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Abstract: TH-OR42

Super-Resolution Microscopy in Clinical Specimens Using Conventional Widefield Microscopes

Session Information

Category: Pathology and Lab Medicine

  • 1700 Pathology and Lab Medicine

Authors

  • Kylies, Dominik, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Zimmermann, Marina, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Haas, Fabian, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Kuehl, Malte Benedikt, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Czogalla, Jan, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Kretz, Oliver, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Tomas, Nicola M., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Wiech, Thorsten, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Kuppe, Christoph, Rheinisch-Westfalische Technische Hochschule Aachen, Aachen, Nordrhein-Westfalen, Germany
  • Kramann, Rafael, Rheinisch-Westfalische Technische Hochschule Aachen, Aachen, Nordrhein-Westfalen, Germany
  • Wong, Milagros N., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Huber, Tobias B., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
  • Puelles, Victor G., Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
Background

Super-resolution microscopy (SRM) enables nanoscale molecular characterization of tissues, but the access to SRM systems is limited, hindering their applicability in the scientific and clinical pathology community. Expansion microscopy and computational image enhancement algorithms like “super-resolution radial fluctuations” (SRRF) are promising alternatives, but do not achieve sufficient resolution when combined with LED-based widefield microscopy (WFM). Here, we introduce expansion-enhanced super-resolution radial fluctuations (ExSRRF), enabling nanoscale molecular SRM in clinical pathology samples using WFM.

Methods

We performed immunofluorescence labeling of tissues, followed by hydrogel embedding, tissue expansion and time-stacked image acquisition with WFM. Subsequent computational processing using the SRRF algorithm yielded super-resolved images. To define the resolution range, nanorulers (synthetic molecules containing two fluorescent dyes at precisely predefined distances) were expanded and imaged in a similar fashion to tissues. Automated image analysis of the slit diaphragm (SD) was performed using a multi-step process including region of interest- and ridge-detection, followed by SD-density and dilatation measurements using both, custom and open-source tools.

Results

In a set of nanorulers, ExSRRF displayed non-overlapping point-spread functions at distances between 120nm and 25nm, thus providing a resolution of at least 25nm. ExSRRF was applied across a broad range of formalin-fixed paraffin-embedded clinical and experimental tissues. In an experimental model of renal ischemia-re-perfusion injury, ExSRRF resolved endoplasmatic reticular dilatation. In human kidney biopsies, ExSRRF resolved normal foot processes (FP) and detected FP effacement as a diagnostic feature of minimal change disease (MCD). In a small case series, ExSRRF resolved the SD and provided quantitative changes and a morphological disease signature of MCD.

Conclusion

ExSRRF is a flexible, scalable, inexpensive, and robust method for the molecular characterization of experimental and clinical specimens and thus has the potential to bridge SRM and both clinical and experimental pathology, enabling universal access to molecular nanopathology.