Three-dimensional structural analysis of papillary thyroid carcinoma nuclei with serial block-face scanning electron microscopy (SBF-SEM)

Abstract

Nuclear morphology of carcinoma cells is critical for the pathological diagnosis of papillary thyroid carcinoma (PTC). However, three-dimensional architecture of PTC nuclei is still elusive. In this study, we analyzed the three-dimensional ultrastructure of PTC nuclei using serial block-face scanning electron microscopy which takes advantage of the high-throughput acquisition of serial electron microscopic images and three-dimensional reconstruction of subcellular structures. En bloc-stained and resin-embedded specimens were prepared from surgically removed PTCs and normal thyroid tissues. We acquired two-dimensional images from serial block-face scanning electron microscopy and reconstructed three-dimensional nuclear structures. Quantitative comparisons showed that the nuclei of carcinoma cells were larger and more complex than those of normal follicular cells. The three-dimensional reconstruction of carcinoma nuclei divided intranuclear cytoplasmic inclusions into "open intranuclear cytoplasmic inclusions" connecting to cytoplasm outside the nucleus and "closed intranuclear cytoplasmic inclusions" without that connection. Cytoplasm with abundant organelles was observed in open inclusions, but closed inclusions contained fewer organelles with or without degeneration. Granules with a dense core were only observed in closed inclusions. Our observations suggested that open inclusions originate from nuclear invaginations, and disconnection from cytoplasm leads to closed inclusions.

Keywords: intranuclear cytoplasmic inclusion; nuclear invagination; papillary thyroid carcinoma; serial block-face scanning electron microscopy; three-dimension; thyroid gland; ultrastructure.

Figure 1

Workflow of serial block‐face scanning electron microscopy (SBF‐SEM) and 3D reconstruction. (a) (i) Resection of the thyroid tissue, (ii) fixation and en bloc staining, (iii) embedding into resin, (iv–v) acquisition of serial electron microscopic images from SBF‐SEM by automatic repeated slicing and imaging, (vi–vii) 3D reconstruction along with segmentation of nuclei. Light microscopy images of papillary thyroid carcinoma (PTC) tissues stained with heavy metals and embedded in resin at low (b) and high (c, d) magnification. P, papillary structure; arrows, PTC cells with intranuclear cytoplasmic inclusions. (e) Serial SBF‐SEM images and representative images (right side). Scale bars: 100 µm (b), 10 µm (c–e).

Figure 2

Papillary thyroid carcinoma (PTC) nucleus showing deep infoldings of the nuclear membrane compared to anormal follicular cell (NFC) nucleus. Representative images of serial block‐face scanning electron microscopy (SBF‐SEM) and 3D reconstruction of a NFC nucleus (a–f, green) and a PTC nucleus (g–l, red). Electron microscopy (a, g), 3D reconstruction (b–f, h–l). Scale bars: 10 µm.

Figure 3

Papillary thyroid carcinoma (PTC) nuclei are complex and larger than the nuclei of normal follicular cells (NFCs). Morphological parameters of nuclei: volume (a), Feret diameter (b), surface area (c), and sphericity (d). Boxes and whiskers show medians along with interquartile ranges and max/min values, respectively.

Figure 4

Morphological parameters of nuclei show distinct patterns between papillary thyroid carcinoma (PTC) cells and normal follicular cells (NFCs). Surface area versus Feret diameter (a), surface area versus volume (b), and surface area versus sphericity (c). Dotted line, regression line; R, correlation coefficient.

Figure 5

3D reconstructions demonstrating an open and a closed intranuclear cytoplasmic inclusion (INCI). Closed INCI scanning electron microscopy (SEM) images (a–k) and their 3D reconstruction (L) shows the INCI (red) is isolated from the perimeter of the nucleus within the nucleoplasm (yellow), but a vacuolar structure is identified between the INCI and the perimeter of the nucleus (e–j, black arrowhead). Open INCI SEM images (m–w) and their 3D reconstruction (X) identifies the connection (arrow) from the INCI to outside the nucleus (X). Scale bars: 10 µm.

Figure 6

Ultrastructure of organelles in intranuclear cytoplasmic inclusions (INCIs). A lysosome (blue arrowhead) and endoplasmic reticulum (red arrowheads) in an open INCI (a, b). The thin connection (arrows) from the INCI to outside the nucleus in an open INCI (c, d). Fewer organelles in a closed INCI (e, f, h). Numerous granules with dense cores (arrowheads) in a granuloplastic INCI (i, j and l). Electron microscopic images (a, b, e, f, i, j). b, f, j are magnified insets of the yellow dot rectangles of a, e, i, respectively. Scale bars: 5 µm.

Figure 7

Hypothetical scheme of intranuclear cytoplasmic inclusions (INCIs) morphogenesis. Nuclear invagination contains cytoplasm with numerous cytoplasmic organelles (a). A thinner connection from inside to outside an invagination forms an open INCI (b). Complete loss of connection leads to a closed INCI (c). A closed INCI may have few organelles (d) or many granules (e).