Label-free multiphoton excitation imaging as a promising diagnostic tool for breast cancer.

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.

Nonlinear Optics with Near-Infrared Excitation Enable Real-Time Quantitative Diagnosis of Human Cervical Cancers.

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.

Practical method of cell segmentation in electron microscope image stack using deep convolutional neural network☆.

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.

Possible etiology of calculi formation in salivary glands: biophysical analysis of calculus.

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.

Clinical investigation of amelanotic malignant melanoma in the oral region.

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.

Ultrastructural analysis of salivary calculus in combination with X-ray microanalysis.

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.

Oral squamous cell carcinoma: electron microscopic and immunohistochemical characteristics.

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.

An ultrastructural study of calcifying odontogenic cyst, especially calcified material.

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.

Ultrastructure of oral sarcoma.

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.