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13Front cover image legend:1. Axonal growth cone of a cortical neuron treated with Netrin-1and labeled with Alexa 488 phalloidin to show actin filamentsin red and anti-tubulin antibodies to show microtubules ingreen. Netrin—1 increases filamentous actin within 30 minutes.Image courtesy of Katherine Kalil.2. A living epithelial cell expressing activated Rac1 GTPase. Thecell was injected with fluorescent tubulin and analyzed bymulti-mode DIC and fluorescence microscopy. Rac 1 activityinduces extensive microtubule growth.Image courtesy of Torsten Wittmann and Clare Waterman-Storer.3. Xenopus spinal cord explant at interface between two matrixproteins, laminin and tenascin. Red shows actin filaments labeledwith Alexa 546 phalloidin and green shows immunocytochemicallabelling of phosphotyrosine. Blue represents highestdensity of deposited laminin.Image courtesy of Timothy Gomez.4. Multicolor labeling of neurons in a mouse cortical slice usingthe "DiOlistic" technique.Image courtesy of Wen-Biao Gan.5. An OCT image of a nail fold.Image courtesy of Johannes de Boer.726546. Dual-mode non-linear optical image of the head of an adult C.elegans. A single ultrafast laser excites both second harmonicgeneration (violet) from contractile muscles and fluorescence(green) from GFP-labeled neurons and autofluorescent granuleswithin the gut.Image courtesy of William Mohler.7. Live CHO cells were labeled with QdotTM 565 (green), 605(orange) and 655 (red) Streptavidin Conjugates. The cells weretreated with a cell-surface biotinyllation reagent before beingincubated with each QdotTM Streptavidin Conjugate. Then thecells labeled in different colors were mixed and examined undera epi-fluorescence microscope.Image courtesy of Xingyong Wu, Quantum Dot Corporation.

2nd Symposium onBiological ImagingNew Dimensions in In Vivo ImagingPromega’s Biopharmaceutical Technology CenterMadison, WIFriday, May 30, 2003Table Of ContentsProgram Introduction Page 1Schedule of Events—Morning Session Page 2Schedule of Events—Afternoon Session Page 3Schedule of Events—Evening Activities Page 4Workshop Abstracts:Nathan O’Connor, Universal Imaging Page 5Kevin Eliceiri and LOCI Group Page 6Poster Abstracts Page 7Lecture Abstracts:Dr. William Mohler Page 11Dr. Johannes de Boer Page 12Dr. Enrico Gratton Page 13Dr. Clare Waterman-Storer Page 14Dr. Marcel Bruchez Page 15Dr. Georgyi Los Page 16Dr. Wen-Biao Gan Page 17Keynote Speaker Abstract:Dr. Mark Ellisman Page 18Vendors Page 19Sponsors Page 20Websites Page 21Notes Page 22

VWelcome to the 2nd Symposium onBiological ImagingNew Dimensions in In Vivo ImagingProgram Introductionisualizing the structure of living organisms has been central to biological studies from theirbeginning, with the goal of explaining the relationship between form and function. However,living organisms are dynamic structures, and in order to understand fundamental biologicalprocesses such as the change in the conformation of a protein, the movement and division of cells,the development of an embryo, or the integrated neural activity in an intact brain, the visualizationof structures in three dimensions that change over time is necessary. Until the advent of modernbiological imaging technology it was not possible to observe directly the molecular and cellularinteractions that underlie essential functions within cells and tissues. Such interactions could only beinferred from fixed specimens. Now, however, with the aid of probes that label intracellularstructures and new imaging devices such as multiphoton microscopy, biologists can observechanges in form related to function directly in living cells and tissues.The current explosion in our knowledge of dynamic events in molecular living organisms hasspurred a renaissance in the use and development of new imaging technology for studying live cellsand tissues. Today, probes can be made that will allow the spatial visualization of expressed geneproducts in a cell or tissue. At the single cell level, biologists can observe the dynamic movements ofintracellular organelles or single vesicles at functioning neuronal synapses. Changes in thecytoskeleton of cells can be visualized directly as they occur when cells divide, migrate or executespecific functions according to their differentiated state. In tissues, the behavior of ensembles of cellsduring fundamental events in development such as gastrulation, neurulation, organogenesis, axonpathfinding and target identification, and synaptic rearrangement can be captured in real time.The symposium will highlight recent developments in in vivo imaging. Lectures and posterpresentations will demonstrate how new imaging tools can be applied to solve a variety of biologicalproblems, ranging across scale from protein structure to the intact brain. Topics covered will include:second harmonic imaging, optical coherence tomography, fluorescence correlation spectroscopy,speckle microscopy, nanocrystal labeling, small molecule reporters, and in vivo two-photonmicroscopy. In addition, interactive workshops on automated image analysis and on software forbiological visualization will be presented.1

Biological ImagingNew Dimensions in In Vivo ImagingSchedule of EventsMorning Session7:45-8:30AM8:30AM8:45-10:45AMRegistration and Continental Breakfast-AtriumWelcome by Ron Kalil, University of Wisconsin-Madison andBob Bulleit, Promega Corporation-AuditoriumWorkshopsAutomated Methods for Light Microscopic Imaging and AnalysisNathan O’Connor, Universal Imaging CorporationRoom 236, two 1-hour sessionsFreeware and Open-Source Software Tools for Biological VisualizationKevin Eliceiri and LOCI Group, University of Wisconsin-MadisonRooms 216/217, two 1-hour sessions10:45-11:30AM11:30-12:30PMPoster Session and Vendor DisplaysLunch-Served buffet style in Atrium. Seating is available in Rooms104/105, 219, 221, 226/227, Cafeteria, or on the terrace (weather permitting).Seating areas are designated by posted door signage.Schedule continued on next page2

RBiological ImagingNew Dimensions in In Vivo ImagingWorkshop AbstractFreeware and Open-Source Software Tools for Biological VisualizationKevin Eliceiri and LOCI Group, University of Wisconsin-MadisonMadison, WIRooms 216/217, two 1-hour sessions8:45-9:45AM and9:45-10:45AMecent developments in non-invasive medical imaging and in vital biological microscopy haveyielded a new form of massive data type - the multichannel, three-dimensional, time-courseimage recording. These multidimensional data sets document the dynamic changes within the fullvolume of a specimen over time, often simultaneously monitoring several different parameters.Examples include time-lapse, three-dimensional, light microscope image recordings and functionalMRI recordings. While the imaging modalities used to generate the data differ greatly, they share thecommon attribute of being multidimensional and are usually represented by large data archives. Tomeet the computational challenge of the effective analysis of these data, several groups, includingours, are developing software visualization tools. Many of these tools are open-source applicationsthat may be readily adapted to different and emerging file formats, and may readily adopt testedsolutions from the graphics programming community. In this workshop we will present and reviewseveral freeware and open-source tools that facilitate the incorporation of new features—demandedby new avenues of biological research—that are beyond the current scope of commercial products.6

Figure 8: Result from the different cameras in the virtual setupFigure 9: Camera positions for virtual frameworkwith a image correlation algorithm. The correlation gives us a value between the rangeof 0 and 1 where 0 is a bad results and 1 is the groundtruth. The table 1 presents thecorrelation values obtained for different camera position, and the figure 1 shows the 3Dmesh generated.6 CONCLUSIONS AND FUTURE WORKWe present a methodology for face reconstruction in a mixed enviroment of activepasivesetup. Structured light shows a quality improvement against the results obtainedwith pasive setups. Time multiplexing codification has the problem of motion betweenthe captured images generating a waving efect in the reconstructions. Even robustalgorithms of point matching for dense depthmaps were tested there were no real improvementin the results. We will try with color or 2D patterns which only requieres11

OBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractOptical Coherence Tomography, An Emerging Imaging Technique in MedicineJohannes de BoerAssistant Professor, Harvard Medical SchoolWellman Laboratories of Photomedicine, Massachusetts General HospitalBoston, MAAuditorium1:10-1:50PMptical Coherence Tomography (OCT) is an optical imaging technique that provides crosssectional images of tissue. The technique is analogous to ultrasound, with better resolution butpoorer depth penetration. The principles of this technique will be explained, as well as functionalextensions such as Doppler OCT, which provides flow sensitivity, and Polarization Sensitive OCT(PS-OCT), which provides sensitivity to polarization properties of tissue. Advancement incomputational power allows for real time acquisition and display of Multi Functional OCT signals,and current state of the art imaging will be demonstrated. Recent results in application of thetechnique in assessing the depth of thermal injury for burn victims, screening for Basal CellCarcinoma and imaging of the retinal nerve fiber layer in ophthalmology for the early detection ofGlaucoma will be discussed. A future outlook for the technology will be given.12

FBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractScanning FCS: Measuring Spatio-Temporal Correlations in the Millisecond Range inLive CellsDr. Enrico GrattonLaboratory of Fluorescence DynamicsUniversity of Illinois at Urbana-ChampaignAuditorium1:50-2:30PMluctuation correlation spectroscopy has become a commonly used methodology to studymolecular processes in solutions and inside cells. Cross-correlation and multichannelcorrelation is also being developed in several laboratories. An important aspect of correlation whichhas not been widely exploited is the capability to detect events which occur either simultaneously orin a given statistical sequence in different spatial locations. Image correlation spectroscopy has inprinciple the capability to provide both spatial and temporal correlations. However, the commonlyused methods used for image acquisition are relatively slow and do not allow the measurement offast correlations, typically in the millisecond scale which are typical of protein diffusion in thecytoplasm of live cells. In this work we describe a variant of the line technique, scanning FCS thatprovides millisecond time resolution in a relatively large area. This technique can provide themobile and immobile fraction of molecules in a region of the cell as well as information on localvelocity gradients.13

FBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractQuantitative Fluorescent Speckle Microscopy in Studies of the Cytoskeleton inCell MigrationDr. Clare Waterman-StorerThe Scripps Research InstituteLa Jolla, CAAuditorium2:50-3:30PMluorescent Speckle Microscopy (FSM) is a technology for analyzing the dynamics ofmacromolecular assemblies. Originally, the effect of random fluorescent speckle formation wasdiscovered with the microtubule cytoskeletal polymer. Since then, the method has been expanded toother proteins of the cytoskeleton such as f-actin and microtubule binding proteins. Newlydeveloped, specialized software for analyzing speckle movement and photometric fluctuation in thecontext of polymer transport and turnover has turned FSM into a powerful method for the study ofcytoskeletal dynamics in cell migration, division, morphogenesis, and neuronal path finding. In allthese settings FSM serves as the quantitative readout to link molecular and genetic interventions tocomplete maps of the cytoskeleton dynamics and thus can be used for the systematic deciphering ofmolecular regulation of the cytoskeleton. We envision that FSM has the potential to become a coretool in automated, cell-based molecular diagnostics in cases where variations in cytoskeletaldynamics are a sensitive signal for the state of a disease, or the activity of a molecular perturbant. Iwill review the origins of FSM, discuss the most recent technical developments and give a glimpse tofuture directions and potentials of FSM.14

SBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractQdot TM Conjugates in Biological ImagingDr. Marcel BruchezPrincipal ScientistQuantum Dot CorporationHayward, CAAuditorium3:30-4:10PMemiconductor quantum dots are emerging as a powerful new tool for fluorescence detection inbiological systems. As a material, these quantum dots have large absorbance cross-sections,high quantum yields, and very narrow and tunable emission spectra, all of which translate to highlysensitive multiplexed detection in biological systems. Using a range of sizes of CdSe materials, wehave prepared quantum dot probes that span the visible wavelength range from blue-green to red.To date, though there have not been robust, simple chemistries that are available for routinemodification of the quantum dots to alter their physical, chemical, or biological properties. We havedeveloped new chemistries based on amphiphilic polymers for functionalizing these nanoparticlesthat are simple, stable and maintain the underlying optical properties. These materials serve as thestarting materials for further refinement and optimization of the colloid and biological properties ofthese materials. We have reduced the non-specific binding of these materials in cellular assays withboth live and fixed cells by chemically modifying the surface with a variety of functional groups.We have used these materials with reduced nonspecific binding to prepare conjugated probes ofbio-molecules with dramatically improved performance in cellular imaging and in-vivo imaging ofisolated cells and in live animals. The performance advantages of these materials in biologicaldetection and multiplexing will be highlighted.15

WBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractSite-Specific Localization of Small Molecule Reporters Within Living CellsDr. Georgyi LosCellular Analysis, Promega CorporationMadison, WIAuditorium4:10-4:50PMith the completion of genome sequencing efforts it is now important to understand how theproducts of these genes function, and most importantly how they function within living cellsto dictate the biology of organisms. The ability to specifically label proteins can help revealinformation about protein functions and dynamics within the complex biochemical environment ofthe living cells. Here we describe a novel technology for covalently tethering functional groups[(FG), e.g. fluorescent dye] to a universal reporting protein (URP) within cells. The URP is a mutantenzyme capable of forming a stable covalent bond to a modified substrate coupled to the functionalgroup. In our initial approach the URP is a halo-alkane dehalogenase from Rhodococcus rhodochrous(DhaA) with a mutation in a critical active site residue. The activity of DhaA cleaves carbon-halogenbonds in aliphatic and aromatic halogenated compounds involving a typical hydrolytic triad. In thisreaction an enzyme-substrate complex is formed by a nucleophilic attack involving Asp106 and theformation of an ester intermediate; His272 activates H 2 0 that hydrolyzes this intermediate releasingproduct from the catalytic center. A point mutation in DhaA involving a substitution ofphenylalanine for His272 impairs the hydrolysis step leading to a stable covalent intermediate withsubstrate and any conjugated FG. This technology allows the labeling of mutant DhaA or mutantDhaA fusion proteins expressed in cells. A significant advantage of this approach is the flexibility tocreate labeled proteins with a potentially wide range of optical properties or other functionalities.The technology can be applied in different cells and organisms allowing a variety of experimentalapproaches to study protein function in living cells.16

TBiological ImagingNew Dimensions in In Vivo ImagingLecture AbstractIn Vivo Two-Photon MicroscopyDr. Wen-Biao GanSkirball Institute, New York University School of MedicineNew York, NYAuditorium4:50-5:30PMhe invention of two-photon microscopy has dramatically enhanced the field of laser scanningmicroscopy. Unlike single-photon excitation used in confocal microscopy, two-photonexcitation depends on nearly simultaneous absorption of two photons by a single fluorophore. Eachphoton used for excitation in two-photon microscopy has approximately half the energy (or twicethe wavelength) of the one in confocal microscopy. Due to the use of longer wavelength light andthe occurrence of two-photon excitation only at the focal point, two-photon microscopy allowsincreased tissue penetration, reduced photo-damage and more efficient fluorescence detection incomparison with confocal microscopy. These advantages are especially important for monitoringthick living tissues because multiple exposures often cause significant photo-toxicity. Asdemonstrated in many recent studies, two-photon microcopy excels in imaging thick biologicalspecimens such as living brains and other intact tissues.We have recently developed a transcranial two-photon imaging technique to study long-termchanges of synaptic structures in transgenic mice expressing Yellow Fluorescent Protein (YFP). Byfollowing identified dendritic spines of pyramidal neurons in the primary visual cortex, we foundthat the overwhelming majority of spines (~96%) remain stable over a one-month interval, with ahalf-life greater than 13 months in adulthood. These results indicate that dendritic spines areremarkably stable in the adult, providing a potential structural basis for long-term informationstorage. In combination with the generation of transgenic animals over expressing fluorescentmarkers, in vivo two-photon microscopy opens exciting new avenues for studying not only basicbrain function, but also long-term changes seen in neurodegenerative diseases such as Alzheimer’sdisease.17

TBiological ImagingNew Dimensions in In Vivo ImagingKeynote Speaker AbstractMulti-Scale Structure and Function in the Nervous SystemDr. Mark EllismanProfessor of Neurosciences and BioengineeringDirector, National Center for Microscopy and Imaging ResearchUniversity of California, San DiegoLa Jolla, CAAuditorium7:30-9:00PMhe activities of the National Center for Microscopy and Imaging Research in San Diego in thedevelopment of novel techniques for 3 dimensional visualization of neuronal structures anddirect identification of their protein constituents in situ will be described. This includes a newtechnique for correlated 4 dimensional light and 3 dimensional electron microscopy. A continuingchallenge to neuroscientists and biomedical researchers in general is the understanding of structureson the scale of 1 nm3 to 10's of µm3, a dimensional range that encompasses macromolecularcomplexes, organelles, and multi-component structures like synapses and the cellular interactionswithin tissues. On the smaller end of this range, structures have traditionally been difficult to studybecause they fall in the resolution gap between technologies, such as X-ray crystallography andelectron microscopy on the lower end, and it is challenging to make correlations between 3D datafrom light and electron microscopy on the upper end. But the continuum of information will have tobe mapped if the results of the molecular revolution, the protein products of sequenced genomes,are to be situated in their proper subcellular, cellular and tissue contexts. The technique of electrontomography, the derivation of 3D structure from a series of 2D electron microscopic images, hasgone a long way towards bridging the resolution gaps. While electron tomography is a useful technique for providing information in this spatial domain, development of new methods to provideselective contrast to molecular constituents, organelle complexes, cells and cell processes arerequired. We recently developed a new molecular-tagging technique to chronicle the development,movement and interactions of proteins as they do their work in living cells. [Gaietta et al., ScienceApril 19, 2002]. This multi-scale molecular tagging technology involves the genetic tagging of aprotein with a small binding area (a tetracystine domain between six to 20 amino-acids long), thatthen interacts with a variety of other compounds. Results with two of these compounds, greenFlAsH and red ReAsH, have been used in sequence to provide the ability to monitor the traffickingof proteins in cells. The red label, ReAsh, can then be used to generate reactive oxygen and drive thedeposition of diamino benzadine for direct EM-level detection of the same protein complex. The useof this new labeling method, other multi-scale staining techniques and new advanced imaginginstruments will be described in the context of work create a Biomedical Informatics ResearchNetwork (BIRN). BIRN is a leading example of a virtual distributed database effort that is federatingmultiscale data about the nervous systems.18

Biological ImagingNew Dimensions in In Vivo ImagingVendorsCarl Zeiss, Inc., LSM 5 Pascal Microscope (Room 215)The LSM 5 PASCAL is a confocal laser scanning microscope for basic research in medicine and biology.Equipped with optimized, user friendly hardware and software, this system delivers excellent two– andthree-dimensional images, especially in fluorescence applications. It is ideal for the acquisition of time series inphysiological experiments as well as for quantitative measurements.Leica Microsystems, TCS SP2 AOBS Microscope (Room 122)The Leica TCS SP2 AOBS integrates multiple innovations of Leica Microsystems R & D into a research systemthat sets new standards for efficient protection of live cells and flexibility. Designed as a system in harmony itbegins with special "confocal" objectives, versatile microscope stands and diffraction limited optics;preconditions for outstanding image quality and a must for meaningful results in live cell research. Milestonesin optical innovation include our multi-laser merge module with programmable control of excitationwavelengths and intensity, beam scanning with our proprietary K scanner and our highly efficient andselective SP prism spectrophotometer for detection of fluorescence emission.Mad City Labs (Room 122)Mad City Labs, Inc is the leading American manufacturer of nanopositioning systems with sub-nanometerprecision at an affordable price. We have the largest product line of nanopositioning systems available andspecialize in custom nanopositioner applications. We deliver the tools for nanotechnology in 30 to 45 days, andprovide the highest level of customer service and satisfaction in the industry. Applications for nanopositionersinclude AFM, NSOM, Scanned Probe Microscopy, fiber positioning, interferometry, single moleculespectroscopy and lithography.Nikon C1 Confocal Microscope System (Room 122)A universal confocal microscope system that is ultra-compact and lightweight yet provides confocal images ofthe highest quality in its class. Highest optical performance; 4-channel simultaneous detection; Four positionpinhole turret; 12 bit imaging; Optical fiber coupling; Intuitive software; Advanced acquisition and analysisfeatures; 50 nm steps along the Z axis; CF160 optical system; Advanced accessories and various lasercombinations are available.The Fryer Company, (Room 122) located in Huntley, Illinois, is one of the largest distributors of NikonBiomedical and Industrial microscopes in the United States. Over the past 40 years, Fryer has become a leadingforce in marketing innovative imaging products throughout the medical, industrial and research fields.19

Biological ImagingNew Dimensions in In Vivo ImagingSponsorsBio-Rad Laboratories, Inc. is a multinational manufacturer and distributor of life scienceresearch products and clinical diagnostics. Its Cell Science Division manufactures a range of laser-microscopeproducts. These include confocal and multi-photon laser scanning imaging systems (Radiance2100) and cellisolation systems (Clonis). The latest version of the Radiance confocal imaging system - the 'Radiance Rainbow'adds spectral discrimination and the ability to reassign (unmix) the emission from overlapping fluorophores.20

Bio-Rad Laboratories—microscopy.bio-rad.comCarl Zeiss, Inc.—zeiss.comLeica Microsystems—leica-microscopy.comLOCI Group—loci.wisc.eduMad City Labs —madcitylabs.comBiological ImagingNew Dimensions in In Vivo ImagingWebsites Associates with this SymposiumNational Center for Microscopy and Imaging Research—ncmir.ucsd.eduNew York University School of Medicine—med.nyu.eduNikon—microscopyU.comPromega Corporation—promega.comPromega R&D—promega-rd.infoQuantum Dot Corporation—qdots.comScripps Research Institute—scripps.eduThe Fryer Company—fryerco.comUniversal Imaging—image1.comUniversity of Connecticut Health Center—uchc.eduUniversity of Illinois at Urbana-Champaign—uiuc.eduUniversity of Wisconsin-Madison—wisc.eduW.M. Keck Laboratory—keck.bioimaging.wisc.eduWellman Laboratory of Photomedicine—mgh.harvard.edu/wellmanSecond Symposium on Biological Imaging—promega-rd.info/bioimage2003/default.asp21

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