Target deletion or holding on sections after enzyme digestion monitored with attenuation-of-sound images

Abstract

Tissues consist of various components and if these can be deleted or reserved, their location and proportion can be detected. Scanning acoustic microscopy (SAM) calculates the attenuation of sound (AOS) through tissue sections to obtain histological images without staining. AOS values are reduced as tissue components break down. Here, we digested specific components in tissues using enzymes and followed the process with AOS imaging over time. In addition, we applied specific dyes and antibodies to inhibit enzyme activity and maintain a specific component in the section. We used specific enzymes to degrade tissues that contain the enzymes substrate, such as collagenase for bone, elastase for skin and arteries, actinase for amyloid-positive cervical arteries and lymph nodes, amylase for the corpora amylacea (CA) of the brain and DNase and haematoxylin for adenocarcinomas. Collagenase digested bone and cartilage to clearly visualise the internal structure. The structural components had characteristic AOS values, which gradually decreased. Elastases break elastic fibres in the skin and arteries differently between young and old individuals. The dermis and tunica media of arteries in the elderly fracture more easily than those of younger individuals. Actinase digested the cervical artery except for amyloid deposits, which were preferentially detected by Congo red staining. Actinase-digested lymphoid cells remained horseradish peroxidase (HRP)-staining positive. Amylase digested some CAs, which became periodic acid-Schiff (PAS) staining negative and diminished in size by electron microscopy observation. Cell nuclei were digested and deleted by DNase except for those stained with HRP. Residual nuclear images of AOS matched those of light microscopy, and haematoxylin staining inhibited DNase digestion of the nucleus. Specific inhibition of enzymes preserved the target cells and materials. SAM observation can monitor the tissue breakdown process, which provides an advantage over light microscopy as no staining is required and exhibits higher sensitivity to detect fragile structures.