Background A scanning acoustic microscope (SAM) uses an ultrasound to image an object by plotting the speed-of-sound (SOS) through tissue sections on screen. Because hard tissues result in great SOS, SAM can provide data on the tissue elasticity and lesions. We have investigated the utility of SAM in evaluating thyroid lesions. Methods Formalin-fixed, paraffin sections of 10-μm thickness were scanned using SAM with a 120-MHz transducer. SOS through each area was automatically calculated and plotted on the screen to provide histological images, and SOS of each lesion was compared and statistically analyzed. Results High-concentrated colloids, red blood cells, muscle, and collagen fibers showed great SOS, while low-concentrated colloids, parathyroids, lymph follicles, and epithelial tissues including carcinomas demonstrated less SOS. SOS of colloids that corresponded to their concentration correlated with follicular function. SAM clearly discriminated structure of thyroid components and its resolution quality was as high as that of low magnification of light microscopy. Thyroid tumors were classified into three groups by average SOS: the fast group consisted of follicular adenomas/carcinomas and malignant lymphomas; the slow group contained poorly differentiated and undifferentiated carcinomas; and the intermediate group comprised papillary and medullary carcinomas. Fragmented colloids, irregular-shaped follicles, and desmoplastic reactions were observed in the invasive area of surrounding carcinomas. Conclusions The SAM imaging method had the following benefits: (1) images were acquired in a few minutes without special staining and were well visualized to discriminate each structure and lesion; (2) structural irregularity of follicular size and shape and desmoplastic reactions, which indicated malignancy, was detected; (3) images reflected tissue elasticity, which was statistically comparable among lesions by SOS; (4) follicular functional Thyroid or Peer Rview O NLY/ ot or Disibution activity was predictable by converting colloid concentration to SOS; and (5) tumor classification was predictable by SOS because more poorly differentiated carcinomas had a tendency to show less SOS. A higher frequency transducer will make it possible to use SAM as another precise histological tool for diagnosing carcinoma. Page 1 of 36 Thyroid or Peer Rview O NLY/ ot or Disibution 1 Histological evaluation of thyroid lesions using a scanning acoustic microscope 1 2 Katsutoshi Miura, M.D., Ph.D. 1 and Hiroyuki Mineta, M.D., Ph.D. 2 3 1 Department of Health Science, Pathology and Anatomy, Hamamatsu University School 4 of Medicine 5 2 Department of Otorhinolaryngology, Hamamatsu University School of Medicine 6 Contact information: 7 Katsutoshi Miura, M.D., Ph.D. 8 Department of Health Science, Pathology and Anatomy, 9 Hamamatsu University School of Medicine 10 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192, Japan 11 Telephone: 81-53-435-2811; Fax: 81-53-435-2800 12 E-mail: kmiura@hama-med.ac.jp 13 14 Hiroyuki Mineta, M.D., Ph.D. 15 Department of Otorhinolaryngology, Hamamatsu University School of Medicine 16 1-20-1 Handa-yama, Higashi-ku, Hamamatsu 431-3192, Japan 17 Telephone: 81-53-435-2252; Fax: 81-53-435-2253 18 E-mail: mineta@hama-med.ac.jp 19 Running title: Scanning acoustic microscope for thyroid lesions 20