Fig 1.
General experimental procedure for super-resolution multiplexed imaging in an expandable hydrogel specimen.
(a) Stepwise description of multiplexed imaging in an expandable hydrogel specimen. The entire experiment can be divided into three main steps: gelation and labeling, multi-round imaging, and image registration. The first step includes the gelation of the biological specimen, the labeling of biological molecules and fiducial markers, and the physical homogenization of the hydrogel–specimen complex. The sequence of such works can differ depending on the type of expansion method used. The second step is multi-round imaging by repeating the cycle of staining and de-staining. The de-staining step includes all signal elimination approaches, such as antibody stripping, DNA elution, or fluorophore bleaching. After acquiring images from each round, the third step, image registration, is performed to match the distorted pixel coordinates finely. (b) Conventional image registration using sparsely labeled markers. Representative sparsely labeled markers are nuclei or blood vessels. An image registered with sparsely labeled markers still includes severe pixel mismatches, especially in the region where fiducial markers are barely displayed. (c) Image registration using dense-labeled markers. Fluorophore NHS-ester staining can be utilized as a densely labeled fiducial marker. Since fiducial markers are densely localized in the entire field-of-view, this approach provides relatively high registration accuracy.
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.
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.
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.
Fig 5.
Registration of multiplexed images via actin staining in expanded cultured cells.
(a) Composite image generated by registering two images acquired from the first and second imaging rounds. Phalloidin-labeled actin fibers were used as fiducial markers. The yellow box displays six-color images, including the DAPI channel. Gray, nucleus; red, actin; cyan, vimentin; blue, laminA/C; yellow, CCP; and magenta, cytokeratin 8/18. (b–c) Magnified views of the boxed region in a. Scale bars: (a) 2 µm; (b–c) 500 nm. All length scales are presented in pre-expansion dimensions. Number of sample N = 1 acquired from a single substrate.