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

Peter Ilgen,Christian Andreas Wurm,Lena-Christin Conradi,Stefan Stoldt,Josef Rüschoff,B Michael Ghadimi,Torsten Liersch,Stefan Jakobs,Anthony W.I Lo

doi:10.1371/journal.pone.0101563

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.

Highlights

The number of human tissue specimen stored in biorepositories was estimated at more than 300 million already 15 years ago in the US alone [1,2]
The sections were stained with DAPI to highlight the nuclei and decorated with antisera against the integral membrane protein HER2 and against the mitochondrial outer membrane protein Tom20, an essential protein expressed in all human cells
Confocal microscopy demonstrated the strong expression of HER2 in the tumor tissue, whereas the surrounding normal stroma cells did not express a relevant amount of HER2 (Figure 1)

Summary

Introduction

The number of human tissue specimen stored in (clinical) biorepositories was estimated at more than 300 million already 15 years ago in the US alone [1,2]. Many of these specimens have been formalin fixed and paraffin embedded, a standard clinical preservation method that is in use for more than a century. The stored and annotated paraffin-embedded tissue may be sectioned, dewaxed, stained and imaged by conventional light microscopy. In classical (fluorescence) microscopy the attainable resolution is limited by diffraction to about 250 nm, restricting the extractable information from a specimen. Several super-resolution microscopy techniques (nanoscopy) have been developed that allow to fundamentally overcome the diffraction limit, enabling far-field microscopy with a substantially improved resolution [3,4,5]

Methods

Results

Conclusion