Highly accurate image registration for 3D multiplexed cyclic imaging using dense labeling in expandable tissue gels
Fig 2
Validation of a dense label-based registration.
(a) Experimental procedure for validation. The image registration accuracy was estimated from sparsely labeled (DAPI) and densely labeled (ATTO 680 NHS-ester) markers. The first round was stained and imaged with a rabbit anti-vGluT1 antibody and an Alexa Fluor 488 (AF 488)-conjugated goat anti-rabbit secondary antibody. Signal of the first round was photobleached and the second round was stained and imaged with anti AF 488 antibody. Images acquired from the first and second round were finally registered through the DAPI staining channel and the NHS-ester staining channel, respectively. (b) Initial region of interest (ROI) with DAPI (blue) and NHS-ester staining (light blue) channels. Target ROI where DAPI signal is barely visible (magenta-highlighted region). (c) Magnified view of post-registered vGluT1 fluorescent signal registered by DAPI and NHS-ester, respectively (first round: red, 2nd round green). (d) Box plot of Pearson correlation coefficient between DAPI registered images (1st–2nd round) and NHS-ester registered images (1st–2nd round) within initial ROI and magnified target ROI. The brain region where validation has been conducted was between CA3 and dentate gyrus regions. Please see S1 Data for individual numerical values of the Pearson correlation coefficient. Scale bars: (b) 20 µm; (c) 1 µm. All length scales are presented in pre-expansion dimensions. Number of sample N = 6, Number of datapoints M = 11 for each sample.