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Welcome to the CNSI Mini Symposium website If you missed the lecture, you can use this resource to view streaming videos of the lectures. Simply click on the speakers name in the navigation bar to browse to their page. You will find biography and abstract information on each speaker and also a link to stream the video. Please note that Windows Media Player is required to view the videos. |
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The foremost experts in the emerging field of "super-resolution optical imaging" will discuss the most recent breakthroughs and their potential to impact life sciences and medicine. Nanoscopy is set to answer some of the most fundamental questions in biology that require molecular-level imaging resolution. Symposium Description: Fluorescence microscopy has become the most popular imaging tool in cell biology. It is non-invasive and allows probing cellular functions and structures in the 3-dimensional space at the submicron scale. Fluorescent light microscopy is however limited in spatial resolution because the smallest possible spot size of light is intrinsically dictated by diffraction (known as Abbe's law). Exciting proof-of-principal experimental breakthroughs are now emerging to beat this diffraction barrier. They are yielding high-resolution images of intracellular structures to obtain ever-better resolution well within 15 nm. Pioneering new concepts in light microscopy are paving the way towards nanoscale microscopy and are ripe to tackle outstanding fundamental questions in biology that lie somewhere at nanometer spatial resolution. This CNSI symposium will address such state-of-the-art "super-resolution" methods as Stimulated Emission Depletion (STED), Structured illumination (I5M), Photoactive localization microscopy (PALM), Stochastic optical reconstruction microscopy (STORM) and 4Pi microscopy by bringing to UCLA the foremost experts in the field. The foremost experts in the emerging field of "super-resolution optical imaging" will discuss the most recent breakthroughs and their potential to impact life sciences and medicine. Nanoscopy is set to answer some of the most fundamental questions in biology that require molecular-level imaging resolution. |
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Mark Bates, Harvard Sub-diffraction limit imaging by stochastic optical reconstruction microscopy |
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Abstract:
We have developed a high resolution fluorescence imaging technique based on nanometer-accuracy localization of individual fluorescent labels. Photo-switchable fluorophores are used, which can be turned on and off using pulses of light. During the imaging process, the fluorophores are switched on and off so that individual fluorophores are distinguishable and can be localized with high precision. After a series of localization cycles the positions of many fluorophores have been determined and these are used to construct an overall image with sub-diffraction limit resolution. Our work is applicable to cell imaging and to in vitro imaging of single molecules. We have demonstrated an imaging resolution of 20 nm using a simple total-internal-reflection fluorescence microscope and low-power continuous-wave lasers. Biography: Mark Bates is a graduate student in Xiaowei Zhuang's lab at Harvard University, pursuing his Ph.D. in Applied Physics. Mark received his B.Sc. degree in Engineering Physics at Queen's University, and his M.Sc. in Physics from McGill University. His research focuses on applications of photo-switchable fluorescent molecules for high resolution microscopy. In 2004 he discovered that a commonly used fluorescent molecule, Cy5, can be switched between its fluorescent state and a dark state using pulses of light. He has applied these results to high resolution imaging as co-inventor of stochastic optical reconstruction microscopy (STORM), a nanometer-scale fluorescence imaging technique. | ||
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Jörg Bewersdorf, Jackson Laboratory Novel biological applications of 4Pi microscopy |
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Abstract:
Providing 100 nm axial resolution, 4Pi microscopy is the only optical microscope technique with ultra-high 3-dimensional resolution commercially available today. The point-spread function of regular confocal microscopy is sharpened ~6-fold along the axial direction by utilizing two opposing objectives in a coherent manner. The feasibility of this interference-based resolution enhancement depends significantly on the optical quality of the specimen, a reason why 4Pi microscopy was initially only applied to imaging of single cells. This presentation includes different biological applications showing the successful imaging of proteins in the cell nucleus, mouse blastocysts and brain tissue sections with 4Pi microscopy. Additionally, it is demonstrated how 4Pi microscopy gains from the use of quantum dots and new 4Pi imaging modes. Biography: Joerg Bewersdorf received his doctoral degree in Physics from the University of Heidelberg, Germany, 2002. Working with Stefan Hell at the Max Planck Institute for Biophysical Chemistry in Goettingen and at Leica Microsystems, he was strongly involved in the development of the Leica TCS 4PI, the first commercially available 4Pi microscope. After 2 years of postdoctoral work at the Max Planck Institute he moved to Bar Harbor, Maine, in summer 2005. As an Associate Research Scientist at The Jackson Laboratory, he now heads the 4Pi laboratory applying ultra-high resolution optical microscopy to biological questions and developing new microscopy techniques. | ||
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Rolf Borlinghaus, Leica Microsystems A Long Tradition of Being Ahead of the Times: Leica Microscopy |
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Abstract:
Biomedical research has many tools and instruments for a wide variety of experiments. Different to other techniques and equipment, microscopy not only attracts researchers by pure performance in terms of numbers and accuracy but the fine art of microscopic imaging and the impressive pictures also contains an esthetic value. Of course, once one starts to look into the so far unseen details of the fabric of life, one immediately tries to see even more details, with even more contrast and color as possible. Leica provides solutions at the cutting-edge of the most advanced technologies. In fluorescence microscopy, especially confocal and related techniques, Leica has introduced many completely new concepts and beneficial designs, which are now standard in biomedical research. At the same time, Leica remains committed to innovation. Biography: Based out of Heidelberg and Mannheim, Germany, Rolf Borlinghaus concentrates his efforts in production management for confocal microscopy at Leica Microsystems. He received his PhD in Biology from the Department of Biophysics at the University of Konstanz in Germany. His latest publication in the Journal of Microscopic Research Technology is entitled "High speed scanning has the potential to increase fluorescence yield and to reduce photobleaching". | ||
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Mats Gustafsson, UC San Francisco |
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Biography:
The Gustaffson research group works on creating and applying new forms of biological light microscopy with new abilities, especially modes that can reach higher spatial resolution than is normally allowed by the diffraction limit. The light microscope plays a large and important role in modern cell biology, because of its unique ability to follow specifically labeled molecular players within the 3D interior of living cells. Its main weakness is its modest resolving power, which is fundamentally limited by diffraction. This limit was thought of as an absolute barrier for more than a century, but it has recently been broken, and some of the ways to break it were developed here. Present and future directions include:
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Harald Hess, Howard Hughes Medical Institute Imaging Intracellular Fluorescent Proteins at Nanometer Resolution |
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Abstract:
We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent proteins molecules were activated, localized (to 2-25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method – termed photoactivation localization microscopy – to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane. Biography: Harald Hess recenetly became Director of Applied Physics and Instrumentation Group at Janelia Farms Research Campus, HHMI . There he is exploring new microscopies, imaging concepts and other instrumentation technologies relevant to its mission of bioimaging. An independent inventor last year he coinvented and developed a new superresolution microscope called PALM. He has also spent 8 years in industry innovating new concepts for both hard disk drive testing and semiconductor manufacture at the nanoscale. Prior to that he spent 10 years at Bell Labratories inventing a variety of new scanned probe microscopies to image magnetic, optical, electrical fields, and energy state densitiy. These opened new insight into vortex states structure in superconductors, quantum hall states, and luminescent constituents of a quantum well. As a postdoc at MIT he conceived of evaporative cooling as the path toward creating Bose-Einstein condenstates. | ||
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Sam Hess, University of Maine Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy (FPALM) |
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Abstract:
Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. In order to control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). Non-fluorescent inactive PA-GFP molecules are activated by laser illumination at 405 nm and then imaged by CCD camera under illumination by an Ar+ ion laser, which also eventually photobleaches those active molecules and removes them from the field of view. Because only a small number of molecules are visible at a given time, their positions can be determined precisely by localization: with only ~100 detected photons per molecule, the localization precision can be as much as ten-fold better than the resolution, depending on background levels. FPALM images of PA-GFP expressed in transfected live and fixed cells are presented. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and demonstrate significant improvement compared to the expected diffraction-limited optical resolution. Biography: Sam Hess earned a Bachelor of Science from Yale University in Physics, graduating summa cum laude , followed by a Master of Science and Ph.D. from Cornell University in the laboratory of Watt Webb, where he studied the photophysics of green fluorescent proteins (GFPs), two-photon excitation, and fluorescence correlation spectroscopy (FCS). His postdoctoral work with Joshua Zimmerberg at the National Institutes of Health focused on the lateral organization of biomembranes and the role of rafts in the life cycle of influenza virus. Sam recently joined the faculty in the Department of Physics and Astronomy at the University of Maine, where he now studies the dynamics of lateral organization in cell membranes, and the single molecule photophysics of GFPs, quantum dots, and fluorescent lipid analogs. The study of membrane rafts has been a strong motivation for the development of ultra-high resolution microscopy. This motivation led to Sam's development of fluorescence photoactivation localization microscopy (FPALM), which can visualize structures on length scales shorter than the diffraction limit by localizing small numbers of photoactivatable molecules at a time. Photoactivatable GFP molecules are activated, localized, and then bleached, and the process is then repeated to obtain information about a large number (>105) of molecules, whose positions can then be plotted to generate an image. | ||
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Stefan W. Hell, Max Plank Institute Far-field Fluorescence Microscopy at the Nanoscale |
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Abstract:
Since the discovery of the diffraction resolution barrier by Ernst Abbe in 1873, the spatial resolution of a focusing (far-field) light microscope has been basically limited to ~200 nm. In this lecture, we discuss physical concepts that have fundamentally broken the diffraction barrier and lead to far-field microscopy resolution at the molecular scale (< 20 nm). Special emphasis is given to STED microscopy 1 and the RESOLFT concept utilizing photoswitchable proteins and organic compounds as molecular markers. Finally, we show applications in which nanoscale fluorescence 'nanoscopy' was key to solving problems in cell biology and beyond 2,3. Stefan W. Hell (43) is a scientific member of the Max Planck Society and a director at the Max Planck Institute for Biophysical Chemistry in Göttingen, where he currently leads the Department of NanoBiophotonics. He is an honorary professor of experimental physics at the University of G??ttingen and adjunct professor of physics at the University of Heidelberg . Since 2003 he has led the High Resolution Optical Microscopy division at the German Cancer Research Center (DKFZ) in Heidelberg, and is a member of the board of directors of the Göttingen Laser Laboratory . Stefan W. Hell received his doctorate in physics from the University of Heidelberg in 1990. From 1991 to 1993 he worked at the European Molecular Biology Laboratory, also in Heidelberg, and followed with stays as a senior researcher at the University of Turku, Finland, between 1993 and 1996, and as a visiting scientist at the University of Oxford, England, in 1994. In 1996 he received his habilitation in physics from Heidelberg, where he teaches physics. In 1997 he was appointed to the Max Planck Institute for Biophysical Chemistry in Göttingen, where he has built up his current research group dedicated to sub-diffraction-resolution microscopy. In 2002, following his appointment as a director, he established the department of Nanobiophotonics. Stefan W. Hell is credited with having both conceived and validated the first viable concept for breaking Abbe's diffraction-limited resolution barrier in a light-focusing microscope. He has published more than 100 original publications in refereed journals and has received several national and international awards, including the Prize of the International Commission in Optics (2000) and the Carl Zeiss Research Award (2002). With his wife Anna he has two sons, Sebastian and Jonathan. | ||