Highly accurate image registration for 3D multiplexed cyclic imaging using dense labeling in expandable tissue gels
Fig 3
Registration of multiplexed images via ATTO 680 NHS-ester staining and photobleaching in an eMAP-processed mouse brain slice.
(a) Experimental schematic of the registration of multiplexed images. Target proteins for each imaging round were stained, imaged and removed with photobleaching treatment. A pair of images from adjacent rounds are registered by the ATTO 680 NHS-ester staining image. (b–r) Seven-round cyclic staining images of an eMAP-processed mouse brain slice. (b) Merged 3D 16-plex image with 30 µm z-stacks. Blue, DAPI; gray, ATTO 680; white, Laminin; cyan, NF-H; yellow, Iba1; green, Lamin B1; orange, vGluT1; orange-red, Homer1; magenta, GFAP; crimson, Synaptophysin; dark purple, SV2A; purple, GM130; pink, SOX2; gold, ABAT; light green, Alpha-tubulin; green-yellow, MBP. (c–r) Single-channel images of the target proteins. Scale bars: (b–r) 20 µm. All length scales are presented in pre-expansion dimensions. Number of sample N = 2 acquired from two independent mouse brain slices.