Research Article: Ribbon scanning confocal for high-speed high-resolution volume imaging of brain

Date Published: July 7, 2017

Publisher: Public Library of Science

Author(s): Alan M. Watson, Annika H. Rose, Gregory A. Gibson, Christina L. Gardner, Chengqun Sun, Douglas S. Reed, L. K. Metthew Lam, Claudette M. St. Croix, Peter L. Strick, William B. Klimstra, Simon C. Watkins, Thomas Abraham.

http://doi.org/10.1371/journal.pone.0180486

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.

Partial Text

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 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. 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. Recent advances in tissue clearing approaches (Sca/e[1], CLARITY[2], CUBIC[3] and DISCO (3[4], i[5], u[6]) have allowed imaging at great depths, making the third dimension obtainable and enabling imaging of whole brains.

Here we demonstrate the potential of ribbon scanning confocal microscopy to rapidly image large-area tissue sections of marmoset brains and reconstruct large-volumes of chemically cleared mouse brains at subcellular resolution. This approach increases the speed of large-area / large-volume confocal microscopy by at least a factor of 10 when compared to commercially available systems. When paired with chemical clearing of tissues, the approach makes the collection of truly high-resolution large-volumes feasible. The novel combination of chemically cleared brains, confocal ribbon scanning and high-NA long working distance objectives allowed us to sample one-half of a brain in nine hours. We also demonstrate a novel use for this technology to study the pathogenesis of encephalitic alphaviruses. We observed the breakdown or occlusion of microvasculature in large regions of the brain associated with advanced virus replication, an observation that would not have been feasible using traditional confocal imaging approaches. We suggest that the speed and utility of this approach (when applied to the brain and other organs) makes it broadly applicable to the study of infectious disease, gene expression, developmental biology and neurobiology.

 

Source:

http://doi.org/10.1371/journal.pone.0180486

 

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