Abstract
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. We describe the first application of multicolor ribbon scanning confocal methods to collect high-resolution volume images of chemically cleared brains. We demonstrate that ribbon scanning collects images over ten times faster than conventional high speed confocal systems but with equivalent spectral and spatial resolution. 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. 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. Visualization of this fundamental impact of infection would not be possible without sampling at subcellular resolution within large brain volumes.
Highlights
Until recently, understanding cellular interaction(s) and connectivity has been hampered by an inability to contextualize interactions within the framework of the whole tissue
We find that the sensitivity and three axis resolution of the device allows us to detect single infected neurons within an entire mouse brain following exposure to the encephalitic Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses
Traditional large area mosaic imaging systems utilize a ‘stop-and-shoot’ modality that requires that sample stage to be precisely positioned for each tile
Summary
Until recently, understanding cellular interaction(s) and connectivity has been hampered by an inability to contextualize interactions within the framework of the whole tissue. Images were generally collected as “representative” snapshots defined by the observer. This is evident within the study of the central nervous system, which is a very large, very complex, integrated, yet compartmentalized network of cells and vasculature. 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.
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