2005 Computerworld Honors Program

	Imaging Technology Center for Light Microscope Imaging & Biotechnology Pittsburgh, PA USA Year: 1996 Status: Award Recipient Category: Science Nominating Company: Eastman Kodak Company
	Automated light microscopes acquire, process, analyze, display and archive 4-dimensional image data on the chemical dynamics that are responsible for the functioning of living cells.
	Background The cell is the basic unit of life. Hundreds of chemical reactions are performed in beautifully orchestrated processes within the living cell to produce the functions of life. Basic cell functions include the duplication of cells, locomotion, production of energy and communication within and between cells. The myriad of chemical reactions are performed with complex temporal and spatial relationships that encode the necessary information to carry-out cellular functions. Normal cells orchestrate the communication of information properly, while disease states such as cancer occur as a result of faulty "information" at the level of the archival information stored in the DNA and/or the alteration of normal chemical reactions. Light microscopes have been used by biologists and clinicians for hundreds of years to view the structures of cells and tissues and to detect some of the chemicals involved in cell functions. Until recently, the light microscope has been primarily a tool to see patterns of structures and chemicals in cells and tissues that are chemically stabilized (fixed). Although a wealth of information has been obtained over the years by studying these dead cells, the complex interplay of chemical reactions that are responsible for life have not been defined. A major step forward was made by combining electronic cameras, digital imaging technologies and robotic controls of microscope components to create a quantitative, machine vision tool to replace the qualitative human observer. Different modes of optical contrast that yield distinct structural and chemical information can be applied to the same cells to assemble detailed dynamic maps of structure and chemistry. The digital information technologies have enabled scientists to extract quantitative and dynamic information from living cells in ways not possible before. The creation of novel, fluorescent chemicals used as "sensors" of the functions of living cells has been the final component of the technologies required to fulfill the goal of investigating the dynamics of life at the cellular level. Fluorescent molecules are usually small organic compounds that have the property of absorbing light of one color and giving off another color. The properties of the fluorescent light that is emitted from these molecules, called fluorescent dyes, can vary depending on the immediate chemical environment of the dyes. It has been possible to construct fluorescent protein "biosensors" by combining the optimal fluorescent dye with specific sites on proteins. These "biosensors" can be incorporated into living cells where they report the temporal and spatial dynamics of the chemical reactions that they were designed to "sense". The automated microscopes are then used to record, analyze, display and archive the 4-dimensional information. Several colored dyes can be used in the same cell so that multiple chemical events can be correlated in time and space. A new class of "dye" has recently been defined that allows specific components to be labeled using advances in genetic engineering. This will make it even easier to construct "sensors". Objectives Our objective has been to develop and integrate the necessary technologies to create an approach (and corresponding tool) that can detect and measure the dynamic chemical events that produce cell functions that are responsible for life. A tool for investigating living cells was necessary to decipher the highly orchestrated and dynamic chemistry of life. Information technologies have been at the heart of the developments, since digital imaging through a light microscope is the key component of this methodology. Description of the Application The application is the automated light microscope used in conjunction with fluorescent "biosensors" that have been incorporated into living cells. The application has been discussed in several reviews [1, 2, 3, 4, 5]. A standard light microscope has been modified so that all of the moving parts are robotic and under computer control. Electronic cameras are used to acquire image data sets in 4 or more dimensions (3-D space, time and different modes of microscope contrast). Digital imaging technologies are used to process, analyze, display and archive the complex data sets. Specific "biosensors" are incorporated into living cells that are then investigated with the automated microscope. Our application can be considered as a functional imaging tool for chemical reactions in living cells. This application is to living cells what x-ray and nuclear magnetic resonance imaging tomography is to whole humans. Since the cell is the unit of life, we are exploring the very mechanisms responsible for life. Figure Captions (for the five slides submitted) The Multimode Microscope is our fourth generation automated microscope. It is capable of acquiring multiple modes of microscopy on live cells, including Reflection Interference Microscopy, 3-D Fluorescence Microscopy, Differential Interference Contrast-Video Enhanced Contrast Microscopy (VEC-DIC), Fluorescence Anisotropy, Fluorescence Recovery After Photobleaching, and Multispectral Fluorescence. The user in SLIDE #1 is running the microscope through the computer interface. The Multimode Microscope, together with a variety of fluorescent probes, enabled the first application of five spectrally distinct biological probes to be analyzed in living cells. SLIDE #2 demonstrates this technology by showing five distinct parameters numbered 1:nucleus, 2:actin analog, 3:endosomes, 4:mitochondria, 5:cytoplasmic volume, and a composite overlay of probes 1-4. Four parameter fluorescence imaging alone with DIC-VEC imaging, was used to correlate the spatial changes of four parameters in live fibroblasts migrating into a "wound". The arrows on the right panels indicate the direction of cell migration. Stress fibers containing actin are absent during late migration (bottom right image) and the mitochondria and endosomes maintained in the perinuclear region during early cell migration (top right image) become distributed throughout the cell after polarized locomotion was initiated. The method of ratio imaging developed by us, has been used to detect patterns of relative concentration of proteins in living cells. SLIDE #3 shows pseudocolored, ratio images of myosin II (a molecular motor) concentration during the dynamic events of cell division (red - regions of high myosin II concentration; blue - regions of low myosin II concentration). Recent results from this study revealed new mechanisms of recruitment and transport of myosin II in forming the cleavage furrow, the structure that generates force for cell division. SLIDE #4 demonstrates one of the new biosensors that has been developed and used to detect the activation of myosin II by attaching a phosphate to a specific site on this motor protein within living cells. This biosensor uses fluorescence resonance energy transfer to detect the level of myosin II phosphorylation. Ratio imaging of donor:acceptor wavelengths during the process of early cell migration detected an increasing gradient of myosin II phosphorylation (activation) (panel A) from the leading edge of the cell to the rear of the cell containing the nucleus (N). The Multimode Microscope, using a rapid acquisition program, also collects a third image in the same live cell at the same time point that can be used to create a second ratio image of the relative concentration of myosin II. The arrow indicates the direction of migration and blue = low levels of myosin II phosphorylation activation (Panel A) or low concentrations of myosin II (Panel B), red = higher levels of myosin II phosphorylation activation (Panel A) or high concentrations of myosin II (Panel B). The results demonstrated that cell locomotion involves the assembly of the myosin II motor at the leading edge, and transport of the contractile machinery into the rear of the cell where the myosin II motor is turned on and force-generating contractions occur. SLIDE #5 depicts the user interface for the fifth generation, automated microscope called the Automated Interactive Microscope (AIM). This is the main AIM window on a Silicon Graphics, Onyx computer. The right side of this window has user-friendly controls for defining the experimental parameters including optical configurations, and all optical-mechanical motion controls. On the left side of this window are two sub-windows for image display. The upper live window depicts a migrating cell whose boundaries are tracked by the "snake" algorithm. The bottom window displays multi-color, 3-D fluorescence images. In the present case, a labeled sea urchin in early development is being investigated. These image display windows can be used with our 5-D viewer tool to observe cellular dynamics. Publications 1) Taylor, D. L., M. Nederlof, F. Lanni, and A. Waggoner. 1992. The new vision of light microscopy. American Scientist 80:322-335. 2) Farkas, D.L., G. Baxter, R. DeBiasio, A. Gough, M. Nederlof, D. Pane, J. Pane, D. Patek, K. Ryan, and D. L. Taylor. 1993. Multimode light microscopy and the dynamics of molecules, cells and tissues. Annu. Rev. Physiol. 55: 785-817. 3) Taylor, D. L., R. DeBiasio, G. LaRocca, D. Pane, P. Post, J. Kolega, K. Giuliano, K. Burton, B.Gough, A. Dow, J. Yu, A. Waggoner, and D.L. Farkas.1994. Potential of machine-vision light microscopy in toxicologic pathology. Toxicologic Pathology 22: 145-159. 4) Giuliano, K. and D.L. Taylor. 1995. Light optical-based reagents for the measurement and manipulation of ions, metabolites, and macromolecules in living cells. Methods in Neuroscience 27: 1-15. 5) Giuliano, K, P. Post, K. Hahn, and D. L. Taylor. 1995. Fluorescent protein biosensors: measurement of molecular dynamics in living cells. Annu. Rev. Biophys. Biomol. Struct. 24: 405-434.
	The application has helped basic and clinical scientists begin to define the mechanisms responsible for normal cell functions, such as how cells control duplication (division), how immune cells attack "foreign cells", and how cells locomote to "fill in" wounded regions of tissue or track down and engulf bacteria and viruses. The application has also stimulated the development of new, high-speed methods for screening the effects of drugs on specific properties of living cells, as well as methods for determining the properties of cancerous cells that make them different from normal cells. How has it affected them? What are its most important benefits? The application has put a completely new tool in the hands of scientists and clinicians. Before this technology was developed, these investigators used chemically fixed, dead, cells to try to understand cellular chemistry. They then attempted to extrapolate the results to explain how cells worked. The new technology allows specific chemical reactions to be studied in living cells, while the cell is functioning. Cells can now be treated as "living test tubes". It has produced a renaissance and revolution in the use of the light microscope in biology and medicine. What impact will it have on society? The information stored in living cells is the most sophisticated information "technology" available to mankind. Cells are remarkable, "living computers". The modern revolution in the biological sciences is based on the the fact that biological information can be deciphered and manipulated at ever higher rates. Biological information falls into three general categories that represent increasing levels of complexity: 1) one-dimensional information in DNA, the digital archive of life, with a four-letter language of nucleotides; 2) the three-dimensional information of proteins, the cellular machines of life, with a twenty-letter language of amino acids; and 3) the multi-dimensional information in living cells, the orchestration of chemical reactions in time and space, with as yet only partially defined language. This last category encodes the information that will unlock the mystery of life and how we can use it. Our application is a critical tool for defining the "language" of cell functions and thus an important gateway to understanding and harnessing living cells. The application uses digital imaging information technology to access the multi-dimensional information of living cells. This knowledge will lead to improvements in human health and create new biotechnology industries. The impact has recently extended into the field of education. Together with the Carnegie Science Center, in Pittsburgh, PA, we developed a planetarium show entitled "Journey into the Living Cell". Our multi-dimensional imaging tools were used to create a group immersive visualization environment (G.I.V.E.) to take students and the general public on an interactive journey to explore the functions of cells. The dynamic chemistry of cells will be presented to more than a million people over the next year.
	The central component of this application has been the development of digital imaging tools to control the automated acquisition of image data sets and then process, analyze, archive, and visualize the chemical reactions that the "biosensors" report. We modified and extended digital imaging technologies for the specific application of studying living cells. New algorithms were created to process, analyze and display 4-dimensional data sets. A fully integrated system of automated control, image acquisition, processing, analysis, display and archiving was developed specifically for the application. In addition, algortihms used in other fields were adapted for use on dynamic image sets of cells. For example, we extended a "snake" boundary detection algorithm, originally developed for another field, to identify and track the boundaries of moving cells. We also developed a ratio imaging approach to map spectral and polarization changes of fluorescence-based sensors in living cells. This latter method converted the microscope into a dynamic spectroscopic tool for measuring changes in cell chemistry. Finally, we developed a "5-D" viewer for analyzing 3-D data sets, in time and at several colors of fluorescence. This tool allows the investigator to view the dynamic interplay of the chemical changes while measurements are made. The combination of using some existing imaging tools borrowed from other fields and developing novel algorithms created the heart of the application.
	Our application is unique and original. We were the first to integrate the development of robotic light microscopes, electronic cameras and imaging sciences with the development of very specific "biosensors" of protein functions based on fluorescence to explore the chemical and molecular dynamics of living cells. Our work has stimulated the use of this technology by an ever increasing number of scientists around the world. We continue to develop the application by pushing the performance boundaries of the computational tools, electronic imaging devices, light optical methods and fluorescence-based "biosensors". We are now building the fifth generation automated microscope using high performance computing and communication. Our present goal is to acquire, process, analyze, display and archive n-dimensional image data sets in real-time. This will open a new avenue for automated, interactive exploration of living cells by scientists. We will then be in a position to manipulate the chemical reactions that we measure, while the reactions are going on. We continue to lead in the extension of the original application. How did your application evolve? What is its background? The application evolved out of the need for biologists to detect and measure the dynamics of living cells in order to define the functions. It became clear that biochemistry, molecular biology and static, fixed cell structural approaches would not solve the problem. Therefore, the necessary technologies were integrated to build the automated light microscope. No existing tool was available to solve the problem. An interdisciplinary team of biologists, chemists, physicists, engineers and computer scientists was assembled to design, build and use the technology to first approach fundamental questions about the mechanisms of cell locomotion. Subsequently, the application has been directed toward questions in developmental biology, neuroscience and applied biotechnology, including drug screening and multicolor fluorescence-based clinical diagnostics.
	The application has exceeded its goals. The original goal was to measure the chemical dynamics of living cells in order to define the mechansim of cell locomotion. However, each of the integrated technologies have all undergone major developments that have extended the power of the application over the last five years. For example, solid state cameras replaced night vision cameras, so the quantitation of the light signals became much simpler and better. High performance computers are making it possible to perform the measurements of chemical events in real time. Finally, our development of the standing wave fluorescence microscope has improved the axial resolution of the fluorescence microscope by a factor of five to ten. Therefore, the quality and type of measurements have been dramatically better than we envisioned five years ago. In addition, the application is now being embraced by the fields of cell biology, developmental biology, neurosciences, immunology, toxicology and pathology, to name a few. Our fourth generation automated microscope is fully operational and is being used daily in biomedical investigations. In parallel, we are building the fifth generation system. It is difficult to define how many people benefit, since the numbers increase yearly. The existing generation technologies have been transferred to industry and various companies make systems and components available to the biomedical community. The plans for the future are to collaborate with a major microscope manufacturer to design the optimal robotic microscope and develop the high performance computing tools to extend the power of the application. In addition, further advances in the construction of biosensors will accelerate the impact of the application. In addition, we are beginning to collaborate with pharmaceutical companies in the design of a system specifically for high throughput drug discovery. We have also initiated studies, in collaboration with cancer biologists, to define differences between normal and cancerous cells, by imaging from the molecular to the full organism (in vivo) level.
	The most important challenge was the integration of the distinct technologies to create a user-friendly tool for biologists. In addition, the first and second generation automated microscopes were based on less than optimal electronic cameras (night vision cameras) and computers that were state of the art for the day, but under-powered for the task. Therefore, the application evolved with the improvement in electronic cameras (cooled charge-coupled devices) and high performance computing. Finally, we have been able to harness the rapid developments in the field of molecular biology to create "biosensors" based on genetic engineering. The earliest stages of the application were limited by financial resources, but sizeable grants, particularly from the National Science Foundation, accelerated our developments. The intellectual resources, including outstanding undergraduate and graduate students, have always been present. The interdisciplinary nature of the project attracted excellent faculty from the university, as well as corporate involvement. In fact, corporate scientists and engineers have played important roles in helping to solve some technical questions. The creation of the Center for Light Microscope Imaging and Biotechnology formed the organizational structure needed for an interdisciplinary program. The early stages of the program required the scientists and engineers from different fields to learn the "languages" of the other areas. This was successfully accomplished through the extensive involvement of undergraduate and graduate students who formed the heart of the effort. The students "trained" their faculty mentors and actually stimulated the involvement of faculty from different departments to serve on thesis committees. A two-day retreat one year ago cemented the communication between the fields.