3-DIMENSIONAL RECONSTRUCTION BY CONFOCAL LASER-SCANNING MICROSCOPY INROUTINE PATHOLOGICAL SPECIMENS OF BENIGN AND MALIGNANT LESIONS OF THEHUMAN BREAST

Confocal laser scanning microscopy (CLSM) has become an exciting new i nstrument because of its increased resolution over conventional wide-f ield microscopy and its high performance three-dimensional (3D) optica l sectioning. Although CLSM has been used extensively in cell biology, few applications have been reported in routine clinical pathology. In this study, 3D reconstruction was performed on routine formalin-fixed , par affin-embedded tissues of normal mammary duct, simple ductal hyp erplasia, intraductal papillary hyperplasia, ductal carcinoma in situ, invasive carcinoma, and lymph node metastatic carcinomas of the human breast by using computer-assisted CLSM in conjunction with a 3D recon struction software package (micro Voxel). The selected specimens were sectioned at 30 mu m, mounted on glass slides, and stained with the DN A fluorescent probe, YOYO-1 iodide. The nuclear DNA and chromatin text ure were clearly demonstrated after pretreatment with RNAase and hydro lysis with 2 N HCl. High quality 3D images were obtained by processing the optical section stacks with volume render and surface display par ameters in microVoxel. 3D morphologic characteristics of different bre ast lesions were examined in various orientations by angular image rot ation. The clearly benign lesions (simple ductal hyperplasia and intra ductal papillary hyperplasia) revealed similar 3D morphologic features , including: (1) smooth nuclear surface and homogeneous chromatin fluo rescence intensity; (2) hyperplastic cell nuclei showing similar shape and volume; and (3) clearcut margin of basement membrane defined by s pindle-shaped myocytes of the ductal outer layer. In contrast, carcino mas displayed remarkably different features in 3D morphology, includin g: (1) irregular nuclear surface; (2) marked nuclear pleomorphism (irr egular, angulated and indented shape of nuclear volume); (3) irregular and coarse chromatin texture; (4) chaotic arrangement of tumor cell n uclei; and (5) absence of myocytes, indicating no clear margin at the site of infiltration of cancer cells. In conclusion, nuclear structure , specifically demonstrated by CLSM of YOYO-1 iodide fluorescently sta ined cells, used in tandem with 3D Volume morphologic reconstruction, may provide a useful research diagnostic tool in pathology.