Highly accurate image registration for 3D multiplexed cyclic imaging using dense labeling in expandable tissue gels
Fig 4
Registration of multiplexed images via ATTO 565 NHS-ester staining and signal unmixing in an eMAP-processed mouse brain slice.
(a) Experimental schematic of the registration of multiplexed images. Target proteins for each imaging round were consecutively stained without signal removal. A pair of images from adjacent rounds are registered by the ATTO 565 NHS-ester staining image. The registered images were unmixed to extract target protein expression. (b–m) Five-round cyclic staining images of an eMAP-processed mouse brain slice. (b) Merged 3D 10-plex image with 20 µm z-stacks. Blue, DAPI; gray, ATTO 565; brown, calnexin; green, lamin B1; yellow, SOX2; cyan, NF-H; red, CALB2; white, laminin; gold, vGluT2; magenta, GFAP. (c–m) Single-channel images of the target proteins. Scale bars: (b–m) 20 µm. All length scales are presented in pre-expansion dimensions. Number of sample N = 6 acquired from two different mouse brain slices.