Recorded at: High-Content Analysis

Digital Course: Drug Discovery & Development: Live-Cell Imaging


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About this Product:

Recent developments in microscopy, fluorescent probes, cell manipulation, and image analysis have pushed the frontiers of live-cell imaging, allowing researchers to study cellular processes in real time and increasing detail. Cambridge Healthtech Institute's Live-Cell Imaging Digital Course includes presentations on novel techniques, including FLIM-FRET microscopy methods for early cancer screening and detecting protein network interactions, Fluorescence by Unbound Excitation from Luminescence for high-content analysis, a system to monitor cells exposed to ionizing radiation, and innovative Tissue Dynamic Imaging.

Product Details:
5 Presentations
110 Slides
Over 132 Minutes
Individual: $345
Site License: $1380

Formats Available:
Digital Download
On Demand


Agenda at a Glance:

Localization of Tryptophan and NADH Interactions Using Three-Photon FLIM-FRET Microscopy

Ammasi Periasamy, Ph.D., Professor, Biology; Director, W.M. Keck Center for Cellular Imaging (KCCI), University of Virginia

Recent reports link tryptophan (TRP) metabolic activity to cancer development and progression. Increased TRP degradation may also occur in early-stage breast and lung cancer. To quantitate the TRP and nicotinamide adenine dinucleotide (NADH) interaction, we developed a novel three-photon excitation (3PE) fluorescence lifetime imaging and Förster resonance energy transfer (FLIM-FRET) microscopy method, able to differentiate tumorigenic from non-tumorigenic human live cells. Based on perturbation studies, where the addition of glycolytic substrates significantly quenches TRP lifetimes in tumorigenic HeLa cells, our results demonstrate the potential use of 3PEFLIM-FRET as a tool for screening in the early stages of cancer.

Monitoring Dynamic Protein Interactions in the Living Cell Nucleus

Richard N. Day, Ph.D., Professor, Cellular and Integrative Physiology, Indiana University School of Medicine

A critical challenge for public health research is to identify the molecular mechanisms that function in the dynamic control of the epigenome. The heterochromatin protein 1 alpha (HP1α) coordinates a network of protein interactions critical for epigenetic regulation during cellular differentiation, but the underlying mechanisms are not well understood. We are using frequency domain fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) to detect steady-state protein interactions involving HP1α. Combined with fluorescence correlation spectroscopy (FCS) to characterize the sub-nuclear protein diffusion, these techniques provide novel insights into protein network interactions inside the living cell nucleus.

FUEL for Thought: A Novel Approach to Detect Spatial Proximity on Mesoscopic Scales in vitro and in vivo Using Luminescence Excitation

Spencer L. Shorte, Ph.D., Director, Plateforme d'Imagerie Dynamique (PFID), Imagopole, Institut Pasteur

Bioluminescence Resonance Energy Transfer (BRET) improves the sensitivity of bioluminescence by red-shifting blue photons, and provides a measure of molecular co-localization at distances of up to 10nm. However, BRET detection methods may overlook long-distance, radiating energy excitation-emission effects that are significant in the bioluminescent detection regime. Fluorescence by Unbound Excitation from Luminescence (FUEL) describes this radiating luminescence effect that excites fluorophores by epifluorescence at distances far beyond 10nm, many microns, or even millimeters away in a manner completely distinct from BRET. Further, we show that detection of FUEL per se is sufficient to provide a detection of long-distance proximity in, and beyond the microscopic range both in vitro and in vivo. By enabling detection of mesoscopic proximity between luminescent and fluorescent probes in the context of living biological tissues FUEL promises utility as a novel tool for high-content analysis in cell and animal models.

Tracking "DNA Repair Centers" in Living Mammalian Cells

Sylvain Costes, Ph.D., Principal Investigator, Cancer and DNA Damage Response, Lawrence Berkeley National Laboratory

Upon DNA damage, nuclear sub-domains are formed in the nucleus. These radiation-induced foci (RIF) are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). We recently hypothesized that protein recruitment occurs in specific nuclear regions called "repair centers." By integrating a small X-ray device with a microfluidics system on a fluorescent light microscope, we can monitor various mammalian cells expressing 53BP1-GFP while being exposed to ionizing radiation. Using novel RIF counting algorithms and cell tracking algorithms, the formation of DNA repair factories will be discussed as a function of cell cycle, cell lineage and cell type.

Dynamic Imaging for High-Content Analysis of Three-Dimensional Tissue

David Nolte, Ph.D., Professor, Physics, Purdue University

An innovative label-free non-invasive imaging technology called Tissue Dynamic Imaging (TDI) extracts high content from live three-dimensional tissue culture responding to pharmaceuticals. Coherence-gated dynamic light scattering captures cellular dynamics through ultra-low-frequency speckle fluctuations that encode a broad range of cellular and subcellular motions as a new form of functional imaging contrast. The motions are altered by different mechanisms of action and generate drug-response spectrograms that act as fingerprints for phenotypic profiling. Applications span from early drug candidate screening to point-of-care and personalized medicine.

Speaker Biographies:

Sylvain Costes, Ph.D., Principal Investigator, Cancer and DNA Damage Response, Lawrence Berkeley National Laboratory 

Sylvian CostesSylvain Costes received his Ph.D. in Nuclear Engineering from UC Berkeley in 1999, and he is a world-renowned expert in high-throughput fluorescence microscopy, image processing and in radiation biology. He has more than 35 peer-reviewed publications, and his work has been cited more than 1,000 times over the past eight years. After his postdoctoral fellowship in the Mathematics Department at UC Berkeley, he joined the National Cancer Institute where he developed novel quantitative tools for fluorescence 4D microscopy. Some of these quantitative tools were integrated into several commercial imaging packages such as Carl Zeiss Imaging AG. When Dr. Costes joined the Berkeley Lab in 2004, he promoted a new work culture, nurturing the integration of engineering practices and physics into state of the art microscopy and molecular biology. Through his integrative approaches he has developed true multidisciplinary programs. For instance, the merging of array technology with chemistry, fluorescent probes, image processing and microscopy has led him to develop a novel platform with the capability to monitor DNA repair in living human cells. This technology has recently been noticed by mainstream media and has led to the creation of the early-stage startup Exogen Biotechnology, Inc., founded by Sylvain Costes and Jonathan Tang in 2012, both scientists at Berkeley Lab. Exogen Biotechnology is now applying this assay to identify patients with high risk of acute radiation sensitivity and monitoring the efficacy of antioxidant diets. Dr. Costes continues his work as a principal investigator at the Berkeley Lab while being involved at Exogen.

Richard N. Day, Ph.D., Professor, Cellular and Integrative Physiology, Indiana University School of Medicine

Richard DayRichard Day is a Professor in the Department of Cellular and Integrative Physiology at the Indiana University School of Medicine. His research focuses on understanding the network of regulatory protein interactions that function to control cell-type specific gene expression. His laboratory group uses biochemical and molecular approaches to define networks of protein interactions that are coordinated by specific transcription factors. These in vitro approaches are complemented by non-invasive live-cell imaging techniques using the many different color variants of the fluorescent proteins. Recent studies from the laboratory have used fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) to quantify steady-state protein interactions inside the living cell nucleus. In addition, fluorescence correlation spectroscopy (FCS) is being used to measure the diffusion of proteins inside the cell nucleus. Together, these techniques provide novel insights into protein network interactions inside the living cell nucleus.

David Nolte, Ph.D., Professor, Physics, Purdue University

David NolteDavid D. Nolte is a Professor of Physics at Purdue University, actively working in the fields of molecular diagnostics and drug screening. He received his baccalaureate from Cornell University in 1981, his Ph.D. from the University of California at Berkeley in 1988, and a post-doctoral appointment at AT&T Bell Labs before joining the faculty at Purdue. He has been elected Fellow of the Optical Society of America, Fellow of the American Physical Society and Fellow of the AAAS. In 2005 he received the Herbert Newby McCoy Award of Purdue University. He has founded two biotech startup companies in the area of diagnostic screening and high-content analysis.

Ammasi Periasamy, Ph.D., Professor, Biology; Director, W.M. Keck Center for Cellular Imaging (KCCI), University of Virginia

Ammasi PeriasamyA key area of my research is focused on the design and development of advanced light microscopy techniques to investigate/monitor exogenous and endogenous protein-protein interactions, intravital imaging and monitoring the physical parameters of normal versus cancer tissues. We developed a 2-color/3-color steady state, confocal, multiphoton, and FLIM based Förster resonance energy transfer (FRET) imaging system for protein localization in living specimens. The important aspect of the FRET-work is the development of a PFRET software package for quantitative FRET data analysis to estimate the nanometer (1-10 nm) distance between the protein molecules in living/fixed cells and tissue for various light microscopy techniques for any combination of fluorophore pairs. This PFRET software is used nationally and internationally by the FRET microscopy users. Dr. Periasamy is an internationally recognized expert in advanced microscopy techniques, particularly in the area of molecular imaging in living cells and tissues. He is one of the pioneers in developing lifetime imaging microscopy for intracellular calcium measurement and later he developed the same methodology for protein-protein interactions and cancer diagnosis. He has published over 100 articles in refereed journals and book chapters. He has given over 100 invited lectures nationally and internationally. Dr. Periasamy has edited three books, served as Chairperson (since 2001) in organizing an annual International conference on Multiphoton Microscopy in the Biomedical Sciences through SPIE, and runs a hands-on training annual workshop (since 2002) on FRET Microscopy at the University of Virginia, Charlottesville during March. Dr. Periasamy is one of the elected "Fellow" members of the SPIE Optical Society.

Spencer L. Shorte, Ph.D., Director, Plateforme d'Imagerie Dynamique (PFID), Imagopole, Institut Pasteur

Spencer ShorteSpencer Shorte is Core Facilities Group Leader at the Institut Pasteur in Paris (Plateforme d'Imagerie Dynamique) and Director of the Imagopole core facilities conglomeration founded in 2005. He received his Ph.D. in Biochemistry at Bristol University, UK, and has extensive experience in the fields of cell physiology, cell biology, and neuroendocrinology. He is an expert in techniques for quantitative imaging, where he has some 25 years applied experience. He has published more than fifty research articles and five patents. He received the 2005 French national engineer of the year in the category of Research Science awarded for his work on microscopic tomography.


About the Conference:

Cambridge Healthtech Institute's High-Content Analysis meeting is the premier event showcasing the latest advancements in HCA applications and technologies. Over the years the technology has matured and its adoption has spread into many areas of compound screening/evaluation and functional analysis. The Tenth Annual High-Content Analysis 2013 meeting will focus on the next steps of technology development, including new assays and probes, more advanced image analysis and data management, and novel biological models for screening. It will also cover case studies and applications in compound screening, cytotoxicity evaluation, stem cell research and functional analysis.