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NeuroZoom™: Figure 1A shows the basic microscope instrument setup that is controlled with NeuroZoom™ software. Up to eight standard glass slides are mounted on the motorized microscope stage. Each slide holds 5-8 cross sections of mouse brain tissue. The slides are automatically analyzed for the location of every section. The sections are then automatically imaged at very high resolution directly into Neurome's database. Figure 1B shows the individual tiles that are imaged as they correspond to each microscope field of view. The tiles are reassembled automatically into a single large-scale mosaic (Figure 1C). Finally, NeuroZoom™ aligns the sections by rotating and translating them into 3D registration to produce a single 3D dataset of the mouse brain.

NeuroZoom™: Very high resolution images are stored in Neurome's database by accurately controlling microscope instrumentation and using sophisticated techniques to stitch the images back together as a complete mosaic. The images available for viewing in the atlas module of Neurome's NeuroPortal™ viewing and access application ranges from macroscopic views of the entire section, to regions of cells, and finally to cellular and even subcellular levels of resolution.

BrainPrint™: 3D visualization of gene expression data in a virtual mouse brain atlas using the BrainPrint™ function. Gene expression levels are correlated with specific colors from a color scale. The Nissl-stained reference brain used to generate the atlas is displayed in panel (B) in all three orthogonal planes. The sagittal and horizontal planes are virtual sections dynamically constructed from the original coronal sections. (A) Quantitative results from in situ hybridization analysis of mRNA expression in two different mouse strains (left) are displayed within the basal ganglia and hippocampus of the brain atlas. The middle panel shows the expression levels from the DBA mouse brain and the right panel shows the DBA / C57 ratio of expression. Scale bars on the right show the expression levels as colors. (C-E) Display of gene expression levels from microarray analysis in various regions of the brain atlas. (C) Expression levels of gene X mRNA in periventricular brain regions (BNST, hypothalamus, and PAG) derived from three different embryonic secondary vesicles. Scale bar on right shows signal intensity levels as colors. (C) Specific brain regions (listed) along the rostrocaudal neuraxis of the atlas template that demonstrate gene expression patterns are color-coded. (D) The regional expression levels of embryonic patterning genes based on hybridization signal intensity in each brain region are shown in the adult mouse brain. (Scale bar for D is in Panel B).

MRM 3D Reconstructions: 3D surface reconstructions generated from image segmentation of the hippocampus (A), as well as the cerebellum (B) and whole brain (C). Figure D shows the 3D surface reconstructions within a 3D volumetric MRM file. Note the brain is undissected and still lies within the mouse head. Modified from Redwine et al., PNAS (2003) 100:1381-1386.

Amyloid 3D reconstruction: 3D reconstruction of amyloid beta distribution. (A) Serial coronal sections immunostained with 3D6 were imaged and compiled into a 3D data file. A surface reconstruction of the hippocampus is shown in yellow. (B) Amyloid beta deposits were segmented by thresholding and are displayed as a 3D reconstruction (red). A surface reconstruction of the hippocampus (yellow) and a single coronal section are shown for orientation. (C) 3D reconstruction of amyloid beta (red) viewed from the posterior aspect of the brain with the hippocampus shown as transparent yellow. Note the extensive deposition in the neocortex and hippocampus, and the central lucency representing the midbrain and caudate-putamen with punctate amyloid beta visible in the frontal cortex and olfactory bulb. (D) Large lakes and ribbons of amyloid beta (cyan) were identified by automated detection of contiguous structures within the 3D reconstruction of amyloid beta (shown as transparent red; same angle of view as C). (E) Amyloid beta sheets (cyan) are visible in the rostral part of the dentate gyrus, shown against a single coronal section, with the surface reconstruction of the hippocampus in transparent yellow. (F) Magnified view of the amyloid beta lakes and ribbons (cyan) in the dentate gyrus (within the transparent yellow hippocampal surface) and extending into the retrosplenial cortex (above). Modified from Reilly et al., PNAS (2003) 100:4837-4842.

Amyloid 3D reconstruction movie: 3D reconstruction of the amyloid deposits, shown in red. Note the extensive deposition in the neocortex and hippocampus, and the central lucency representing the midbrain and caudate-putamen with punctate amyloid visible in the frontal cortex and olfactory bulb.

Amyloid 3D reconstruction movie: Large lakes and ribbons of amyloid. These are colored blue, and were identified by automated detection of contiguous structures within the 3D reconstruction.

TOGA®: TOGA® (TOtal Gene expression Analysis) is a patented method of identifying and determining the concentration of nearly all of the genes active in a sample cell or tissue. TOGA¨ automatically identifies and simultaneously measures all genes expressed in any cell or tissue sample and provides an inventory of gene activity in any biological sample. The combination of the TOGA¨ robotic assay and the integrated TOGA Portalª informatics suite allows the researcher to assemble data from a single experimental comparison of any gene or set of genes across hundreds of other tissue types, time points, and conditions.

TOGA Automation: TOGA is performed on robotics as shown in pictures from the Neurome TOGA laboratory. The process proceeds clockwise: here, starting in the upper left hand corner, a liquid handling robot sets up enzymatic reactions in small 96-well plates, followed by their distribution via a robotic arm to PCR machines. The PCR amplified products in the plates are placed into capillary electrophoresis instruments shown in the lower right hand picture where fluorescent data is generated and transferred automatically to the TOGA data center shown in the bottom left hand corner.

Photoconversion Chamber: The diOlistic method has been optimized by Neurome to label multiple individual neurons within a single post-fixed mouse brain slice (Wu et al, 2003). These diOlistically labeled neurons are then photoconverted using a closed conversion chamber, shown in Figure A, with an oxygen-enriched environment to oxidize the fluorescently labeled neurons into a permanent signal with sufficient signal for transmitted light brightfield microscopy (TLBM) imaging and cell reconstruction at the higher-magnification required for dendritic spine detection. Figure B shows confocal laser scanning microscopy imaged dendritic spines before photoconversion. Figure C shows the TLBM-imaged dendritic spines after photoconversion. Note the qualitative resemblance of the individual spines (arrows). Overall, many cells can be photoconverted in a single pass for higher throughput. This protocol is more powerful than the conventional methods, which are limited to the photoconversion of only one neuron at a time. This overall protocol in use at Neurome produces neurons with sufficient detail under TLBM conditions to visualize dendritic spines (Wu et al., 2003).

DiOlistics: Neurome's DiOlistic method can robustly label individual neurons in the subfields of the hippocampal slice. After a high-throughput confocal-laser microscopic imaging protocol (Wu et al., 2004), individual neurons can be quickly 2-D digitized and 3-D displayed for dendritic arbor analysis and subsequently, photoconverted for high-magnification spine analysis.

Vaccine Technology: In situ hybridization image of intestinal Peyer's Patch probed for gene associated with M cells.

 

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