Figure 2: Validation of MAGNIFY in multiple tissue types. (IMAGE)
Caption
Example of (a) pre-expansion images of human kidney imaged at 60× and processed with SOFI compared to the same field of view (b) post-expansion with MAGNIFY taken at 40×. Magenta, DAPI; Orange, anti-alpha-actinin 4 (ACTN4); Blue, vimentin. Post expansion images are maximum intensity projected over 25 frames in z. (c-e) Root mean square (RMS) length measurement error as a function of measurement length for pre-expansion versus post expansion images for (c) DAPI, (d) ACTN4, and (e) Vimentin. Solid line, mean of channel; shaded area, standard error of mean (s.e.m); n = 5 technical replicates; average expansion factor, 8.64× (s.e.m 0.24). Example of (f) pre-expansion images of human prostate imaged at 60× and processed with SOFI compared to the same field of view (g) post-expansion with MAGNIFY taken at 40×. Magenta, DAPI; Green, Anti-ATPase Inhibitory Factor 1 (ATPIF). Post expansion images maximum intensity projected over 3 frames. (h-i) RMS length measurement error as a function of measurement length for pre-expansion versus post expansion images of (h) DAPI, and (i) ATPIF. Solid line, mean of channel; shaded area, s.e.m.; n = 4 technical replicates; average expansion factor, 10.38× (s.e.m 0.57). (j-o) Validation of MAGNIFY across multiple human tissue types. FFPE samples of human tissue were imaged at 40× (top left). Images were taken at 60×and processed with SOFI (bottom left). The white box indicates the field of view of the higher magnification images. The samples were then processed with the MAGNIFY protocol, and the same fields of view were imaged post-expansion in water at 10× (top right) and 40× (bottom right). Post expansion images were projected over 4-17 z slices. Magenta, DAPI; Green, ATPIF; Blue, Cytokeratin Pan Type I/II. Expansion factors in water were (j) Colon: 8.85×, (k) Breast: 9×, (l) Uterus: 8×, (m) Placenta: 8.75×, (n) Thymus: 10.00×, (o) Thyroid: 10.59×. (p-r) Example 3d images of human tissues: (p) kidney (Expansion factor 8.68×). Magenta, DAPI; Orange, ACTN4; Blue, WGA. (q) colon (Expansion factor 9.67×). Magenta, DAPI; Green, ATIPF; Blue, Cytokeratin Pan Type I/II. (r) Uterus (Expansion factor 8×). Magenta, DAPI; Green, ATIPF; Blue, Cytokeratin Pan Type I/II. Zoomed in regions indicated by dashed white box. Scale bars (yellow indicates post expansion images): (a) 5 μm; (b) 5 μm (physical scale post expansion: 40.75 μm; expansion factor: 8.15×); (f) 5 μm; (g) 5 μm (physical scale post expansion: 51.9 μm; expansion factor: 10.38×); (j-o) top: 10 μm; bottom: 1 μm; (p-t) 5 μm. Scale bars are all in biological scale.
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