STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue

Abstract

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Immunofluorescence labeling of HER2 positive paraffin-embedded rectal cancer tissue.

Confocal overview image of a region of a HER2 positive rectal cancer tissue section labeled with DAPI (blue) to highlight the nuclei and decorated with antisera against Tom20 (fire) and HER2 (green). (A) overlay, (B) Tom20, and (C) HER2. Scale bars: 25 µm.

Figure 2. STED super-resolution microscopy of mitochondria in the rectal Muscularis externa demonstrates high structural preservation of the stored paraffin-embedded tissue.

STED recordings were performed on 2 µm thick dewaxed sections cut along the longitudinal axis of the rectum. (A) Left: STED overview image of a region of the inner circular layer of the rectal Muscularis externa decorated with an antiserum against Tom20. Right: Magnifications of the areas in the indicated dashed squares showing the distribution of TOM clusters within the mitochondria. (B–E) STED images of tissue sections decorated with antisera against Tom20 (B), Mic60 (mitofilin) (C), aconitase (D), and cyclophilin D (E). In each panel the confocal (top, left) and the corresponding STED image (top, right) is displayed. Bottom: Magnification of the STED image as indicated by a dashed square. Note the different distributions of the four proteins within the mitochondria. Scale bars: 20 µm (A, left); 1 µm (A, right) and (B–E, top); 200 nm (B–E, bottom).

Figure 3. Comparison of (A) Hematoxylin-Eosin-, (B) immunohistochemistry-, and (C) immunofluorescence-labeling on three consecutive 2 µm thick tissue sections of a HER2 positive paraffin-embedded human rectal cancer.

Note that the three consecutive sections cover the same region in the tissue. The images were taken with diffraction-limited widefield (A,B) or confocal (C) microscopy. (D,H) and (E,I): corresponding magnifications out of (B) and (C), respectively, at sites indicated by the arrows. (F,G,J,K): comparison of diffraction-limited confocal microscopy with STED super-resolution microscopy on sectioned stored tumor tissue. Shown are magnifications of the areas indicated by the dashed squares. Top-right corners: confocal imaging. Left-bottom area: STED imaging. The arrows point to HER2-positive vesicle-like structures that are resolved in the STED images. Scale bars: 1 mm (A–C), 50 µm (D,E,H,I), 2 µm (F,J), and 500 nm (G,K).

Figure 4. STED super-resolution microscopy of archived human tissue samples stored for up to 17 years in a clinical repository.

Representative images of tumor tissues stored at room temperature for less than 1 year (A), 11 years (B) or 17 years (C), were sectioned, dewaxed, decorated with an antiserum against Tom20 and imaged. Left: Representative confocal images. The same color table was used for the three images in order to visualize the relative staining efficiencies. Middle/Right: Comparison of STED (middle) and confocal (right) microscopy of tissue sections of different age. Here, the color tables were adjusted to the signal intensities obtained. Scale bars: 10 µm (left) and 1 µm (middle, right).