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Speakers
Chiara Stringari
Chiara Stringari is a researcher at the French National Center for Scientific Research (CNRS) at the Laboratory for Optics and Biosciences (LOB) at École Polytechnique, France. She obtained her PhD at the European Laboratory for Non-Linear Spectroscopy in Florence, Italy and she joined the LOB in 2014 after a Postdoc at the Biomedical Engineering Department of the University of California, Irvine.
Besides her interest in non-linear optics (NLO) and instrumentation development she is also committed to an interdisciplinary research approach. Her main focus is the development and implementation of label-free multiphoton microscopy methods for biophotonics and biomedical applications. Her major contributions include the development of two-photon fluorescence lifetime microscopy (2P-FLIM) of intrinsic biomarkers for metabolic imaging of living tissues and polarization-resolved second harmonic generation (SHG) and third harmonic generation (THG) microscopy. Her current research focuses on the development of fast FLIM methods for functional metabolic imaging of large tissues and NLO based methodologies for probing myelin distribution in healthy and pathological brain tissues in a multi-scale manner.
David Jameson
David Jameson received a BS in Chemistry from Ohio State University and a PhD in Biochemistry from the University of Illinois. His thesis advisor was Gregorio Weber, considered to be the father of modern fluorescence spectroscopy. He carried out postdoctoral research first at the Universidad de Paris-Sud working with Synchrotron Radiation and then returned to Gregorio Weber’s laboratory to continue postdoctoral research. He next joined the Pharmacology Department at the University of Texas Southwestern Medical Center as an Assistant Professor after which he joined the Biochemistry and Biophysics Department at the University of Hawaiʻi. He is presently a Professor in the Department of Cell and Molecular Biology in the John A. Burns School of Medicine. His research has always involved application of fluorescence methodologies to study protein-protein and protein-ligand interactions both in vitro and in live cells.
This lecture will present an overview of significant historical developments in fluorescence and fluorescence microscopy. Observations of the phenomenon we now call fluorescence dates back hundreds of years and some of the more important observations and developments in this area will be discussed. The early development of fluorescence microscopy will be examined as well as events leading to modern Fluorescence Lifetime Imaging Microscopy (FLIM). Finally an overview of Phasor Analysis, which is very prevalent in modern FLIM studies, will be presented.
Ammasi Periasamy
Dr. Periasamy was graduated from Annamalia University with M.S. degree in Physiscs. Later M.S. and Ph.D. at the Biomedical Engineering, Indian Institute of Technology-Madras, Chennai, India. Dr. Pereiamsy acquired his postdoctoral training at the Bioengineering department, University of Washington, Seattle, in the area of Bioimaging. Currently Dr. Periasamy is working as a Professor of Biology and Biomedicla Engineering. He is the director and founder of the WM Keck Center for Cellular Imaging, a unvieristy of Virginia Imaging Center.
Dr. Periasamy is an internationally recognized expert in the development of advanced optical microscopy techniques for single cellular imaging and analysis in living cells, tissue, and animal. A key area of Dr. Periasamy’s research is focused on the design and development of optical methodologies to investigate/monitor exogenous and endogenous molecular interactions. He has has recently developed a FLIRR (Fluorescence Lifetime Redox Ratio) to investigate metabolism and mitochondrial dysfunction in cancer cells and tissues. The main goal of the project is to monitor the early detection of prostate cancer (PCa) and correlate with PSA. The clinical sample tests were encouraging.
Dr. Periasamy is one of the pioneers in the development of fluorescence lifetime imaging microscopy (FLIM) for measuring the oscillations in cytosolic and nuclear free calcium in single intact living cells. He has developed a 2- and 3-color steady state, confocal, multiphoton, and FLIM based Förster resonance energy transfer (FRET) imaging systems for protein localization in living specimens. He has published more than 184 articles in refereed journals, book chapters and proceedings. He has more than 200 invited lectures nationally and internationally. Dr. Periasamy has edited three books, series book editor on cellular and clinical imaging (11 books), Chairperson in organizing an annual international conference on Multiphoton Microscopy in the Biomedical Sciences through SPIE (since 2001) and runs a hands-on training annual workshop on FLIM, FRET and metabolic imaging Microscopy at the University of Virginia, Charlottesville, during March (since 2002). Dr. Periasamy was elected “Fellow” member of the SPIE Optical Society in 2012.
Abstract
Mitochondrial Structure Function Related to Cancer-tauSTED and Two-photon FLIRR Microscopy
Most biological functions are in the nanosecond time domain. Investigation of any small sensitive changes can be detectable or monitored using two-photon fluorescence lifetime imaging microscopy (2p-FLIM) techniques. FLIM assays are of particular interest in measuring responses to treatment in various cancer pathologies, cancer being a metabolically heterogeneous pathology, which generates energy by oxidative phosphorylation (OXPHOS) and (often preferentially) by glycolysis. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture auto-fluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. 2-photon FLIM to track changes in mitochondrial metabolism via NAD(P)H/FAD lifetimes and relative abundances. Single wavelength 2p excitation simplifies FLIRR (Fluorescence Lifetime Redox Ratio) imaging, data analysis using artificial intelligence (machine learning), decreasing the total imaging time, avoids motion artifacts and increases temporal resolution. 2p-FLIRR and super resolution microscopy will precisely help us to investigate the role of mitochondrial structure -function relationship in cancer using tauSTED microscopy and how this relationship gets modified in aggressive forms of cancer for metastasis.
Melissa C. Skala
Melissa Skala is the Carol Skornicka Chair at the Morgridge Institute for Research and a Professor of Biomedical Engineering and Medical Physics at the University of Wisconsin - Madison. She is a fellow of the Professional Society for Optics and Photonics Technology (SPIE), Optica, and the American Institute for Medical and Biological Engineering (AIMBE). Her lab develops biomedical optical imaging technologies for cancer research, cell therapy, and immunology with support from the National Institutes of Health and the National Science Foundation. She serves as an editorial board member at Cancer Research and the Journal of Biomedical Optics. She received her BS from Washington State University, MS from the University of Wisconsin – Madison, and PhD from Duke University
Abstract
My lab develops non-invasive optical imaging approaches to unravel dynamic relationships between immune cell function and metabolism at a single cell level within living samples. Immune cell function is closely coupled to cell metabolism, and significant heterogeneity exists between individual immune cells. However, current techniques to measure metabolism in single immune cells require sample destruction or the introduction of reporters that may alter the native context. Fluorescence lifetime imaging microscopy of the endogenous metabolic co-enzymes NAD(P)H and FAD, or optical metabolic imaging (OMI), provides intrinsic sources of metabolic contrast within single cells, which can be used to monitor metabolic features of immunity within living systems. I will discuss applications of OMI and machine learning models in cell manufacturing for adoptive cell transfer therapy in cancer patients. Here, single cell metabolic imaging with OMI provides unique insights into cell subpopulations and manufacturing conditions that improve outcomes in cancer.
Alex Walsh
Alex Walsh is an associate professor in the Biomedical Engineering Department at Texas A&M University. Shereceived her MS and PhD degrees in Biomedical Engineering from Vanderbilt University where she received education and training in biophotonics, multiphoton microscopy, label-free microscopy, quantitative image analysis, and laser tissue interactions. She completed her post-doctoral training at the Air Force Research Lab studying the biophysical effects of infrared light. Currently, her lab seeks to improve human health through the development and application of label-free optical technologies. Her work is supported by the NIH NIGMS through a Maximizing Investigator’s Research Access (MIRA, R35) award, the Air Force Office of Scientific Research, and the Cancer Prevention and Research Institute of Texas.
Abstract
Leveraging Autofluorescence Lifetime Imaging for Live Cell Measurements of Metabolism
Cellular metabolism, the process by which cells generate energy, is dysregulated in many diseases and pathologies including cancer, neurodegeneration, and diabetes. Current biochemical assays for metabolism are limited to either cell-destructive protocols, such as mRNA analysis, or measure readouts from collective cell populations, such as oxygen consumption assays. Yet metabolic measurements with high resolution of live cells are important since cellular heterogeneity is known to drive disease progression, cancer metastasis, and resistance to therapies. Fluorescence lifetime imaging of the metabolic coenzymes, reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD), provides a label-free method to interrogate cellular metabolism. Both coenzymes, NAD(P)H and FAD, exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime. However, correlations between lifetime measurements and metabolic phenotypes have remained elusive. We are creating and testing machine learning models that can identify metabolic phenotypes of individual cells from fluorescence lifetime metrics and images. Additionally, we are increasing the accessibility of fluorescence lifetime imaging by creating improved analysis tools and automated methods for single-cell segmentation of autofluorescence images.
My lab develops non-invasive optical imaging approaches to unravel dynamic relationships between immune cell function and metabolism at a single cell level within living samples. Immune cell function is closely coupled to cell metabolism, and significant heterogeneity exists between individual immune cells. However, current techniques to measure metabolism in single immune cells require sample destruction or the introduction of reporters that may alter the native context. Fluorescence lifetime imaging microscopy of the endogenous metabolic co-enzymes NAD(P)H and FAD, or optical metabolic imaging (OMI), provides intrinsic sources of metabolic contrast within single cells, which can be used to monitor metabolic features of immunity within living systems. I will discuss applications of OMI and machine learning models in cell manufacturing for adoptive cell transfer therapy in cancer patients. Here, single cell metabolic imaging with OMI provides unique insights into cell subpopulations and manufacturing conditions that improve outcomes in cancer.
Steven Vogel
Dr. Vogel received his B.S. from the City College of New York in 1978 and his Ph.D. in Biochemistry and Molecular Biophysics from Columbia University in 1989, where he studied G-proteins in the nervous systems of Aplysia and squid with Jimmy Schwartz. During a postdoctoral fellowship with Joshua Zimmerberg at NIDDK and NICHD, he investigated the calcium dependence of exocytosis in sea urchin eggs and discovered heterogeneity for calcium responses in a population of secretory vesicles. Dr. Vogel next became an assistant and subsequently a tenured associate professor at the Medical College of Georgia (1997-2003), where he studied the mechanism of exocytosis - endocytosis coupling. He joined NIAAA as an investigator in 2003, and became a senior investigator in 2013. His laboratory develops new fiber optic and microscope-based assays using FRET, fluorescence polarization, and fluorescence correlation spectroscopy to study protein-protein interactions in living cells, focusing on the structure and function of CaMKII holoenzyme. Dr. Vogel is also a faculty member for the annual University of Virginia FRET Workshop
Abstract
Quantum coherent excitonic coupling between fluorescent proteins under physiological conditions
Quantum Biology seeks to determine how biological mechanisms exploit quantum behaviors such as superposition and entanglement. Several biological processes are thought to utilize quantum behaviors including photosynthesis, animal migration, olfaction, and some enzymatic mechanisms. How living systems overcome the dephasing influences of hot and wet biological environments that allow quantum behaviors to manifest is poorly understood. We hypothesize that if a quantum behavior offers life an evolutionary advantage, protein evolution will both preserve and perfect structures that facilitate quantum interactions under physiological conditions. LanYFP, avGFP, and dsRED are fluorescent proteins (FPs) isolated from marine organisms which diverged in pre-Cambrian oceans over 541 million years ago. While they have < 30% sequence homology, and their spectra, absorption coefficients, and quantum yields differ, they do share a common 𝛽-barrel protein structure. Here we show that despite their divergent evolution tandem dimers (TDs) derived from these FPs all show quantum coherent coupling behavior under physiological conditions. Circular dichroism spectroscopy revealed Davydov splitting, a manifestation of quantum excited-state superposition, with excitonic coupling strengths ranging from 113–357 cm-1. Supporting exciton excited-state entanglement, photon-antibunching indicated TDs are all single photon emitters despite being comprised of two functioning chromophores. Venus antibunching is observed even at 20 nm separation, consistent with coherent energy transfer. Solid-state interferometry measures longer than expected coherence times ranging from 96–120 fs. Our findings suggest that the conserved 𝛽-barrel structure is the common attribute supporting the conserved coherent FP excitonic coupling under physiological conditions. Since excited state superposition is a key component required for quantum sensing, we hypothesize that 𝛽-barrel FP structures found in diverse marine animals might function to support quantum sensors for detecting weak electro-magnetic fields.
Luke D. Lavis
Luke Lavis grew up in rural Southern Oregon in the shadow of the Cascade Range. He received his BS in Chemistry in 2000 from Oregon State University where he performed synthetic organic chemistry research in the laboratory of James White. After a four-year stint in industrial R&D at Molecular Probes and Molecular Devices, Luke joined Ronald Raines’s laboratory at the University of Wisconsin–Madison, designing and building novel fluorescent dye tools to image biomolecular trafficking in living cells. He received his PhD in 2008 and then moved to Janelia. At Janelia, Luke has been focused on constructing novel fluorogenic tools to enable high-resolution imaging and sophisticated neurobiological experiments.
Abstract
Designing brighter dyes for single-molecule microscopy and beyond
Specific labeling of biomolecules with bright, photostable fluorophores is the keystone of fluorescence microscopy. An expanding method to label cellular components utilizes genetically encoded self‑labeling tags, which enable the attachment of chemical fluorophores to specific proteins inside living cells. This strategy combines the genetic specificity of fluorescent proteins with the favorable photophysics of synthetic dyes. Over the past decade, our laboratory has pioneered novel organic chemistry methodologies for synthesizing small-molecule fluorophores. Through this work, we have elucidated structure–activity relationships, culminating in the development of dyes with enhanced quantum efficiency and photostability across the entire visible spectrum. These fluorophores seamlessly integrate with self-labeling tags, facilitating sophisticated microscopy techniques, such as single-molecule and super-resolution modalities. Our chemistry also enables further modifications to fine-tune spectral and chemical properties for experiments in increasingly complex biological samples such as intact organisms.
Hailey A. Parry
Hailey A. Parry, PhD, is an IRTA Postdoctoral Fellow in the Muscle Energetics Laboratory at the National Heart, Lung, and Blood Institute of the NIH in Bethesda, MD. She completed her doctoral training in exercise physiology with Dr. Andreas Kavazis at Auburn University. Hailey is continuing her training by learning to perform novel electron and light microscopy techniques. Specifically, she is interested in understanding how mitochondrial metabolism changes relative to subcellular regional location in skeletal muscle in health and disease
Abstract
Subcellular distribution of NADH pools in skeletal muscle mitochondria
New research suggests neighboring mitochondria can simultaneously participate in metabolic synthesis and breakdown processes. This suggests high, regional-specific regulation of these metabolic processes. However, there is limited knowledge of the spatial distribution of mitochondrial enzymatic activity in energy-demanding tissue such as the skeletal muscle. This research explores how mitochondrial metabolic processes depend on subcellular location in a skeletal muscle cell. Fluorescent lifetime imaging microscopy was performed to measure the unbound and bound NADH pools in isolated skeletal muscle fibers. Mitochondrial NADH pools were then determined based on their proximity to capillaries. Preliminary data shows that paravascular mitochondria (PVM), located directly next to capillaries, have a higher mean lifetime (p = 0.0003) compared to interfibrillar mitochondria (IFM), located in the middle of the cell. These data indicate a greater influence of bound NADH lifetime on the PVM NADH pool. Perhaps most interestingly, the lifetime values of the bound NADH pools in the PVM and IFM are different, implying the different pools of mitochondria have specific metabolic processes. Together, these data demonstrate the regional specificity of mitochondrial NADH pools in skeletal muscle cells.
Rozhin Penjweini
Rozhin Penjweini is a scientist at the National Institutes of Health (NIH) with an interest in developing and using different multiphoton imaging techniques to deepen understanding of cellular metabolism and oxygenation. Being able to image both intracellular and interstitial oxygenation and metabolism is a paramount objective, as it permits physiological and pathological metabolic processes to be tracked in real time. Multiphoton redox and metabolic imaging methods may have broad applicability in the study of diseases and therapeutics.
Abstract
Lifetime imaging of cellular metabolic processes
Mitochondria are not only responsible for producing energy by oxidative phosphorylation (OXPHOS), but they also play a critical role in the control of metabolism, reactive oxygen species (ROS) generation, apoptosis, cell proliferation and differentiation. Whether increasing mitochondrial respiration reduces O2 availability in other cellular compartments (altering other O2-dependent phenomena) remains to be explored. Using fluorescence lifetime imaging of a targetable, genetically encoded oxygen [O2] sensor (Myoglobin-mCherry), we spatially mapped oxygen partial pressure (pO2) in mitochondria and nuclei in single cells cultured under different glucose and O2 concentrations, as well as under proliferation and differentiation conditions. The change of ROS was monitored via detection of metMyoglobin using lifetime imaging of a sandwich probe, EYFP-Myoglobin-mCherry. In combination with another fluorescence lifetime imaging tool, the free/bound ratios of key metabolic co-factors, (NAD(P)H and FAD+), we show that pO2 gradients are linked to, and reflect, the functional metabolic rewiring in mitochondria occurring during hypoxia, glucose starvation and cell differentiation. Despite the rapid diffusion of gases, a possible role for mitochondria as modulators of nuclear O2 concentration and sequelae is plausible, especially in confined regions
Philip Bleicher
Philip Bleicher started his training as a Molecular Biotechnologist at the Technical University in Munich (TUM). He then moved to the Physics Department (TUM) for his PhD studies, working under Prof. Andreas Bausch to study the dynamics of the actin cytoskeleton. After completing his thesis and obtaining a fellowship at the NHLBI, he continued his research in the Molecular Physiology lab of Dr. James Sellers, studying the contractility of myosins in reconstituted, biomimetic actin arrays.
Abstract
Non-muscle myosin 2 (NM2) plays a vital role in mediating cellular tension, driving essential processes such as morphogenesis and cellular motility. While contractile filaments of NM2 have been observed and kinetically characterized both in vitro and in vivo, certain biophysical properties - specifically, their tension transfer to the actin cytoskeleton - remain poorly understood. To investigate this, we utilized recombinant NM2 with an embedded FRET tension sensor and reconstituted micropatterned actin bundles (elongated by the formin mDia1) in a sarcomere-like arrangement. We quantified the motile properties of myosin filaments that contained either NM2-A or NM2-B paralogs, as well as co-filaments containing both paralogs during tension generation using TIRF microscopy. Furthermore, we measured the forces exerted by these filaments using FLIM. Our findings reveal that, although NM2-A is faster than NM2-B and shows a tendency to sever and retract tense actin bundles, both paralogs produce comparable tension. We validated this result by incorporating a FRET sensor into the anchoring mDia1, which indicated equivalent tension transmitted through the bound actin bundles. Lastly, by expressing the tension sensor mDia1 in U2OS cells, we demonstrate that live-cell tension measurements of cortical actin are achievable using this probe.
Kandice Tanner
Kandice Tanner received her doctoral degree in Physics at the University of Illinois, Urbana-Champaign. She trained as a Department of Defense Breast Cancer Post-doctoral fellow jointly at University of California, Berkeley and Lawrence Berkeley National Laboratory. Dr. Tanner integrates concepts from molecular biophysics and cell biology to understand organ specific metastasis . In recent years, she has received the 2023 Max Planck-Humboldt medal, 2023 ASCB E.E. Just award and the 2021 Arthur S. Flemming award. She is also a Fellow of the American Physical Society and the American Society of Cell Biology
Abstract
Microenvironment regulation of metastasis
In the event of metastatic disease, emergence of a lesion can occur at varying intervals from diagnosis and in some cases following successful treatment of the primary tumor. Genetic factors that drive metastatic progression have been identified, such as those involved in cell adhesion, signaling, extravasation and metabolism. However, organ specific biophysical cues may be a potent contributor to the establishment of these secondary lesions. We combine a novel preclinical model of metastasis with a suite of biophysical tools to elucidate the role of tissue biophysical properties of in the establishment of metastatic lesions in vivo. Specifically, I will discuss our efforts to determine what physical cues influence disseminated tumor cells in different organ microenvironments using in vitro and in vivo preclinical models such as 3D culture systems and zebrafish
Program (Draft)
Time | Drescription | Presenters |
---|---|---|
8:00 AM | Registration & Coffee | |
8:45 AM | Welcome and Introduction | Richard Childs, NHLBI DIR SD, Chris Combs, Gary Laevsky |
9:00 AM | History of FLIM | David Jameson |
9:40 AM | Metabolic trajectories | Chiara Stringari |
10:20 AM | CAR-T metabolism | Melissa Skala |
11:00 AM | Break | |
11:15 AM | NADH/FAD+ FLIRR | Ammasi Periasamy |
11:55 AM | Vendor Talk | Becker & Hickl |
12:15 PM | Lunch/Posters/Vendor Tables | |
1:15 PM | Taxol damaged mitos | Rozhin Penjweini |
1:35 PM | Metastasis | Kandice Tanner |
2:05 PM | FRET | Steve Vogel |
2:35 PM | Vendor Talks | Nikon/PicoQuant:Leica:Zeiss |
3:05 PM | Dye designs | Luke Lavis |
3:35 PM | Break | |
3:50 PM | Vendor Talks | Axiom:Yokogawa::Hamamatsu:MKS |
Bruker:Prior:Toptica:MKS:Horiba:3i | ||
4:35 PM | Tension | Walter P Bleicher |
4:50 PM | Myocytes | Hailey Parry |
5:05 PM | Vendor Talks | ISS:Abberior:FAES |
5:30 PM | Metabolic Assays | Alexandra Walsh |
6:10 PM | Prospects | Chris Combs/Jay Knutson |
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Registration requried for the Inaugural Quantitative Fluorescence Lifetime Intensity Microscopy (QFILM) Meeting at National Institutes of Health held on November 7th, 2024
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