Postembedding Iodine Staining for Contrast‐Enhanced 3D Imaging of Bone Tissue Using Focused Ion Beam‐Scanning Electron Microscopy

Author:

Ayoubi Mahdi12,Weinkamer Richard1,van Tol Alexander F.12,Rummler Maximilian1,Roschger Paul3,Brugger Peter C.4,Berzlanovich Andrea5,Bertinetti Luca6ORCID,Roschger Andreas7ORCID,Fratzl Peter1ORCID

Affiliation:

1. Department of Biomaterials Max Planck Institute of Colloids and Interfaces 14476 Potsdam Germany

2. Berlin‐Brandenburg School of Regenerative Therapies (BSRT) Charité Campus Virchow‐Klinikum D‐13353 Berlin Germany

3. Ludwig Boltzmann Institute of Osteology Hanusch Hospital of OEGK and AUVA Trauma Centre A‐1140 Vienna Austria

4. Department of Anatomy Center for Anatomy and Cell Biology Medical University of Vienna A‐1090 Vienna Austria

5. Center of Forensic Science Medical University of Vienna Sensengasse 2 A‐1090 Vienna Austria

6. B CUBE—Center for Molecular Bioengineering Technische Universität Dresden 01307 Dresden Germany

7. Department for Chemistry and Physics of Materials Paris Lodron University of Salzburg 5020 Salzburg Austria

Abstract

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high‐resolution imaging techniques. In the case of bone, focused ion beam‐scanning electron microscopy (FIB‐SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin‐embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB‐SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization.

Publisher

Wiley

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