors and sort cells are expensive (~$500K), complicated, hazardous, large and bulky. For these reasons, flow cytometers are often shared amongst labs, leading to conflicts in sample handling and scheduling. With a growing market expected to reach over $3 billion by 2015, flow cytometry can address a diverse array of biomedical challenges. However, there are no high performance flow cytometers with cell sorting capabilities that are affordable for any lab. Despite the increasing demand for affordable cell sorters, the flow cytometry industry faces fundamental limits in the current technology and its evolutionary path. The current system architectures are highly inefficient in accommodating and fully utilizing the increasing number of available fluorescent colors, sensitivity, ease of use, and sorting capabilities. We propose an accessible, affordable, and high performance flow cytometry technology that will allow any scientist to perform cell analysis and sorting in their own laboratory. In this Lab-to-Market Phase II program, we propose to dramatically improve our flow cytometric cell sorting platform, the WOLF Cell Sorter, including extending the dynamic range of the fluorescent detection, implementing real time cell sorting verification, expanding chip volume manufacturing, and validating the system with early adopters such as induced pluripotent stem cell researchers excited by the unique features of our system. These technology and business goals will have a direct impact on basic research and clinical applications in human health and disease.

DESCRIPTION (provided by applicant): Portable Lab-on-a-chip Flow Cytometer: Prototype and Application Development NanoSort RESEARCH & RELATED Other Project Information 7. PROJECT SUMMARY The lab-on-a-chip NanoSort device proposed here advances the achievements of the Phase I Beta Prototype project. This device will be appropriate for commercial success by improving access to and reducing the cost and complexity of sorting flow cytometry. Furthermore, it will expand the applications that this device can serve, including translational and clinical point-of-care flow cytometry applications for diagnostics, prognosis, and personalized medicine. Having completed key milestones in the Phase I program, such as mas- producible injection molded chips, advanced signal detection and processing and key performance metrics, we propose to extend the capabilities to compete with best-in-class devices. Our Aims for this project are to create a device (NanoSort-1 or NS-1) that will have improved performance parameters of 6-color detection with a single photomultiplier tube (PMT), 4-way sorting using low shear stress piezoelectric (PZT) actuators, and a robust digital signal processing algorithm (Color-Space-Time (CoST) implementation that allows 10,000 events per second detection and sorting. In addition to these innovations, we will construct the device using current good manufacturing processes (cGMP) to assure that this device will be UL and CE certified and scalable to 10,000 units per year. We will validate this device with consultation from established researchers at UCSD and device manufactures (Becton-Dickinson and Life Technologies).

This INSPIRE award is partially funded by the Genetic Mechanisms and Cellular Dynamics and Function Programs in the Division of Molecular and Cellular Biosciences, by the Plant Genome Research Program in the Division of Integrative Organismal Systems, and by the Division of Emerging Frontiers in the Directorate for Biological Sciences, and also by the Biotechnology, Biochemical, and Biomass Engineering Program in the Division of Chemical, Bioengineering, Environmental and Transport Systems in the Directorate for Engineering.

Epigenetic modifications to the DNA and protein components of chromatin are important determinants of gene expression, and dynamic epigenetic changes are thought to underlie the ability of organisms to rapidly respond to environmental perturbations. Much of what is understood about such regulation has been obtained through so-called ensemble approaches, which determine epigenetic modifications from cells isolated from mixed tissue types; thus, the data represent an average of epigenetic and gene expression profiles of multiple cells. Recent technical advances have made it possible to obtain DNA sequence information and gene expression profiles for single cells, but it is not yet possible to obtain epigenetic information from single cells. The ability to monitor dynamic epigenetic profiles in single cells would revolutionize the field of epigenetics by enabling hitherto impossible studies of stochasticity, stability, and heritability of epigenetic responses. This project seeks to develop and implement an innovative pipeline for controlled hormone and stress perturbation of plant cells, followed by single-cell analysis of genome-wide DNA methylation profiles. An integrated micro- and nano-fluidic device will be built to enable single-cell isolation from specific cell types and subsequent chromatin sorting. Then DNA methylation information will be extracted from the isolated cells via new kinetic analysis of real-time sequencing data produced by ultrasensitive FRET-based single molecule sequencing. If successful, this new technology will enable analysis of epigenomic profiles of single cells, thus making it possible to address fundamental questions about epigenetic regulation in a revolutionary new way, not only in plants, but also in mammalian and microbial cells.

In addition to the scientific impact, the project will have broad educational impacts by offering opportunities for cross-disciplinary training at the interfaces of molecular biology and nano-engineering. Postdoctoral and graduate students will be involved directly in carrying out the research, and the technology developed in the project will be incorporated into hands-on laboratory training modules for both graduate and undergraduate students.

DESCRIPTION (provided by applicant): Single cell biology has offered a new path to obtain biological insight that has been masked by the ensemble average from a large cell population. Studying biology at the single-cell level will not only enhance our understanding of the complicated biological mechanisms but also help produce new therapy and drugs to cure human diseases. To support single cell analysis, new tools of extraordinary precision, flexibility, and capability have to be developed. Although microfluidic device has been demonstrated as a highly promising platform for investigating single cells, one serious limit of today's microfluidic single-cell device is that cells in microfluidic environment is very different from cells in standad culture environment and even more different from cells in physiological environment. We propose to develop a universal microfluidic single-cell dispensing and conditioning device to addresses this important limitation. The device has an innovative architecture that integrates different functions, including single cell detection, capturing, conditioning, and release, on a microfluidic platform. The proposed device will operate in a close-loop fashion, requiring no user intervention once the user application is specified, due to the field-programmable-gate-array (FPGA) control and coordination of all the functions together. Besides these unique features, the proposed device can be manufactured at very low cost, fully automated, compatible with industrial standards, and expanded into array format for ultrahigh throughput. The proposed device is intended to become a tool that will be widely used on a daily base by researchers performing fundamental and clinical research benefiting from single cell studies. Since single-cell biology prevails in many areas of biology and medicine, the proposed device is anticipated to satisfy a major need in biomedicine with increasing demands in the future. If developed successfully, the device will facilitate and accelerate discoveries in cancer and stem cell research, new cell therapy, drug screening and testing, infectious and genetic diseases, cell-cell interactions, and cell signaling. The device will also become an invaluable tool for the development of medical devices and assays dealing with rare cell populations such as assays for circulating tumor cells (CTCs), cancer stem cells and personalized medicine.

DESCRIPTION (provided by applicant): This proposal describes a novel method for rapidly counting neutrophils in a patient's blood sample. Neutropenia, characterized by an abnormally low number of neutrophils that serve as the primary defense against infections, is a common side effect of chemotherapy. Without prompt medical attention, the condition of neutropenia may become life threatening. Since the majority of oncology patients (about 1 million each year in the US) are treated on an outpatient basis, the number of neutrophils must be closely and frequently monitored to allow timely treatment in the event of neutropenia and associated infections. Furthermore, patients undergoing antiproliferative chemotherapy with neutropenia are particularly sensitive to nosocomial (hospital) infections, causing 99,000 deaths each year. Therefore, in consideration of the interest of cancer patients, as well as the cost and efficiency of our healthcare system, the ability for self-administered neutrophil test at the patient's residence is particularly important and attractive. Today's complete blood count devices have been designed for central medical facilities. They are very expensive and too complicated to operate by patients. Even the simper blood test devices for point-of-care applications are designed for use in physician's office and unsuitable for patient-administered testing at home. To address this important unmet need, we propose a unique and innovative device for patient-administered neutrophil testing at home. Our neutrophil counting device takes advantage of cellular properties manifested specifically when flowing through microfluidic channels. Supported by recent publications, the combined information of cell size and deformability can determine the equilibrium positions of flowing cells in a microfluidic channel due to fundamental fluidic dynamic properties. We invented a space-time coding technique to encode the forward scattering signal of each travelling cell. By decoding the signal, we obtain the velocity and position of each individual cell with a very high accuracy (0.1 um). Since the cell position within a microfluidic channel is directly related to the cell volume and deformability, we have found an innovative method to classify white blood cells, particularly neutrophils. Our device and analysis method possesses the following salient features: (1) it is minimally invasive, requiring only 5 microliters of blood (a similar amount to a typical blood glucose test); (2) straightforward sample preparation and test procedures that can be performed by persons without medical training; (3) accurate test results that are easy to understand and compliant to medical standards; (4) low cost; and (5) high portability. We therefore envision that the successful development and commercialization of the device will bring tremendous benefits to cancer patients because the device can greatly reduce the risk of hospital infection, cut down healthcare costs, improve outcomes during chemotherapy due to close monitoring of neutropenia, and minimize the inconvenience and pain for unnecessary hospital and emergency room visits.

Non-technical Description:
The San Diego Nanotechnology Infrastructure (SDNI) is set up as a national nanotechnology research and education infrastructure to serve the country?s needs for advancing research, facilitating technology commercialization, supporting entrepreneurship, developing strong and competitive work force, enhancing K-12, college and graduate education, and promoting diversity and inclusion. By accomplishing its missions, SDNI will become a key contributor to the pursuit of scientific research and the national health, prosperity, and security. SDNI offers unique tool sets, skills, technical support, mentorship, and services to produce a myriad of innovative materials and devices. These unique capabilities will help the nation to gain competitive advantages in areas critical to the nation?s economy and security, including artificial intelligence (AI), advanced manufacturing, quantum information science (QIS), and 5G/6G communications. The SDNI will also play a pivotal role in research pursuits that align with NSF?s 10 Big Ideas for the future, with particular focus on supporting and growing convergent research, enhancing science and engineering through diversity, and seeding innovation. To develop a more diverse and productive scientific workforce, the SDNI is committed to developing a systematic and executable outreach and education program to promote STEM. Built upon a pilot program that has shown feasibility through very positive responses from all stakeholders, including students, teachers, and administrators from school districts with high minority student populations, SDNI?s proposed outreach efforts will bring nanotechnology to the science curriculum of middle and high schools in southern California first and then across the country, through collaborations with other sites in the NNCI network.

Technical Description:
As part of the National Nanotechnology Coordinated Infrastructure (NNCI), the SDNI offers technical strengths in the areas of Nano/Meso/Metamaterials, NanoBioMedicine, NanoPhotonics, and NanoMagnetics. SDNI?s strategic goals are to (1) Provide infrastructure that enables transformative research and education and leverages San Diego?s innovation ecosystem, which includes major research institutes and over 2,000 companies employing more than 60,000 scientists and engineers; (2) Accelerate the translation of discoveries and new nanotechnologies to the marketplace; (3) Become a key contributing member of the NNCI Network to support and advance the nation?s nanotechnology infrastructure, and (4) Collaborate with the California Board of Education and local school districts to develop education and outreach programs to promote STEM efforts in high school and community colleges, especially at schools with high populations of underrepresented minority (URM) students. Because nanotechnology is a foundational technology with applications across disciplines, SDNI will continue to expand its capabilities, optimize its operations, and actively recruit and engage new and nonconventional users to advance discoveries in scientific areas of national priority. In particular, we expect the SDNI will play a crucial role in the advancement of convergent research to help create breakthroughs in areas of human machine interfaces, exploration of the universe, facilitating revolutions based on quantum physics, and enhancing science and technology by broadening participation in STEM. Discoveries made by users of the SDNI will have the potential to create transformative change in fields critical to the future of human society and national interests.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

? DESCRIPTION (provided by applicant): Fluorescence-activated-cell-sorting (FACS) or flow cytometry enables clinicians and researchers to quantitatively characterize the physical (cell size, shape, and granularity) and biochemical (DNA content, cell cycle distribution, cell surface markers, and viability) properties of cells, however FACS devices do not produce an image of the cell. Increasing sophistication of research assays now rely on the collection of cells based on their phenotypical and spatial characteristics; with the capabilities offered only by microscopic imaging cytometers having severe limits in throughput and lacking cell isolation. We propose an innovative, low cost design to combine the merits of FACS and microscopic imaging cytometry without the limits of each, offering the biomedical research and clinical community a unique tool to address the needs for current and emerging applications. The key innovation is based on a significant extension of the spatial-coding algorithms our team demonstrated in the past years. In the proposed design, we create a special filter with a matrix of periodic slits in front of each PMT detector. The resulting PMT signal is composed of the multiplexed cell signals modulated by the filter, which can subsequently be deconvolved to produce fluorescence and scatter generated from different areas of the cell: the image. In this Phase I program, we will integrate imaging technology into our existing WOLF Cell Sorter to produce the very first imaging cytometer with cell sorting capabilities. Since we use conventional, non-pixelated detectors (e.g. PMTs) found in conventional flow cytometers, this technology is compatible with existing flow cytometer architectures allowing for wide use. Equipped with cell imaging capabilities, researchers can track many important biological processes by analyzing not only the intensity but the localization of certain proteins within cytosolic, nuclear, or cell membrane domains and subdomains. With the rapidly developing capabilities of handling big data, images of millions of single cells in a flow cytometer provide vast resources for research and disease analysis, and rapid growth has been predicted in the market of such high-content imaging cytometer cell sorters for emerging applications such as precision medicine. We believe the proposed design is a major breakthrough that can potentially revolutionize the field of flow cytometry, and its impact and ramifications on both fundamental biomedical research and clinical applications can be tremendous.

Title:
Transient Induced Molecular Electronic Spectroscopy (TIMES) for study of protein-ligand interactions

Project Goal:
To test protein-ligand interactions, the key mechanisms for most drugs, without any external disturbances caused by molecular labelling or immobilization.

Nontechnical Abstract:
Protein ligand interaction is one of the most fundamental and widely studied areas in biology and chemistry because of their close relation to many diseases and disease therapies. Nearly all diseases including cancer, neural disorders, infectious diseases, cardiovascular diseases, renal diseases, metabolic diseases, immune diseases, etc. are connected to the abnormality of protein-protein or protein-ligand interactions. Unfortunately, all the current methods for in-vitro protein-ligand tests use either labels (often fluorescent) or ligand immobilization, both of which can produce substantial disruptions and introduce artifacts. These limitations have caused significant delay and cost increase in drug development. A new method is proposed and investigated to fundamentally solve the above problems, allowing precise in-vitro tests of the binding strength and kinetics of proteins ligand interactions to accelerate the process of drug discovery. The work can have significant impact on public health and the country's healthcare by shortening the time and saving the cost for drug discovery. The proposed research will also contribute to education and training of new generation of scientists for multi-disciplinary research involving biology, biochemistry, biophysics and engineering. The research also includes significant outreach activities to engage middle and high school students and underrepresented minority students to advance the STEM efforts.

Technical Abstract:
Since a protein and ligand have a large molecular weight and size difference (e.g.,100 kDa versus 1 kDa), linking organic fluorescent molecules to a protein or ligand can perturb or even change the binding properties. Similarly, the bioactivity of protein depends on its 3D configuration. Immobilization of the molecules limits its degree of freedom for binding, thus often yielding results different from reactions in physiological conditions. Lacking a precise method of in-vitro detection of protein-ligand interactions without any external disturbances has presented a major challenge for mid to late stage drug tests. Drug companies have often conducted unnecessary and unsuccessful human trials while the failures should have been detected earlier if a precise in-vitro test method exists. The most significant scientific and technological contributions of the proposed research will be the development of the method of Transient Induced Electronic Molecular Spectroscopy (TIMES) that allows label-free and immobilization-free detection of protein-ligand interactions. The TIMES method measures the signal caused by the dipole moment change when protein and ligand form protein-ligand complex. When integrated with ultralow noise electronics and microfluidics, the new µTIMES system will enable researchers to collect unprecedented rich information with high timing resolution and enhanced signal-to-noise ratio. This method will be applied to quantitative studies of protein-ligand and protein-aptamer interactions by measuring the dissociation constant and reaction kinetics.

iFACS: Imaging Florescence Activated Cell Sorter to sort cells based on images NanoCellect Biomedical, Inc. RESEARCH & RELATED Other Project Information 7. PROJECT SUMMARY Advances in reagents (e.g. CRiSPR) and analytical tools (e.g. flow cytometers) have improved the ability to alter and characterize cellular phenotypes. Ultimately, many key applications in biomedicine require efficient and accurate isolation of cell populations according to features contained in high content images. Unfortunately, microscopic laser microdissection systems have a throughput that is too slow to be practical in many applications; while the existing flow cytometers that can sort cells (fluorescence-activated cell sorters or FACS) provide only size, internal complexity, and fluorescence intensity information and lack the rich data of imaging. Another critical limitation is that the existing flow cytometers that can image, cannot sort cells. NanoCellect has made a highly affordable FACS to increase access to this important high-throughput tool for cell analysis. Here we propose to enhance our existing low-cost FACS with the ability to image cells and sort them based on image features. This will allow users to pursue new strategies in drug screening and mechanism of action research; as well as work with suspension cell lines, such as those that dominate the recent advances in immuno-oncology. In Phase I research, we have demonstrated the world's first imaging flow cytometer with cell sorting capabilities (iFACS) in a unique design of space-time coding with an optical spatial filter. The approach adds negligible cost to the system for the desirable features of cell imaging and sorting. To fully realize the enormous potential of the design and to meet the demands for most applications, in Phase II we will develop high-throughput image-based cell sorting with innovative image-guided gating schemes supported by machine learning and interactive user/machine interface. Essentially, image-based flow cytometry gating uses similar cell isolation criteria as the techniques of laser capture microdissection or cell aspiration to isolate cells of interest, with 10,000X throughput improvements to 1000+ cells per second. We envision such unique capabilities will become common, default features for tomorrow's users as the tool becomes as intuitive and ubiquitous as fluorescent microscopy. The proposed iFACS will be transformative and benefit numerous biomedical applications, such as isolation of cells based on organelle translocation, cell cycle analyses, detection and counting of phagocytosed particles, and protein co-localization, to name just a few.

High-throughput single-cell sorting and kinetic analysis of secreted particles NanoCellect Biomedical, Inc. RESEARCH & RELATED Other Project Information 7. PROJECT SUMMARY Advances in reagents and analytical tools have improved characterization of cellular phenotypes. However, most studies are still performed on cell populations that are morphologically and genetically homogeneous, yet have important biological heterogeneity; with individual cells having unique phenotypic or `omic profiles. This leads to an average measurement of individual cells, none of which may actually be average. Single-cell analysis can define the scope of cell-to-cell variations within a population and provide in-depth analysis of single, exceptional cells in contexts such as stem cell differentiation or cancer, where these cells can give rise to clonal populations that drive the biology. While novel tools such as multiparameter flow cytometry, mass cytometry, and image-based cytometry provide in-depth analysis of individual cells, these technologies only provide a single time point ?snapshot? and lack downstream molecular analysis. This project will develop for commercialization an integrated cell-sorting, cell dispensing, and single-cell positioning device to allow single cells to be tracked and measured over time. In addition, we will demonstrate a commercial prototype device that allows transfer of individual cells with desired phenotypic characteristics to 1536-well plates for downstream analysis in a standard format. This integrated system will be benchmarked with cells derived from glioblastoma that has been shown to have important exosome biological characteristics.

Cells from living animals are very small, and yet measuring them and manipulating them are critical for producing new medical therapies. There are encouraging new methods to understand and control cells one at a time, but there really isn't now the technology to measure and control individually the thousands or millions of cells needed for a therapy. New approaches to individually measure and control cells using multiple nanoscopic devices are sorely needed. This award will study a method to control and measure many cells simultaneously. The base technology is a high-density platform of nanoscopic wires that interact with the cells in a culture system. The scalable nanomanufacturing of nanowire devices will make it possible to build "nanolab-on-a-chip" machines. Such tiny "laboratories", combined with a patient's own growing cells could create low-cost, predictive drug-screening platforms to accelerate drug discovery and personalized treatments. The project provides training opportunities for undergraduate, high school, and under-represented minority students in interdisciplinary research in materials science, engineering, and medicine. It augments and improves the course curriculum, and fosters a robust translational exchange with industry partners.

The project aims to overcome the barriers in developing a nanowire array-based system that enables multi-use, non-destructive, high-sensitivity measurements in 3D networks that are not possible with patch-clamp, automated patch, or microelectrode array techniques. Human-derived neurons and cardiomyocytes, which are highly relevant human models for drug screening, are studied. The project explores nanoimprint lithography as a scalable nanomanufacturing method to develop a wafer-scale nanowire neurophysiology platform scalable to 8000 simultaneous data points for 250 wells with 32 nanowire electrodes each. This scalable fabrication method enables the integration of nanowires in high densities and large numbers in integrated systems that comprise on-chip acquisition and digitization electronics and microfluidic drug intervention channels and wells. Furthermore, new architectures of multiple height nanowires are devised for screening the effects of drugs from 3D neuronal and cardiomyocyte networks and fully integrate readout electronics with the nanowire sensors. Finally, all components on a single, low cost platform scalable to 1820 wells and 115,840 simultaneous measurement points are monolithically integrated and the platform validated with a panel of drugs at the Sanford Burnham Prebys Medical Discovery Institute and UC San Diego. These technical innovations should enable non-destructive intracellular potential measurements across the depth of a tissue.

3D Imaging Flow Cytometer NanoCellect Biomedical, Inc. RESEARCH & RELATED Other Project Information 7. PROJECT SUMMARY Fluorescence-activated-cell-sorting (FACS) or flow cytometry enables clinicians and researchers to quantitatively characterize the physical (cell size, shape, granularity) and biochemical (DNA content, cell cycle distribution, cell surface markers, and viability) properties of cells, however FACS devices do not produce an image of the cell. Increasing sophistication of research assays now rely on the collection of cells based on their phenotypical and spatial characteristics; with the capabilities offered only by microscopic imaging cytometers having severe limits in throughput and lacking cell isolation. We propose an innovative, low cost design to combine the merits of FACS and microscopic 3D imaging cytometry without the limits of each, offering the biomedical research and clinical community a unique tool to address the needs for current and emerging applications. The key innovation is based on a significant extension of the spatial-coding algorithms our team demonstrated in the past years. In the proposed design, we create a special filter with a matrix of periodic slits in front of each PMT detector. The resulting PMT signal is composed of the multiplexed cell signals modulated by the filter, which can subsequently be deconvolved to produce fluorescence and scatter generated from different areas of the cell: the image. In addition, we add sweeping structured light with known positions over time to deconvolved information in the Z-axis, as well, allowing 3D image formation. In this Phase I program, we will integrate 3D imaging technology into our existing WOLF Cell Sorter to produce the very first 3D imaging cytometer with cell sorting capabilities. Since we use conventional, non-pixelated detectors (e.g. high-speed PMTs) found in conventional flow cytometers, this technology is compatible with existing flow cytometer architectures allowing for wide use. Equipped with cell imaging capabilities, researchers can track many important biological processes by analyzing not only the intensity but the localization of certain proteins within cytosolic, nuclear, or cell membrane domains and subdomains. With the rapidly developing capabilities of handling ?big data?, 3D images of millions of single cells in a flow cytometer provide vast resources for research and disease analysis, and rapid growth has been predicted in the market of such high-content imaging cytometer cell sorters for emerging applications such as precision medicine. We believe the proposed design is a major breakthrough that can potentially revolutionize the field of flow cytometry, and its impact and ramifications on both fundamental biomedical research and clinical applications can be tremendous.

Preterm birth, preeclampsia, and fetal growth restriction are major causes of fetal and maternal morbidity and mortality. There is increasing evidence that dysregulation of placental function is associated with these disorders, and that the pathologic processes leading to them long precede their clinical manifestations. Therefore, the ability to accurately, rapidly, and safely detect various forms of placental dysfunction early in pregnancy may enable discovery and clinical use of novel biomarkers of pregnancy complications. Currently available strategies for prediction of these adverse pregnancy outcomes suffer from low sensitivity and specificity and/or are unfeasible for broad implementation, due to the need for expensive equipment, specialized expertise, or long turnaround times. This project aims to develop a magnetic nanosensor-based platform for measurement of analytes in the maternal blood that reflect placental function, which can be used to discover novel predictive biomarkers and make them universally available. The project team includes the expertise in maternal fetal medicine (Dr. Laurent), biomarker discovery (Drs. Laurent and Boniface), assay design (Drs. Lo and Boniface), and biosensors and instrument design (Drs. Hall and Lo). Aim 1 will focus on development of a magnetic immunoassays (MIAs) targeting known biomarkers of placental function (PAPP-A, ?HCG, AFP, INHA, sFLT and PLGF) and two novel analytes from a clinical mass-spectrometry-based assay for prediction of spontaneous preterm birth developed by Sera Prognostics (IGFBP4 and SHBG), and validation of these MIAs against existing clinical ELISA- and mass spectrometry-based tests. Aim 2 will include development, testing, and validation of magnetic nanosensor-based miRNA assays (MamiRAs), which will be adapted from a highly sensitive and specific optical PCR-free miRNA assay developed by the Lo group. Replacing the fluorescent quantum dots in the optical assay with magnetic nanoparticles will enable the MamiRAs to be read on the same device as the MIAs for proteins. After initial proof-of-concept studies, we will develop MamiRAs that interrogate a novel set of 21 miRNAs in maternal serum that Dr. Laurent has found to be highly predictive of placental dysfunction. If successful, this project will produce a versatile magnetic nanosensor-based platform for sensitive and specific quantification of a variety of analyte types, which will not only enable development of novel strategies for management and treatment of pregnancy complications, but can also be applied to other clinical and biological systems. Moreover, the platform is amenable to miniaturization, which will enable clinical tests to be performed at the point-of-care, allowing the treating physician to have immediate access to the test results, and enabling testing, interpretation and management/treatment recommendations to be done at the same clinic visit. The ability to test and treat in the same visit will improve the efficiency of the system and eliminate the risk of delayed treatment or loss-to-followup.

SUMMARY The primary goal of the proposed research is to demonstrate a high throughput flow cytometer system that can sort cells based on high-content 3D cell image features. For each single cell flowing in a microfluidic channel, the system will produce cell tomography from spatially resolved fluorescent and scattering signals at a rate of 1000 cells/s. Each multi-parameter 3D cell image will be reconstructed, hundreds of image features will be extracted, and cells with their spatial features meeting the user-defined criteria will be sorted (3D image-guided cell sorting). Essentially the proposed system combines the merits of high throughput cell analysis and sorting capabilities of a fluorescence-activated cell sorter (FACS) with a high-content 3D imaging microscope to offer researchers and clinicians unprecedented features and capabilities to analyze, classify, and isolate cells at single cell resolution. The invention of this tool is anticipated to transform cell phenotype studies, greatly accelerate cell type discoveries, and enhance studies of highly heterogeneous biological samples such as tumors and brain. To realize such ambitious goal, we will take several innovative approaches. To produce high-quality 3D cell images for individual cells travelling fast in a flow channel, we invent a camera-less imaging system using a design that combines scanning structured light excitation and the scheme of confocal detection, which transforms 3D spatial information into temporal signals at the output of high-speed photomultiplier tubes (PMTs). For cell sorting mechanism, we adopt a microfluidic chip/cartridge design with an on-chip piezoelectric actuator to sort cells without causing flow jitters that can disrupt imaging of cells passing the optical imaging area. To achieve real-time image processing and image feature extraction, as well as handling the transport and storage of the large amount of 3D cell image data, we propose an electronic system consisting of a field programmable gate array (FPGA) module and graphics processing unit (GPU), having the FPGA process PMT signals, cell detection, segmentation and image reconstruction, and sorting decision control while having the GPU extract hundreds of 3D image related features and define sorting criteria (i.e. 3D image- guided gating) in parallel. To evaluate the performance of the system, we will perform experiments to sort cells based on the properties of protein translocation and trafficking, spot counting, organelle tracking, and features that help understand the disease biology and drug development. The proposed instrument will offer biomedical community a powerful tool to advance phenotype studies and cell type discoveries, and to link gene expression studies to cell phenotypic characteristics at single cell resolution and high throughput. The impact of the research will be significant and profound.