First application of three-dimensional light sheet fluorescence microscopy to human testicular tumors: New perspectives in histopathology.

Abstract

Background: Testicular tumors are among the most frequently diagnosed cancers in young men. The consequences of this diagnosis are orchiectomies, severely restricting fertility. For these young patients, a comprehensive diagnostics would be desirable, achieving a refined diagnosis and improved therapeutic patient stratification.

Objective: The aim of this study was to use three-dimensional (3D) light sheet fluorescence microscopy (LSFM) to analyze a complete testicular tumor punch at subcellular resolution-allowing a detailed diagnostic assessment of the entire punch.

Materials and methods: Tissue punches (3  and 5 mm diameter) were taken from paraffin blocks of four miscellaneous testicular tumors. After deparaffinization and clearing using benzoic acid/benzyl benzoate, a label-free LSFM autofluorescence imaging was performed. In addition, TO-PRO-3 nuclear stain was applied to several punches. After the scan, the samples were embedded in paraffin again and physically sectioned for conventional planar histology.

Results: Based on the specific autofluorescence, not only the general morphology of the tumor tissue was identified in LSFM datasets, but also diagnostic features like infiltrations, papillary and pagetoid tumor cell formations, germ cell neoplasia in situ and azoospermia. Subcellular characteristics such as vacuolated cytoplasm and pleomorphic nuclei could be detected at maximum magnification. After nuclear staining, virtual H&E sections were reconstructed from the LSFM data and tomographically visualized across the entire punch. Subsequent histology and immunohistochemistry after LSFM analyses is possible.

Discussion: LSFM analysis of testicular tumors enables the detailed 2D/3D analysis of an entire tumor punch for assessment of relevant tumor characteristics due to its intrinsic fluorescence or with specific nuclear staining.

Conclusion: LSFM provides the technical basis for the analyses of complete testicular tumor biopsies, thus maximizing the spatial morphological and anatomical information. The subcellular 3D imaging of the tumor has the potential to identify new cancer imaging biomarkers that have additional diagnostic and prognostic value for patients.