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Generally, a relatively low resolution overview is acquired which is used to guide collection of a higher resolution subimage, with a different objective or intermediate zoom optics (for example the LaVision system). Light sheet methods, in which the sample is illuminated laterally with a thin sheet(s) of light, allows rapid collection using sCMOS detectors. To satisfy Nyquist sampling, as resolution increases, the number of fields are essentially cubed according to the change in magnification and Z sampling, compounding the time to acquire high-resolution volumes. Commonly, complex multipanel images are stitched on 3 axes which takes significant time following collection. Even when utilizing confocals with fast resonant scan heads the process remains slow and takes 2–5 seconds between positions.
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Once cleared, the most time limiting step to collecting high resolution confocal images has been the need to collect single two dimensional snapshots using a stop-and-shoot approach in which the stage is moved, the scanning is initiated, the image is collected and the process is repeated followed by image stitching. Recent advances in tissue clearing approaches (Sca/e, CLARITY, CUBIC and DISCO (3, i, u) have allowed imaging at great depths, making the third dimension obtainable and enabling imaging of whole brains. At every level of resolution, from the single neuronal synapse to the anatomically isolated but interconnected functional compartment, there is a fundamental need to define complexity as a continuum such that cell development, position, interaction, and death are understood within the context of the neighboring cells, and vasculature. This is particularly evident within the study of the central nervous system, which is a very large, very complex, integrated, yet compartmentalized network of cells and vasculature. Images were generally collected as “representative” snapshots defined by the observer. Until recently, understanding cellular interaction(s) and connectivity has been hampered by an inability to contextualize interactions within the framework of the whole tissue. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. 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.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported by grants from the: Defense Threat Reduction Agency, HDTRA1-15-1-0047 NIAID, R01 AI09543605 NINDS, R01 NS24328, P30 NS076405 and NIH Office of the Director, P40 OD010996. Received: MaAccepted: JPublished: July 7, 2017Ĭopyright: © 2017 Watson et al. PLoS ONE 12(7):Įditor: Thomas Abraham, Pennsylvania State Hershey College of Medicine, UNITED STATES (2017) Ribbon scanning confocal for high-speed high-resolution volume imaging of brain. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.Ĭitation: Watson AM, Rose AH, Gibson GA, Gardner CL, Sun C, Reed DS, et al. This reveals that the destruction or collapse of large regions of brain micro vasculature may contribute to the severe disease caused by Venezuelan equine encephalitis virus.
![r multipanel raser avoid overlap r multipanel raser avoid overlap](https://i.stack.imgur.com/Ezim9.png)
Further, using this technology, we reconstruct large volumes of mouse brain infected with encephalitic alphaviruses and demonstrate that regions of the brain with abundant viral replication were inaccessible to vascular perfusion.
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We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. Whole-brain imaging is becoming a fundamental means of experimental insight however, achieving subcellular resolution imagery in a reasonable time window has not been possible.