Michelle Digman, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Educate UCI researchers on how to measure FRET ratiometrically and via use of FLIM.

DESCRIPTION (provided by applicant): This proposal seeks to acquire the first and currently only commercial microscope that has been specifically designed to have the improved sensitivity and reduced electronic noise necessary for monitoring the behavior of proteins in living cells (e.g. ligand binding receptors or proteins forming inclusions in degenerating cells). In combination with analysis methods developed at the LFD (Laboratory for Fluorescence Dynamics, UCI), this instrument reaches new levels of detection and allows measurements that were previously not possible. These capabilities are having a strong impact on the research endeavors at UCI and in particular are enabling several medically relevant projects to proceed. For example, Dr. Lee, breast cancer;Drs. Glabe and Marsh, neurodegeneration, Dr. Nelson, cancer profiling, Digman, cell migration, Gratton, protein trafficking in cells just to name a few. The acquisition of a Zeiss LSM 710 by the Optical Biological Core (OBC) facility at the Developmental Biology Center (DBC) represents an essential goal that would enable users to apply state-of-the-art live cell imaging methods to numerous emerging and fundamental biological problems. The permanent addition of this instrument at the OBC would allow multiple users access to vastly improved detectors that have significant higher signal to noise ratio, availability of multiple laser lines for one and two- photon image experiments or simultaneous imaging, moveable emission wavelength selection for broad or narrow collection and non-descanned detection. In addition, the twin gate beam splitter and high- transmission dichroic wheels allow users to use an array of laser wavelengths for fluorescence excitation. Since four lines can be used simultaneously, the complete system allows for maximum photon collection. The instrument will also have a FLIM detection unit. A microscope with all these features is not available on campus. In addition, an advanced feature of the LSM 710 not found in other commercial instruments includes the application of the Raster-scan Image Correlation Spectroscopy (RICS) technique to cells, a technique developed by Zeiss in collaboration with Dr. Enrico Gratton and Dr. Michelle Digman, the PI of this proposal and the scientific director of the OBC. We developed the RICS, Number and Molecular Brightness (N&B) and phasor FLIM techniques and have made them accessible for the standard commercial microscope. We thus have the expertise and enthusiasm to apply these techniques to an array of different problems in the areas of Medical Sciences, Biochemistry, Pharmacology, Nanotechnology, and Biology as illustrated in the "projects" section of this proposal. This application seeks funds to purchase an LSM 710 to replace the instrument that has been on temporary loan and that has proven to be essential for many projects and in high demand. PUBLIC HEALTH RELEVANCE: The acquisition of the first available commercial instrument capable of high sensitivity, dynamic and quantitative imaging of proteins and fluorescent particles in cells will greatly enable progress on a large number of NIH funded projects ranging from cancer profiling to the study of growth factor ligands interacting with receptors in tissues. The microscope will be housed in a facility that is available to all and that currently has users from 60 PI laboratories in 20 departments across 4 schools. The sensitive filter-less detector and the use of two-photon excitation with FLIM capabilities will allow studies of molecular interactions in living cells and in tissues.

? DESCRIPTION (provided by applicant): This proposal aims to establish a national short course in Big Data Image Processing & Analysis (BigDIPA) intended to increase the number and overall skills of competent research scientists now encountering large, complex image data sources derived from cutting edge biological/biomedical research approaches. Extraction of knowledge from these imaging sources requires specialized skills and an interdisciplinary mindset. Yet effective training opportunities of this sector of the Big Data science community are glaringly underappreciated and underserved compared to other big data fields such as omics. UC Irvine is ideally suited to host a short course to address this thematic training deficit on account of the synergistic colocalization between multiple facilities, renowned for development of numerous advanced imaging techniques, and the outstanding instructional environment provided by faculty with collaborative expertise in biological image processing and computer vision, bioinformatics and high performance computational approaches. Specifically, our BigDIPA proposal assembles an interdisciplinary alliance of faculty experts that can leverage the preeminent imaging resource facilities, such as the Laboratory of Fluorescence Dynamics (LFD) and the Beckman Laser Institute, and fuse these to ongoing campus big data initiatives, e.g. UCI's Data Science Initiative, to create a top-rated training course designed for senior graduate students, postdoctoral researchers, faculty and industry scientists from diverse scientific disciplines who have nascent interests and needs to handle BIG DATA sources beyond their current level of competency. The course theme is focused to utilize discreet examples drawn from the analysis of complex data acquired from different microscopy imaging modalities employed to investigate dynamics in cellular and tissue processes, including signal transduction networks, development, neuroscience and biomedical applications, and that hereto where hidden or inaccessible to standard methods of analysis. Participants will be guided along the complete acquisition- processing-analysis pipeline through exposure to a coherent progression of topics and issues typically encountered when handling BIG DATA. We believe this training approach will therefore be attractive to a broad and significant untapped pool of researchers from the biological disciplines, biomedical engineering, systems biology, math, biophysics, computer science, bioinformatics and statistics who possess some, but not all, of the requisite competencies to effectively traverse the BD2K landscape. We have designed the course such that skills and experience gained by trainees will be transferable to their own research interests. The BigDIPA course format will combine didactic lectures on the theory and foundational frameworks that underpin each step, with practical instruction on implementation and hands-on tutorials in image acquisition, large data handling, basic scripting of computational tools, image processing on high performance computing architectures, as well as feature extraction, evaluation and visualization of results. The course is designed to offer an intense learning experience delivered in a compact time frame, and opportunities to foster interdisciplinary interactions through small team exercises. Participants will also be encouraged to take advantage of pre-courses - separate and distinct training opportunities not funded by this proposal - that will be coordinated to directly precede our course. This unique format provides multiple benefits: it provides an efficient mechanism to address individual participant training deficiencies to permit a more productive experience in the BigDIPA course, adds no-cost mutual benefits to independent but synergistic programs, and facilitates recruitment of applicants who frequently feel interested but intimidated due to a perceived lack of prior adequate training. Beyond providing an intensive on-site training course, all course materials (lecture notes, video lectures and tutorials), tutorial exercises, open source software resources and sample datasets will be made freely available through on-line distribution to maximize outreach and encourage additional contributions of curated training resources solicited from the community.

The genetic information in DNA, which is packaged in the form of chromatin, is protected from damage by multiple DNA repair pathways, and understanding how this critical task of DNA repair is performed is the long-term goal of this research. The project will examine real-time changes in chromatin in response to DNA damage in individual living cells with unprecedented spatial and temporal resolution, and thereby determine the significance of chromatin restructuring during DNA repair. In addition to advancing knowledge of DNA damage response and repair mechanisms, the project will develop and apply several innovative technologies that will likely be of great interest to other researchers. The PI and co-PI are committed to disseminating these techniques broadly in the research community. The multidisciplinary nature of this project will provide a unique and valuable training opportunity for graduate students and postdoctoral fellows as well as undergraduate and high school students. They are expected to learn fundamental and cutting-edge techniques relevant to studying DNA repair mechanisms in mammalian cells, including high-resolution imaging.

The chromatin response to DNA damage is highly dynamic and must be regulated to maintain the appropriate architectural organization that promotes high-fidelity DNA repair and preserves genomic integrity. How these chromatin structural dynamics are coordinated with DNA repair is not well understood. The PIs will develop multiplexing novel fluorescence correlation methods including pair correlation spectroscopy (pCF) and nanoimaging tracking techniques to analyze how DNA damage affects chromatin dynamics and how that in turn dictates DNA repair pathway choice. These phenomena will be investigated in response to relatively simple DNA breaks or complex DNA damage lesions induced in different cellular contexts. Aim 1 includes monitoring changes in chromatin access at the damage site, interrogating the relationship to DNA repair pathway choice, and determining responsible factors. Aim 2 involves high-resolution multiplexed analyses of chromatin dynamics at defined DNA double-strand break sites, and investigating the interplay between DNA repair and transcription. An overall goal is also to develop new methods that yield 4D and nanoscale views of chromatin dynamics in single mammalian cells when DNA is damaged, for a quantitative understanding of how the cellular machinery assesses the damage and decides how to repair its genome.

Abstract The latest figure is that around 1.5 million Assisted Reproductive Technology (ART) cycles are performed each year worldwide, with an estimated 350,000 babies born. One of the critical steps during the IVF process is the selection of high-quality embryos for uterine transfer. This selection is currently based largely on defined morphological criteria and physical characteristics of the blastocyst. While such criteria have proven to be useful in improving implantation rates, assessment of the reproductive potential of individual embryos is not sufficient. Therefore, IVF centers often perform simultaneous transfers of multiple embryos that can result in multiple pregnancies, thus increasing the risk of preterm delivery and the death or lifelong disability of neonates. As the number of assisted reproduction cycles worldwide is increasing, improvements in our ability to predict embryo viability is urgently needed. Development of more qualitative and objective means for assessing embryo quality and viability that are safer and faster could provide significant advances in IVF by enabling singleton embryo transfers rather than the implantation of multiple embryos in order to increase the likelihood of a successful pregnancy. Given the limitations of morphologic evaluation, several technologies have been explored for the assessment of embryo viability. These include the measurement of metabolites in embryonic culture media along with genomic and proteomic profiling of the embryos themselves. Spectroscopic approaches have also been utilized to measure the amount of metabolites that arise during pre-implantation development. However, these approaches are time- consuming and require highly-trained personnel to analyze the complex data. Here we describe the application of a phasor-FLIM (Fluorescence-Lifetime Imaging Microscopy) approach, which is a ?non- invasive? live imaging approach capable of measuring endogenous autofluorescent metabolites within living embryos undergoing in vitro culturing. The approach captures information on the metabolic energy sources utilized by pre-implantation embryos as readout of embryo quality and viability.