Intranuclear birefringent inclusions in paraffin sections by polychromatic polarization microscopy

Abstract

Intranuclear birefringent inclusions (IBI) found in various cell types in paraffin-embedded tissue sections have long been considered to be a tissue processing artifact, although an association with biological processes has been suggested. We applied polychromatic polarization microscopy to image their spatial organization. Our study provides evidence that IBI are caused by liquid paraffin-macromolecular crystals formed during paraffin-embedding procedures within cells and potentially reflect an active transcriptional status.

Figure 1

Spatial patterns of IBI captured by PPM. (a) Extracted PPM signals of intranuclear inclusions overlaid with Hoechst-stained fluorescence image for nuclei visualization; (b) endometrioid carcinoma tissue section by PPM after deparaffinization showing a cluster of IBI; (c) IBI and basement membrane collagen PPM signals in kidney tissue section stained with hematoxylin for nuclei visualization and imaged using PPM; (d) Crystalloid structure of the IBI—nuclear inclusions captured in FFPE unstained tissue sections imaged by PPM with digital background correction; (e) gallery of nuclei segmented from Hoechst-stained renal cell carcinoma tissue imaged by fluorescence and overlaid with IBI acquired by PPM. All images acquired with Olympus BX63 microscope equipped with an Olympus DP80 camera using the CellSens acquisition software. Objectives: (b,c) Olympus UPlanApo 100x, (a,d) additional ×2 magnification lens used, (e) Olympus LUCPlanFLN 20x, with Olympus U_FUNA DAPI fluorescence filter cube. Original images acquired at a resolution of 1360 × 1024 px, resized and put into multi-panel figure using the GIMP image editor.

Figure 2

IHC studies to identify IBI-positive cell types and biological processes: (a) IBI present in CD68-positive macrophages (arrow) but also in CD68-negative cells (hepatocellular carcinoma); (b) IBI present in CD20-positive B cells (arrow) but also in CD20-negative cells (renal cell carcinoma); (c) PAX2 marker-specific B cells transcription factor—on renal cell carcinoma tissue section; (d) PAX8 marker-specific renal cancers transcription factor—on renal cell carcinoma tissue section. All images acquired with Olympus BX63 microscope, DP80 camera, objective Olympus Ach 60 × and PPM modality. Original images acquired at a resolution of 1360 × 1024 px, resized and put into multi-panel figure using the GIMP image editor.