Immunofluorescence Tomography: High-Resolution 3D Reconstruction by Serial-Sectioning of Methacrylate Embedded Tissues and Alignment of 2D Immunofluorescence Images

The study presented immunofluorescence tomography as a high-resolution 3-D reconstruction method using methacrylate embedding and serial-sectioning. This technique involved aligning 2-D immuno-stained images into stacks for 3-D rendering, utilizing Butyl-Methyl Methacrylate (BMMA) plastic for excellent tissue preservation and easy de-plasticization. The method achieved high axial Z-resolution with 2 µm thick sections, allowing imaging by various modalities without the need for tissue clearing. It offered advantages over confocal microscopy, such as minimized antibody penetration and light scattering issues, and allowed for re-probing of sections without structural loss. The approach was cost-effective, using less expensive microscopes and enabling indefinite section storage. Immunofluorescence tomography was highlighted as a powerful tool for quantifying cell sub-populations in 3-D, applicable to tissues like the mammary gland, cornea, and hair follicle.