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      Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

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          Abstract

          We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.

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          Most cited references 12

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          Clustered intrinsic connections in cat visual cortex.

          The intrinsic connections of the cortex have long been known to run vertically, across the cortical layers. In the present study we have found that individual neurons in the cat primary visual cortex can communicate over suprisingly long distances horizontally (up to 4 mm), in directions parallel to the cortical surface. For all of the cells having widespread projections, the collaterals within their axonal fields were distributed in repeating clusters, with an average periodicity of 1 mm. This pattern of extensive clustered projections has been revealed by combining the techniques of intracellular recording and injection of horseradish peroxidase with three-dimensional computer graphic reconstructions. The clustering pattern was most apparent when the cells were rotated to present a view parallel to the cortical surface. The pattern was observed in more than half of the pyramidal and spiny stellate cells in the cortex and was seen in all cortical layers. In our sample, cells made distant connections within their own layer and/or within another layer. The axon of one cell had clusters covering the same area in two layers, and the clusters in the deeper layer were located under those in the upper layer, suggesting a relationship between the clustering phenomenon and columnar cortical architecture. Some pyramidal cells did not project into the white matter, forming intrinsic connections exclusively. Finally, the axonal fields of all our injected cells were asymmetric, extending for greater distances along one cortical axis than along the orthogonal axis. The axons appeared to cover areas of cortex representing a larger part of the visual field than that covered by the excitatory portion of the cell's own receptive field. These connections may be used to generate larger receptive fields or to produce the inhibitory flanks in other cells' receptive fields.
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            Overview of telepathology, virtual microscopy, and whole slide imaging: prospects for the future.

            Telepathology, the practice of pathology at a long distance, has advanced continuously since 1986. Today, fourth-generation telepathology systems, so-called virtual slide telepathology systems, are being used for education applications. Both conventional and innovative surgical pathology diagnostic services are being designed and implemented as well. The technology has been commercialized by more than 30 companies in Asia, the United States, and Europe. Early adopters of telepathology have been laboratories with special challenges in providing anatomic pathology services, ranging from the need to provide anatomic pathology services at great distances to the use of the technology to increase efficiency of services between hospitals less than a mile apart. As to what often happens in medicine, early adopters of new technologies are professionals who create model programs that are successful and then stimulate the creation of infrastructure (ie, reimbursement, telecommunications, information technologies, and so on) that forms the platforms for entry of later, mainstream, adopters. The trend at medical schools, in the United States, is to go entirely digital for their pathology courses, discarding their student light microscopes, and building virtual slide laboratories. This may create a generation of pathology trainees who prefer digital pathology imaging over the traditional hands-on light microscopy. The creation of standards for virtual slide telepathology is early in its development but accelerating. The field of telepathology has now reached a tipping point at which major corporations now investing in the technology will insist that standards be created for pathology digital imaging as a value added business proposition. A key to success in teleradiology, already a growth industry, has been the implementation of standards for digital radiology imaging. Telepathology is already the enabling technology for new, innovative laboratory services. Examples include STAT QA surgical pathology second opinions at a distance and a telehealth-enabled rapid breast care service. The innovative bundling of telemammography, telepathology, and teleoncology services may represent a new paradigm in breast care that helps address the serious issue of fragmentation of breast cancer care in the United States and elsewhere. Legal and regulatory issues in telepathology are being addressed and are regarded as a potential catalyst for the next wave of telepathology advances, applications, and implementations.
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              A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

               ,  ,   (2009)
              In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                8 March 2013
                : 8
                : 3
                Affiliations
                [1 ]Olympus America, Center Valley, Pennsylvania, United States of America
                [2 ]Olympus Corporation, Hachioji, Tokyo, Japan
                [3 ]Allen Institute for Brain Science, Seattle, Washington, United States of America
                [4 ]David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America
                [5 ]Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, California, United States of America
                [6 ]Olympus Soft Imaging Solutions GmbH, Münster, Germany
                Emory University, United States of America
                Author notes

                Competing Interests: Olympus America Inc. provided microscope equipment but was not a funder of, and did not influence the outcome of, the study. The VS120 WSI scanning system was used. Yoshihiro Kawano and Christopher Higgins are employed by Olympus America, Yasuhito Yamamoto by Olympus Corporation and Tobias Schilling by Olympus Soft Imaging Solutions GmbH. Thomas Geer of Olympus America provided advice and counsel and Tatsuo Nirei of Olympus Engineering prepared the prototype stage modifications used in this study. Lorne D. Davies, Olympus America Inc. and I have filed a US patent application entitled: WIDE FIELD MICROSCOPIC IMAGING SYSTEM AND METHOD (publication number US2012/0092477 published on April 19 2012, date of filing October 18, 2010). Olympus Corporation introduced a darkfield illuminator in October 2012. However, this commercially available product is not the same as our prototype. There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

                Conceived and designed the experiments: YK CH YY HK. Performed the experiments: YK CH JN. Analyzed the data: YK CH AB HWD HK. Contributed reagents/materials/analysis tools: YK CH HK AB HWD HK TS. Wrote the paper: YK CH HK AB HWD HK TS.

                Article
                PONE-D-12-27242
                10.1371/journal.pone.0058344
                3592912
                23520500

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 12
                Funding
                No current external funding sources for this study.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Neurological System
                Neuroanatomy
                Biotechnology
                Bioengineering
                Computational Biology
                Computational Neuroscience
                Histology
                Immunology
                Immunologic Techniques
                Immunofluorescence
                Neuroscience
                Computational Neuroscience
                Molecular Neuroscience
                Engineering
                Signal Processing
                Image Processing
                Physics
                Condensed-Matter Physics
                Optics
                Electromagnetic Radiation
                Visible Light
                Science Policy
                Technology Development

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