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Histopathological diagnosis is the ultimate method of attaining the final diagnosis; however, the observation range is limited to the two-dimensional plane, and it requires thin slicing of the tissue, which limits diagnostic information. To seek solutions for these problems, we proposed a novel imaging-based histopathological examination. We used the multiphoton excitation microscopy (MPM) technique to establish a method for visualizing unfixed/unstained human breast tissues. Under near-infrared ray excitation, fresh human breast tissues emitted fluorescent signals with three major peaks, which enabled visualizing the breast tissue morphology without any fixation or dye staining. Our study using human breast tissue samples from 32 patients indicated that experienced pathologists can estimate normal or cancerous lesions using only these MPM images with a kappa coefficient of 1.0. Moreover, we developed an image classification algorithm with artificial intelligence that enabled us to automatically define cancer cells in small areas with a high sensitivity of ≥0.942. Taken together, label-free MPM imaging is a promising method for the real-time automatic diagnosis of breast cancer.

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Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection.Significance:This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye.

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Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection. Significance: This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye.

Segmentation of three-dimensional (3D) electron microscopy (EM) image stacks is an arduous and tedious task. Deep convolutional neural networks (CNNs) work well to automate the segmentation; however, they require a large training dataset, which is a major impediment. In order to solve this issue, especially for sparse segmentation, we used a CNN with a minimal training dataset. We segmented a Cerebellar Purkinje cell from an image stack of a mouse Cerebellum cortex in less than two working days, which is much shorter than that of the conventional method. We concluded that we can reduce the total labor time for the sparse segmentation by reducing the training dataset.

Oral malignant melanoma is extremely rare and carries a poor prognosis. The treatment of choice remains controversial. We retrospectively studied 35 patients with primary malignant melanoma of the oral cavity between 1970 and 2001 to define the clinical features of this disease and evaluate treatment methods. The main variables studied were clinical findings, response to therapy, and outcome. Surgery with complete macroscopic resection was performed at the primary site in 13 patients (surgery group) and radiotherapy was done without surgery in 17 (non-surgery group). The 5-year cumulative survival rate was 15.4% in the surgery group, 35.3% in the non-surgery group, and 21.8% overall. Distant metastasis was present in 64.7% (11/17) of the non-surgery group and 76.9% (10/13) of the surgery group. Improved outcome in oral malignant melanoma requires the development of new therapies and the prevention of distant metastasis.

We have investigated thermoelectric properties of oxygen-included iron-silicide thin films prepared by facing target sputtering for applying them to thermoelectric devices. The highest Seebeck coefficient of 17.84 μV/K are obtained. It is the first attempt to investigate thermoelectric properties of oxygen-included iron-silicide films.

Squamous cell carcinomas of the tongue (n = 49), consisting of 21 cases with cervical lymph node metastasis and 28 nonmetastatic cases, were examined by electron microscopy with special emphasis on tumor cell attach-ment. No difference of tumor size (T classification) or pathological findings between the metastatic group and the nonmetastatic group was found. The metastatic cases had numerous microvilli and a small number of desmosomes regardless of the width of the intercellular spaces. The nonmetastatic cases had few microvilli in relatively wide intercellular spaces, or, in the cases in which numerous microvilli were present in the narrow intercellular spaces, the tumor cells were connected by a large number of desmosomes. Transferrin receptor, which is a marker of cell proliferation, was localized in the cell membrane by immunohistochemistry and especially in microvilli by immunoelectron microscopy. It is suggested that microvilli might be related to the proliferation and the metastasis of squamous cell carcinoma cells.

This study investigated the tissue response associated with dental titanium implants. The mandibular third and fourth premolars and first molar of three adult beagle dogs were extracted bilaterally. Healing was then allowed for 3 months. Six titanium implants were placed in the mandibles of a dog. Three weeks after the implantation, mandibular sections containing the implants were retrieved with the use of a bone saw and investigated by light and electron microscopy, X-ray microanalyzer, and electron diffraction. Scanning electron microscopic observation showed titanium particles on the implant-bone interface, and investigation by microanalyzer revealed titanium not only on the implant-bone interface but also in the bone tissue. Transmission electron microscopic observation and investigation by electron diffraction showed titanium in the bone matrix and cells other than macrophages. In this study, titanium particles from the dental implant were recognized morphologically in the surrounding bone tissue. Thus, study of the influence of titanium particles on the human body is needed.

A rare case of lentigo maligna in the oral cavity was investigated by light and electron microscopy. Cutaneous lentigo malignas often develop to malignant melanomas. However, the electron microscopic examination revealed that even though there were a large number of melanosomes, most of them were late stage and had membrane structure, and positive staining with HMB-45 was not recognized. From our findings, it is difficult to conclude that oral lentigo malignas develop malignant melanomas, and thus further studies are needed.

The response to radiation of the SSM-1 cell line, derived from oral malignant melanoma, was investigated by survival curve and by light and electron microscopic abservation. Survival curve of the cells was investigated by the colony formation assay. X-irradiation with 2, 4, and 8 Gy was performed against the cell line. Morphological changes of the cells at 6, 24, 72, and 120h after irradiation were examined by both light and electron microscopy. The survival curve of the SSM-1 cells showed higher radiosensitivity than that of cutaneous melanoma cell lines. Giant cells and multinucleated cells were found only 120h after 8Gy irradiation. Ultrastructural observation revealed changes in nucleus and melanosomes; melanosomes increased in number at 120h after 8Gy irradiation. Further alterations after irradiation were noticed primarily in the nucleus. Radiosensitivity of the SSM-1 cell line derived from oral malignant melanoma was higher than that of cutaneous melanoma cell lines. The results of this study may support the concept that radiotherapy is effective for oral malignant melanoma.

A rare case of angiosarcoma in the oral cavity was investigated by light and electron microscopy. Immunohistochemical findings and the recognition of Weibel-Palade bodies supported the opinion that this tumor was a malignant hemanigoendothelioma. However, electron microscopic observation revealed various tumor cells resembling endothelial cells, pericytes, and undifferentiated mesenchymal cells, suggesting that the origin of this tumor might be undifferentiated mesenchymal cells.

We have treated a patient with a nodular amyloidosis in the center of the dorsum of the tongue. Ultrastructural observations of human amyloidosis have been reported by many investigators, but there have been few reports of nodular amyloidosis of the tongue. Ultrastructural investigation showed a number of amyloid fibrils around and in the plasmocytoid cells, and severe pressure atrophy was found in many stromal cells, especially in endothelial cells.

In several studies, numerous microvilli were found on the surfaces of malignant tumor cells which had high growth potentials, and some tumor-related antigens were identified on these microvilli. Transferrin receptor (TfR) is expressed in proliferating cells and regarded as a tumor marker. In human carcinoma cell lines (HeLa cells, KB cells, and HSC-7 cells), significantly more microvilli were observed in the subconfluent phase than in the confluent phase. Also, TfR was found along the plasma membranes, especially well developed microvilli, coated pits, and coated vesicles in both phases by the immunoelectron microscopic method. These results indicate that the proliferative ability of the tumor cells might correlate with the development of microvilli.

A rare case of infantile fibrosarcoma in a mandible was investigated by light and electron microscopic observation. The usual features of malignancy may not accurately predict malignant behavior in infantile fibrosarcoma. We investigated the correlation between the clinical- and the morphological features of the tumor. All tumor tissues were highly cellular, and the tumor cells were often arranged in a herring bone-like pattern. Most of them were spindle-shaped. Electron microscopic observation revealed the rough endoplasmic reticulum, whose constituent cisternae were dilated and contained amorphous or granular material, resembling macrophages. These ultrastructural findings might be related to the good clinical course of the presented case.

A rare variety of malignant melanoma in the maxilla was studied by electron microscopic observation and an immunohistochemical investigation of the transferrin receptor (TfR). A number of low-stage melanosomes were seen, and the TfR was found in both the cell membrane and the cytoplasm.

1978年より1988年までの11年間に東京医科歯科大学歯学部第一口腔外科を受診した舌扁平上皮癌1次症例 (177例) のうち, T1 (56例), T2 (80例) 症例の生検標本を病理組織学的 (WHO分類, Y-K分類) に2検索し, 後発頸部リンパ節転移 (以後, 後発頸転移と略) との関係を検討した。後発頸転移は, WHO分類, Y-K分類とも Grade が進むにつれ増加していた。治療法別にみると, 後発頸転移は, 外科療法群に比べ放射線療法群に多かった。放射線療法群では, WHO分類 Grade IIIならびにY-K分類 Grade 4C, 4Dに後発頸転移例が多い傾向であったが, 外科療法群では, 一定の傾向はみられなかった。

Rare varieties of malignant fibrous histiocytoma occurring in the maxilla are reported in this study.We observed 3 cases of malignant fibrous histiocytoma by light-and electron-mlcroscopy and examined the histiogenesis.Fibroblast-like cells, histiocyte-like cells, primitive mesenchymal cells, multinucleated giant cells and xanthomatous nucleated giant cells and xanthomatous histiocytes were recognized, and it was suggested that these tumors might be of primitive mesenchymal cell origin. When the clinical course is relatively good, myxoid type and giant cell type were shown and a number of fine granular materials were found in the stroma and rough endoplasmic reticulum.

Amelanotic malignant melanoma rarely occurs in the oral cavity. We encountered and treated patient with a nodular amelanotic malignant melanoma arising in the maxilla. An 84-year-old woman was referred to our hospital because of a painless, pedunculated, nonpigmented tumor in the center of the anterior alveolar region of the maxilla. The clinical diagnosis was a benign tumor, and the lesion was excised. However, the histopathological diagnosis was malignant fibrous histiocytoma.Three months later, a small, partially pigmented tumorous lesion appeared in the same region. The tumor was resected, and minute quantities of melanin granules were observed in some tumor cells histologically. The specimen was diagnosed as amelanotic malignant melanoma. We performed immunohistochemical studies, and positive immunoreactivity for HMB-45 was found in each specimen, confirming the diagnosis.Although the patient subsequently underwent rotation radiotherapy to the maxilla, recurrence was found in the nasal cavity and ethmoidal sinuses 13 months after radiation therapy. The tumor grew rapidly, and the patient died 2 years 5 months after presentation.

A clinico-statistical and follow-up study was made in 100 cases of mandibular condylar fracture in the First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University. The following results were obtained;1. There were 62 males and 38 females. The ages of the patients at the time of injury ranged from 0 to 80, and the average was 29. 5 years old.2. Eighty-five cases were treated within 2 weeks after injury.3. Of all cases, 85 cases were unilateral fracture, and 15 were bilateral fracture. Direct fracture of the condyle was observed only in 5 cases.4. Fracture position and form were determined according to the classification described by Kubo. Mandibular cervical dislocation was most frequently observed.5. All cases were treated with a conservative therapy; intermaxillary fixation, chin cap, mouth opening training, etc.6. In the results of follow-up examination by mail, 18.5% of all patient had a restriction of the mouth opening, 40.7% had a pain, and 64.8% had a joint sound. In unilateral fractures, 39.1% of the non-fractured joint had a joint sound.7. The analytical results on the fracture type and present symptoms revealed that the patients with no displacement fracture had little dysfunction. On the contrary, those who had a displacement fracture had more problems. In conclusion, a preserved anatomical structure of TMJ proved to be the most important factor to heal mandibular condylar fracture.

Sialolithiasis is one of the common diseases of the salivary glands. It was speculated that, in the process of calculi formation, degenerative substances are emitted by saliva and calcification then occurs around these substances, and finally calculi are formed. However, the exact mechanism of the formation of calculi is still unclear. In this study, we identify some possible etiologies of calculi formation in salivary glands through biophysical analysis. Calculi from 13 patients with submandibular sialolithiasis were investigated by transmission electron microscopy, scanning electron microscopy, X-ray microanalyzer, and electron diffraction. Transmission electron microscopic observation of calculi was performed in the submandibular gland (n = 13). In 3 of the 13 cases, a number of mitochondria-like structures and lysosomes were found near calcified materials. Scanning electron microscopic examination of these materials revealed that there were lamellar and concentric structures and that the degree of calcification was different among the calculi. X-ray microanalysis disclosed the component elements in the calculi to be Ca, P, S, Na, etc., and the main constituents were Ca and P. The calcium-to-phosphorus ratio was 1.60-1.89. Analysis of the area including mitochondria-like structures, lysosomes, and the fibrous structures by electron diffraction revealed the presence of hydroxyapatite and calcified materials. It is speculated that mitochondria and lysosomal bodies from the ductal system of the submandibular gland are an etiological source for calcification in the salivary gland.

Purpose Amelanotic oral malignant melanoma (AOMM) is a rare tumor that is difficult to diagnose. We studied the clinical and pathologic features of nine cases of this tumor to define diagnostic criteria and estimate prognoses for 2 different types of AOMM. Patients and methods Nine patients with 2 different types of primary AOMM were examined between 1970 and 2002. The histopathology of surgical specimens was studied, uncertain diagnoses were supported by immunohistochemical reactions, and electron microscopy and prognoses were reviewed retrospectively. Results AOMM without radial growth phase may be particularly difficult to diagnose correctly without immunohistochemical assistance. Tumors consisted of a mixture of polygonal and spindle cells in different ratios in tumors with and without radial growth phase. The life span ranged from 3 months to 6 years 3 months, and all 9 patients died of the tumor. In 7 of the 9 cases, distant metastases were found. Conclusions AOMM without radial growth phase may be misdiagnosed as epulis or squamous cell carcinoma. Questionable lesions, particularly maxillary and palatal lesions, must be biopsied for histopathologic and possibly immunohistochemical examinations followed by rapid treatment. The prognosis of AOMM was poor.

Sarcoma of the oral region is extremely rare and ultrastructural studies of the tumor are limited in number. We collected oral sarcomas, such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, alveolar soft-part sarcoma, solitary plasmacytoma, and osteosarcoma, and performed ultrastructural studies of these tumors. The value of these studies for an understanding of the biological behavior of the tumors was then investigated. In these studies, electron microscopic examinations of oral sarcoma were of assistance in our attempt to establish correct diagnosis and histogenesis. Data from the studies of oral sarcoma by light microscopy, electron microscopy, and immunohistochemistry should be accumulated.

We developed new ultrasonic tips for apicoectomy with retrograde gutta-percha root filling. To investigate the cutting efficency and safety of the ultrasonic tips, before clinical application we carried out an experiment on enlargment of the root canal of human extracted teeth. The results, showed that enlagement of the root canal with the ultrasonic system could be performed rapidly and accurately compared with manual enlargement using angle reamers. Our ultrasonic tips did not cause any problems for teeth during the experiment. We believe that our new ultrasonic tips are simple and convenient for clinical application.

Oral squamous cell carcinoma is the most common oral malignancy, and we performed electron microscopic and immunohistochemical investigation of the tumor. In patients with cervical metastasis, microvilli were developed and a small number of desmosomes were found, regardless of the width of the intercellular spaces. In patients without the metastasis, few microvilli were found in relatively wide intercellular spaces, or numerous microvilli were found in narrow intercellular spaces, and a large number of desmosomes were shown. However, these findings were different from those of tumors that had received radiotherapy, in which numerous microvilli and a small number of desmosomes were found in the nonmetastatic cases. Transferrin receptor, which is a marker of cell proliferation, was localized on the cell membrane, especially in microvilli. Ultrastructural similarity between the primary tumor and the metastatic tumor was recognized, however, the features of microvilli, desmosomes, and the intercellular spaces differed between them in most cases. It is suggested that microvilli might be related to the metastasis of oral squamous cell carcinoma cells. Immunohistochemically, the protein expression of p53 and pRb2/p130 was related to the clinical course of the patients with oral squamous cell carcinoma; the mechanism of the synthesis of these proteins should be investigated in order to understand the biological behavior of the tumor.

The ultrastructural features of calcification in a case of calcifying odontogenic cyst (COC) were studied. Scanning electron microscopic examination of the inner parts of the cyst wall revealed many short microvilli, and X-ray microanalysis of the high-density masses in the intercellular parts showed prominent calcium peaks, which meant that these masses were calcified materials. On transmission electron microscopic observations, many calcifications exhibited a distinctive ring formation around the periphery of a central core that consisted of an amorphous structure. These calcifications were observed with necrotic remnants of nuclear material and many identifiable mitochondria, thin fibers, and epithelial cells. The cytoplasm of ghost cells consisted of numerous short electron-dense tonofilament bundles. Needle-like structures were shown in the tonofilament bundles. X-ray diffraction analysis showed that the needle-like crystals were hydroxyapatite. It is suggested that calcification in a COC may be related to degenerative mitochondria and tonofilament bundles of ghost cells.

Malignant melanoma in the oral region is reviewed in its clinicopathological aspects. Clinically the melanomas were classified into five types; however, there were no histopathological differences according to these clinical types. Electron microscopic observation of the melanomas and primary culture cells derived from oral malignant melanoma revealed that, in patients whose prognoses were relatively good, many mature-stage melanosomes were found. Immunohistologically, there was a positive reaction for transferrin receptor and the expression of pRb2/p130 was found in only 2 of 13 patients, who are still alive after periods of over 14 years. The radiosensitivity of a cell line derived from human oral malignant melanoma was greater than that in cell lines derived from human cutaneous melanoma and with radiation, the number of melanosomes increased. There are very few clinical cases of oral malignant melanoma and very few cell lines derived from oral malignant melanoma, and so findings in these patients and these cell lines should be accumulated in order to clarify the biological behavior of oral malignant melanoma.

We previously reported that pRb2/p130 gene, one of the Rb family members, was immunohistochemically abundantly expressed in well-differentiated oral squamous cell carcinomas, whereas in undifferentiated ones the expression was low. Oral malignant melanoma is extremely rare, however the prognosis is poor because it tends to locally invade tissue or metastasize and its biological behavior appears to be different from cutaneous malignant melanoma. The present study dealt with the expression of pRb2/p130, Rb, p53, and p16 in 13 cases of malignant melanoma of oral mucosa as revealed by immunohistochemical staining. The stage classification of the 13 patients was as follows; stage II: eight patients, stage III: three patients, and stage IV: two patients. pRb2/p130 was expressed in only two stage II-cases, neither of which have shown any evidence of recurrence or metastasis for over 14 years. Positive staining for Rb was found in three cases consisting of one stage II-case, one stage III-case, and one stage IV-case. p53 was expressed in two cases, one a stage II and the other a stage IV. Positive staining for p16 was found in seven cases consisting of four stage II-cases, two stage III-cases, and one stage IV-case. pRb2/p130 may be inversely correlated with the malignancy of oral malignant melanoma, but further study is needed.

In order to study the efficiency of radiation therapy for human oral malignant melanoma, radiosensitivity for SSM-1 cell line derived from oral malignant melanoma was investigated by colony formation rate, survival curve, light microscopic observation, ultrastructual observation and immunocytochemical study with HMB-45. Survival curve of the SSM-1 cells was higher than that of cutaneous melanoma cell lines for radiosensitivity. Uniform masses of SSM-1 cells were found even with high dose irradiation. Giant cells and multiple nuclei cells were found after high dose irradiation. By ultrastructural observation, various nucleoi, chromatins and nucleus structures were found after low dose irradiation. Immunocytochemically, hot nodules of SSM-1 cells were found to have HMB-45 antibody, and the hot nodules increased after irradiation, suggesting increased number of melanosomes. By ultrastructural observation, the number of melanosomes was found to have increased after low dose irradiation. Radiosensitivity of SSM-1 cell line derived from oral malignant melanoma was higher than that of cutaneous melanoma cell lines, and the results of this study might support that radiotherapy is helpful for oral malignant melanoma.

We examined the ultrastructures of the tongue squamous cell carcinomas which received radiotherapy and compared the metastatic cases with the non-metastatic cases. The results were as follows: 1. The non-metastatic cases were clinically T1N0 or T2N0 and histologically Grade I or II of the WHO classification. Electronmicroscopic observation of these cases revealed that numerous microvilli and a small number of desmosomes were found and the cell attachment seemed to be weak. These findings were different from those of the non-metastatic cases which received surgery. 2. In the metastatic cases of which the primary lesions were controlled, microvilli were not developed and a number of desmosomes were shown as compared with those in the non-metastatic cases. However, cell atypia and dispersion of the nuclear chromatin were clearly recognized.

Ultrastructural studies of salivary calculi were performed. Scanning electron microscopic examination of the calculi revealed lamellar and concentric structures. Granular or globular structures and pyramid structures were found on the surface of the calculi, and in some cases a scaly structure corresponding to fiber and bacteria was recognized. X-ray microanalysis showed the main constitutes of the calculi to be Ca and P. Transmission electron microscopic examination revealed a fine fibrous structure near the degenerated organelles, and analyses of the structure by electron diffraction revealed hydroxyapatite. Calcification was found around the degenerative organelles in the form of lipid-like structures, mitochondria, lysosomes, and microbial structures. Judging from our results, as one of the processes leading to calculi formation, it is speculated that degenerative substances are emitted by saliva, by some phenomenon, and calcification around these substances then occurs, contributing to the formation of calculi.