A modified Spalteholz technique with preservation of the histology

The Spalteholz technique for producing clear macroscopic specimens was introduced 100 years ago, and is still in use today because it immediately provides transparent three-dimensional specimens. The classical clearing procedure with peroxide severely damages formalin-fixed tissue and inhibits further investigation on a histological level. We, therefore, wanted to make a transparent specimen by a modified Spalteholz method and chose the human larynx as an example of a large specimen. Its blood vessels were injected with gelatin, the specimen treated with acetone and peracetic acid and subjected to freeze substitution. After complete dehydration, benzyl benzoate and dimethylphthalate were used for impregnation. The cleared specimen satisfactorily revealed the laryngeal blood vessels and such structures as muscles, cartilage and the thyroid gland. The histology of the laryngeal glands, thyroid tissue, and infrahyoid muscle fibres was preserved. Tissue shrinkage became apparent with subsequent steps of the clearing procedure. We conclude that our modifications maintain the transparency of the specimen and allow histological investigation. The herein described technique thus constitutes an improvement of the classical Spalteholz technique.