(A) Images of human skin samples, both taken by a conventional camera (i, iv) and via fluorescence microscopy (ii-iii, v-vi) when stained with DAPI (gray) and antibodies against vimentin (green) and smooth muscle actin (gene name, ACTA2) (magenta). The samples were digested with 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hour (i-iii) or 2 hours (iv-vi). (i andiv) Conventional camera (i.e., non-fluorescent) photographs of human skin-hydrogel hybrid samples in PBS (~2x expansion), after digestion for 0.5 hour (i) or 2 hours (iv). (ii) Widefield fluorescent image from the sample of (i) pre-digestion. (iii) Post-expansion wide-field fluorescent image of the sample of (ii), with a dashed orange box highlighting regions with autofluorescence in the DAPI channel and distorted vimentin networks post-expansion. (v) As in (ii), for the sample in (iv) pre-digestion. (vi) As in (iii) for the sample of (v) with a dashed orange box highlighting regions with autofluorescence in the DAPI channel. (B) As in (A), except that the samples were digested with 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 25 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl, at 60°C for 0.5 hour (i-iii) or 2 hours (iv-vi). Note that inBi andBiv the samples look quite transparent, due to the heavy digestion. (C) Photographs of human liver samples digested with a 1 mM EDTA-based solution – specifically, 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hour (left) or 2 hours (right). (D) Photographs of human liver samples digested with a 25 mM EDTA-based solution – specifically, 8 units/mL proteinase K solution containing 25 mM Tris (pH 8), 25 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl at 60°C for 0.5 hours (left) or 2 hours (right). Note that the samples ofD are significantly more transparent than those ofC. (E) Widefield fluorescent image of a human liver sample stained with DAPI (gray) and an antibody against smooth muscle actin (gene name, ACTA2) (magenta) prior to the ExPath process. (F) Post-expansion wide-field fluorescent image of the sample ofE, digested with a 1 mM EDTA-based solution for 1 hour. White dashed line outlines an out-of-focus region caused by distortion. (G) Wide-field fluorescent image of a human liver sample stained with DAPI (gray) and an antibody against smooth muscle actin (gene name, ACTA2) (magenta) prior to the ExPath process. (H) Post-expansion wide-field fluorescent image of the same sample as inG. The sample had been digested with a 25 mM EDTA-based solution for 0.5 hour. (I) Post-expansion confocal image of human lymph node tissue with invaded breast cancer stained with DAPI (blue) and an antibody against vimentin (green), and treated with a 1 mM EDTA-based solution for 3 hours. (J) Post-expansion confocal image of a different sample from the same tissue used inI, treated with a 25 mM EDTA-based solution. (K) Post-expansion confocal image of normal human kidney tissue fixed with acetone, stained with an antibody against collagen IV (magenta), and treated with 0.1 mg/ml Acryloyl-X prior toin situ polymerization. Cracks are indicated by white arrows. (L) Post-expansion confocal image of a different sample from the same tissue used inK, treated with 0.03 mg/ml Acryloyl-X prior toin situ polymerization. Scale bars (yellow scale bars indicate post-expansion images:Aii andiii, 9.2 μm (physical size iniii: 40 μm, expansion factor: 4.33).Av andvi, 9.4 μm (physical size invi: 40 μm, expansion factor: 4.28).Bii andiii, 9 μm (physical size iniii: 40 μm, expansion factor: 4.41).Bv andvi, 8.9 μm (physical size invi: 40 μm, expansion factor: 4.51).E andF, 119 μm (physical size inF: 500 μm, expansion factor: 4.22);G andH, 109 μm (physical size inH: 500 μm, expansion factor: 4.58). (I-L) 40 μm, physical size.

(A) Super-resolution structured illumination microscopy (SR-SIM) image of a HeLa cell stained with DAPI and an antibody against α-tubulin. Blue, DAPI; green, α-tubulin. (B) Post-expansion image of the same sample acquired with a spinning disk confocal microscope. (C andD) Root mean square (RMS) length measurement error as a function of measurement length for ExPath vs SIM images (blue solid line, mean of DAPI channel; green solid line, mean of α-tubulin channel; shaded area, standard deviation; n = 3 samples from separate cultures). Scale bars: (A) 4 μm, (B) 4 μm (for yellow scale bar, physical size post-expansion, 19.5 μm; expansion factor: 4.89).

(A) Brightfield image of unstained formalin-preserved human lymph node tissue with invaded breast cancer. (B-E) Widefield fluorescent images of the same sample as inA in four fluorescent channels (listed below). (F) Bright field image of tissue from the same block as inA, stained with H&E. (G-J) Widefield fluorescent images of the sample ofF in four fluorescent channels. Filters for each fluorescent channel (Semrock, Rochester, NY):B andG: excitation 628 ± 20 nm, emission 692 ± 20 nm;C andH: excitation 586 ± 10 nm, emission 628 ± 19 nm;D andI: excitation 482 ± 9 nm, emission 520 ± 14 nm;E andJ: excitation 387 ± 6 nm, emission 409 nm and longer. Images from a given fluorescent channel are adjusted to have the same contrast. (K) Brightfield and widefield fluorescent images of tissue from the same block as inA, stained with H&E, after each step of ExPath processing. Images from a given fluorescent channel are adjusted to have the same contrast. Samples may be especially bright after the heat treatment and gelation steps due to the buffer compositions present. Scale bars:A-J, 500 μm;K, 40 μm (for yellow scalebars, post-expansion, physical size 184 μm; expansion factor: 4.58).

Left panel, widefield fluorescent images of acetone fixed human kidney sections with and without heat treatment in citrate buffer. Right panel, zooms into regions corresponding to the regions indicated by the dashed yellow rectangles in the left panels. Scale bars: 1 mm (left panel), 200 μm (right panel). Abbreviations: ACTN4, actinin-4; ColIV, collagen IV.

(A-D) Post-expansion widefield images of a human acetone fixed kidney section stained with DAPI and antibodies. Blue, vimentin; green, actinin-4; red, synaptopodin; gray, DAPI. (E) Merged image of (A-D). (F andG) Magnified regions showing actinin-4 (F) and synaptopodin (G) zoomed into the white dashed squares ofB andC. (H) Profiles of fluorescent intensity taken along the white dashed line cuts ofF andG. Green, actinin-4; red, synaptopodin. Scale bars: 1 μm (4.5 μm physical size, expansion factor 4.5).

A. Post-expansion widefield image of a FFPE normal human kidney sample treated with a citrate antigen retrieval method (20 mM sodium citrate, pH 8.0), with highlighted region (boxed line) magnified in panelB.C. Post-expansion widefield image of a FFPE normal human kidney sample treated with a Tris-EDTA antigen retrieval method (10 mM Tris base, 1 mM EDTA solution, 0.05% Tween 20, pH 9.0), with highlighted region (boxed line) magnified in panelD.E. Post-expansion confocal image of a FFPE normal human kidney sample treated with a citrate antigen retrieval method, with highlighted region (boxed line) magnified inF.G. Post-expansion confocal image of a FFPE human kidney sample with minimal change disease treated with a citrate antigen retrieval method, with highlighted region (boxed line) magnified inH. All the samples were stained with DAPI (gray) and antibodies against vimentin (blue), actinin-4 (green) and collagen IV (magenta). Scale bars:A,C,E andG, 40 μm (physical size).B,D,F, andH, 8 μm (physical size).

Frozen kidney sections fixed with acetone were stained with DAPI and antibodies. Blue, vimentin; green, actinin-4; magenta, collagen IV; gray, DAPI. Scale bars are indicated in images. For ExPath images, only physical size is indicated. Expansion factors are listed inSupplementary Table 4.

Computational detection and segmentation of the nuclei is significantly more accurate in expanded samples as compared to pre-expanded samples: examples of normal breast, usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS). For the “expert annotation” and “automated segmentation” columns: green filled nuclei are nuclei segmented by the expert or the automated segmentation algorithm, respectively (red circles indicate nucleus outlines, which are not visible in the ExPath row because the resolution is too high and thus the outline is barely visible). In the “automated vs expert” column: green filled nuclei, true positives; red filled nuclei, false negatives; blue filled nuclei, false positives (note that when the automated segmentation yielded larger outlines than the expert, this is expressed as a blue “halo” around the green).

Supplementary Figures 1–9 and Supplementary Tables 1–15 (PDF 3190 kb)