title: Three-dimensional microscopic analysis of clinical prostate specimens.
abstract: OBJECTIVE
  Microscopic evaluation of prostate specimens for both clinical and research purposes is generally performed on 5-μm-thick tissue sections. Because cross-sections give a two-dimensional (2D) representation, little is known about the actual underlying three-dimensional (3D) architectural features of benign prostate tissue and prostate cancer (PCa). The aim of this study was to show that a combination of tissue-clearing protocols and confocal microscopy can successfully be applied to investigate the 3D architecture of human prostate tissue.
  
  
  RESULTS
  Optical clearing of intact fresh and formalin-fixed paraffin-embedded (FFPE) clinical prostate specimens allowed us to visualize tissue structures up to a depth of 800 μm, whereas, in uncleared tissue, detection of fluorescence was only possible up to 70 μm. Fluorescent labelling with a general nuclear dye and antibodies against cytokeratin (CK) 5 and CK8-18 resulted in comprehensive 3D imaging of benign peripheral and transition prostate zones, as well as individual PCa growth patterns. After staining, clearing, and imaging, samples could still be processed for 2D (immuno)histochemical staining and DNA analysis, enabling additional molecular and diagnostic characterization of small tissue specimens.
  
  
  CONCLUSIONS
  In conclusion, the applicability of 3D imaging to archival FFPE and fresh clinical specimens offers unlimited opportunities to study clinical and biological topics of interest in their actual 3D context.
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.|Erasmus Optical Imaging Centre, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Martin E
authors.initials: ME
authors.lastname: van Royen
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Esther I
authors.initials: EI
authors.lastname: Verhoef
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Charlotte F
authors.initials: CF
authors.lastname: Kweldam
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.|Erasmus Optical Imaging Centre, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Wiggert A
authors.initials: WA
authors.lastname: van Cappellen
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.|Erasmus Optical Imaging Centre, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Gert-Jan
authors.initials: GJ
authors.lastname: Kremers
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.|Erasmus Optical Imaging Centre, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.affiliation: Department of Pathology, Erasmus Medical Centre, Rotterdam, the Netherlands.
authors.forename: Geert J L H
authors.initials: GJ
authors.lastname: van Leenders
mesh_terms: D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D016612:Paraffin Embedding; D011471:Prostatic Neoplasms / Q000473:pathology*; D013194:Staining and Labeling
keywords: fluorescence microscopy; pathology; prostate; three-dimensional (3D); tissue clearing