A Computational Framework for Ultrastructural Mapping of Neural Circuitry
A thin 200-nm section was probed with anti-AGB IgGs after excitation of the rabbit retinal GC layer , visualized by silver-intensified immunogold detection , captured on a SyncroscanRT montaging system (182 nm/pixel), and aligned with Syncroscan software (A and C) and ir-translate/ir-grid-refine (B and D). At low magnification, both images appear perfect, but at near pixel level, many small defects emerge in the Syncroscan-aligned mosaic (arrows in [A and C]) that include 200–2,000-nm image shifts and image blurring (box). By using the raw image tiles and their metadata, ir-translate and ir-grid-refine create defect-free mosaics. While the image shifts shown in (A) are irrelevant (indeed invisible) for image display, they are highly corrupting in mathematically sensitive procedures such as clustering and multimodal alignments with ssTEM datasets. (A and B) are bright-field images and (C and D) are contrast-stretched Laplacian filtered images that enhance discontinuities and clearly show alignment defects. The circle in image (D) represents a lysosome of approximately 200 nm diameter. Its contrast is better preserved in the ir-translate and ir-grid-refine mosaic. Scale = 20 μm.