Detailed circuit maps of neural networks and their synaptic connections between/among neurons of the central nervous system (CNS) are needed to advance CNS studies. This need is particularly acute in the emerging field of "connectomics" that requires a very large number of high-resolution three-dimensional microscopy wide-field images to visualize and analyze CNS neural circuitry. Instruments such as Focused Ion Beam Serial Block Face Scanning Electron Microscopy (FIB-SBFSEM) and serial section transmission electron microscopy (ssTEM) achieve high-resolution three-dimensional images and provide the resolution required to trace fine dendrites and synapses. However, Serial Block Face Scanning Electron Microscopy (SBFSEM) is the only three-dimensional microscopy technique that can provide wide-field images. A single instrument that provides high-resolution three-dimensional wide-field images is acutely needed. The principal investigator (PI) in concert with JEOL Inc. will take the first crucial step to meet that need by improving a thru-the-lens (TTL) backscatter detector that will increase the backscattered electron image (BEI) resolution and increase image acquisition speed. Intellectual merit: Improvements in the TTL backscatter detector will greatly advance neuroscientists' ability to 1) identify specific spinal neuron phenotypes and 2) gain insights into the 'impenetrable jungles' of central nervous system regions, thus overcoming obstacles identified over 100 years ago. Broader Impacts: TTL backscatter detector improvements will: 1) permit mapping of basic synaptic microcircuits of some regions of the central nervous system; 2) be used to train next generation Hispanic scientists in the emerging field of connectomics; 3) meet 30 Puerto Rican neuroscientists' need for access to instrumentation; upon successful completion of the beta test and with development and marketing, the instrument will 4) meet the need of many, many neuroscientists engaged in connectivity and other CNS studies. Dissemination: The PI will offer Hispanic neurobiology graduate students and 30 neurobiology faculty members training on the new instrument. The PI's laboratory provides its research images to industry which uses them in its brochures, appropriately crediting the PI and funding agents. This arrangement will continue and expand. The TTL backscatter detector will create opportunities to exploit stunning visual images as a way of enhancing awareness of new developments in science. Towards this goal we will: 1) use images generated from these projects as artwork in UPR-RP class and conference rooms; 2) submit articles to the UPR-RP alumni magazine and to Inventio, a bi-lingual campus publication that presents research and scholarly achievements to the larger business and industry community and to the general public; and 3) use the images to persuade local newspapers and magazines to do more stories on basic science. We hope to use riveting and beautiful images to capture the imagination and interest of the lay public. Finally, through a collaboration with the mouse Biomedical Imaging Research Network (mouse BIRN) in the Laboratory of Neuro Imaging (LONI) at University of California Los Angeles (UCLA), we use the Mouse BIRN Atlasing Toolkit (MBAT), a platform independent image viewer with the ability to simultaneously present multiple images from multiples sources on a single viewing "canvas". With MBAT, local and online images can be compared and explored side-by-side. MBAT allows almost anyone with a modest personal computer and internet connection to view virtual sections of our image data on our website (

Project Report

14.00 Normal 0 false false false EN-US X-NONE X-NONE Photon-based and electron-based serial block-face imaging technologies allow the automated generation of three-dimensional (3D) nanoscale images and the automated analysis of the resulting teravoxel or petavoxel data sets. These imaging technologies have great promise in realizing the full potential of "connectomics and synaptomics", two emerging fields in neuroscience defined by the high-throughput generation of data sets about neural connectivity and the subsequent mining of those data sets. However, existing instrumentation, reagents, and approaches do not adequately overcome problems associated with using electrons as the imaging light source. Charging, radiation/heat damage, ion beam- and electron-induced surface contamination/artifacts, sectioning/ion beam milling artifacts, chemical contrast, and electron diffusion continue to hinder scientists in achieving the consistent image quality and resolution required to fulfill electron-based serial block-face imaging technologies’ potential to advance circuit neuroscience. Thus, without new instrumentation, reagents, and approaches neither the promise of new imaging technologies nor the potential of connectomics to advance circuit neuroscience will be realized. Intellectual merit: The goal of the project was to improve the electron yield in the through-the-lens-detector (TLD) by applying an electric potential to the sample stage to produce an electrostatic lens to help focus the lower energy back-scattered electrons into the TLD. This improvement reduced the electron beam energy and improved imaging parameters such as resolution and contrast. This improvement and newly developed reagents and methods have greatly advanced our ability to: 1) identify specific spinal neuron phenotypes and 2) gain insights into the "impenetrable jungles" of central nervous system regions, thus overcoming some obstacles identified over 100 years ago that hinder achievement of the goals of initiatives such as the "Brain Research through Advancing Innovative Neurotechnologies". Broader Impacts: The developments achieved here have allowed us to begin: 1) mapping the basic synaptic microcircuits of a simple and unique spinal motor circuit we use as a testbed for developing and testing technology described in the project proposal; 2) training a small cadre of next generation Hispanic interdisciplinary scientists in the emerging field of connectomics/synaptomics; 3) collaborating with industry to address critical barriers for which funding is scarce, i.e., the development of reagents and technology, and 4) meeting the need of many, many neuroscientists engaged in connectivity studies.

National Science Foundation (NSF)
Division of Biological Infrastructure (DBI)
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Joyce Fernandes
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University of Puerto Rico-Rio Piedras
San Juan
United States
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