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Ductal carcinoma in situ with microinvasion (DCISM) is a challenging subtype of breast cancer with controversial invasiveness and prognosis. Accurate diagnosis of DCISM from ductal carcinoma in situ (DCIS) is crucial for optimal treatment and improved clinical outcomes. However, there are often some suspicious small cancer nests in DCIS, and it is difficult to diagnose the presence of intact myoepithelium by conventional hematoxylin and eosin (H&E) stained images. Although a variety of biomarkers are available for immunohistochemical (IHC) staining of myoepithelial cells, no single biomarker is consistently sensitive to all tumor lesions. Here, we introduced a new diagnostic method that provides rapid and accurate diagnosis of DCISM using multiphoton microscopy (MPM). Suspicious foci in H&E‐stained images were labeled as regions of interest (ROIs), and the nuclei within these ROIs were segmented using a deep learning model. MPM was used to capture images of the ROIs in H&E‐stained sections. The intensity of two‐photon excitation fluorescence (TPEF) in the myoepithelium was significantly different from that in tumor parenchyma and tumor stroma. Through the use of MPM, the myoepithelium and basement membrane can be easily observed via TPEF and second‐harmonic generation (SHG), respectively. By fusing the nuclei in H&E‐stained images with MPM images, DCISM can be differentiated from suspicious small cancer clusters in DCIS. The proposed method demonstrated good consistency with the cytokeratin 5/6 (CK5/6) myoepithelial staining method (kappa coefficient = 0.818). [ABSTRACT FROM AUTHOR]

Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor lesion that has the potential to progress to invasive pancreatic cancer, and early and rapid detection may offer patients a chance for treatment before the development of invasive carcinoma. Therefore, the identification of PanIN holds significant clinical importance. In this study, we first used multiphoton microscopy (MPM) combining two‐photon excitation fluorescence and second‐harmonic generation imaging to label‐free detect PanIN and attempted to differentiate between normal pancreatic ducts and different grades of PanIN. Then, we also developed an automatic image processing strategy to extract eight morphological features of collagen fibers from MPM images to quantify the changes in collagen fibers surrounding the ducts. Experimental results demonstrate that the combination of MPM and quantitative information can accurately identify normal pancreatic ducts and different grades of PanIN. This study may contribute to the rapid diagnosis of pancreatic diseases and may lay the foundation for further clinical application of MPM. [ABSTRACT FROM AUTHOR]

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer‐related deaths in China. Rapid and precise evaluation of tumor tissue during lung cancer surgery can reduce operative time and improve negative‐margin assessment, thus increasing disease‐free and overall survival rates. This study aimed to explore the potential of label‐free multiphoton microscopy (MPM) for imaging adenocarcinoma tissues, detecting histopathological features, and distinguishing between normal and cancerous lung tissues. We showed that second harmonic generation (SHG) signals exhibit significant specificity for collagen fibers, enabling the quantification of collagen features in lung adenocarcinomas. In addition, we developed a collagen score that could be used to distinguish between normal and tumor areas at the tumor boundary, showing good classification performance. Our findings demonstrate that MPM imaging technology combined with an image‐based collagen feature extraction method can rapidly and accurately detect early‐stage lung adenocarcinoma tissues. [ABSTRACT FROM AUTHOR]

Collagen fibers play an important role in the progression of liver diseases. The formation and progression of liver fibrosis is a dynamic pathological process accompanied by morphological changes in collagen fibers. In this study, we used multiphoton microscopy for label‐free imaging of liver tissues, allowing direct detection of various components including collagen fibers, tumors, blood vessels, and lymphocytes. Then, we developed a deep learning classification model to automatically identify tumor regions, and the accuracy reaches 0.998. We introduced an automated image processing method to extract eight collagen morphological features from various stages of liver diseases. Statistical analysis showed significant differences between them, indicating the potential use of these quantitative features for monitoring fibrotic changes during the progression of liver diseases. Therefore, multiphoton imaging combined with automatic image processing method would hold a promising future in rapid and label‐free diagnosis of liver diseases. [ABSTRACT FROM AUTHOR]

This paper describes a methodology to differentiate morphea from lichen sclerosus based on examination with multiphoton microscopy (MPM) composed of two‐photon excited fluorescence (TPEF) and second harmonic generation (SHG). Subcellular‐resolution images were acquired by MPM from unstained lesion tissues then process spectral analysis to quantify the TPEF and SHG signals. Moreover, U‐Net was employed to segment elastic fiber in TPEF images to combine with collagen fiber in SHG images for precise fiber quantification. Predictions of segmentation showed excellent performance on several evaluation indicators. The mIoU, mPA, and F1 score reach 0.8516, 0.9281, and 0.941. The quantitative analysis demonstrated the increase of collagen fibers in morphea compared to that in lichen sclerosus cases. Meanwhile, the great diminution of elastic fiber in the dermis of lichen sclerosus was depicted based on MPM imaging. Thus, MPM was comparable to the histopathological examination and our experimental results accurately distinguish between morphea and lichen sclerosus. [ABSTRACT FROM AUTHOR]

The tumor microenvironment (TME) is now recognized as an important participant of tumor progression. As the most abundant extracellular matrix component in TME, collagen plays an important role in tumor development. The imaging study of collagen morphological feature in TME is of great significance for understanding the state of tumor. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), can be used to monitor the morphological changes of biological tissues without labeling. In this study, we used MPM for large-scale imaging of early invasive breast cancer from the tumor center to normal tissues far from the tumor. We found that there were significant differences in collagen morphology between breast cancer tumor boundary, near tumor transition region and normal tissues far from the tumor. Furthermore, the morphological feature of eight collagen fibers was extracted to quantify the variation trend of collagen in three regions. These results may provide a new perspective for the optimal negative margin width of breast-conserving surgery and the understanding of tumor metastasis. [ABSTRACT FROM AUTHOR]

Neoadjuvant treatment is often considered in breast cancer patients with axillary lymph node involvement, but most of patients do not have a pathologic complete response to therapy. The detection of residual nodal disease has a significant impact on adjuvant therapy recommendations which may improve survival. Here, we investigate whether multiphoton microscopy (MPM) could identify the pathological changes of axillary lymphatic metastasis after neoadjuvant chemotherapy in breast cancer. And furthermore, we find that there are obvious differences in seven collagen morphological features between normal node and residual axillary disease by combining with a semi‐automatic image processing method, and also find that there are significant differences in four collagen features between the effective and no‐response treatment groups. These research results indicate that MPM may help estimate axillary treatment response in the neoadjuvant setting and thereby tailor more appropriate and personalized adjuvant treatments for breast cancer patients. [ABSTRACT FROM AUTHOR]

Accurate identification of axillary lymph node (ALN) status is crucial for tumor staging procedure and decision making. This retrospective study of 898 participants from two institutions was conducted. The aim of this study is to evaluate the diagnostic performance of clinical parameters combined with collagen signatures (tumor‐associated collagen signatures [TACS] and the TACS corresponding microscopic features [TCMF]) in predicting the probability of ALN metastasis in patients with breast cancer. These findings suggest that TACS and TCMF in the breast tumor microenvironment are both novel and independent biomarkers for the estimation of ALN metastasis. The nomogram based on independent clinical parameters combined with TACS and TCMF yields good diagnostic performance in predicting ALN status. [ABSTRACT FROM AUTHOR]

Worldwide, the most common malignancy in women is breast cancer. At present, the gold standard for breast cancer diagnosis, namely pathological analysis, cannot reach the subcellular level, and is unable to provide more physiological information. Multiphoton microscopy (MPM) has its unique advantages, such as label‐free detection, high resolution, low damage, deep imaging depth, low photobleaching and phototoxicity, and so on, which make up for the deficiences of traditional methods. This review mainly introduces the principle of MPM and its applications in breast diseases, especially in monitoring the occurrence, progression, metastasis, and therapy response of tumor. At the same time, the performance and progress of MPM combined with nanomaterials in breast cancer treatment are summarized. Finally, we propose the clinical challenges and future research directions of MPM. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) has been widely used for imaging microstructure of biological tissues. In this article, we used MPM to investigate the microstructure changes of normal and cancerous human gastric muscular layer in transverse and longitudinal sections. The results displayed different patterns of microstructure changes of smooth muscular tissue, cell morphology and interstitial fibers in transverse and longitudinal sections, being similar to standard histopathological images but without the need for tissue processing. Our study demonstrated that MPM can bring more detailed complementary information on tissue architecture through observing transverse and longitudinal sections of tissues, which are the important pathological information when the pathologists diagnose the gastrointestinal lesions. These observations indicate that MPM could be an important potential tool to provide real-time pathological diagnosis for gastric cancer in the future. SCANNING 38:357-364, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The continuing development of nonlinear optical imaging techniques has opened many new windows in biological exploration. In this study, a nonlinear optical microscopy—multiphoton microscopy (MPM) was expanded to detect tumor response in rectal carcinoma after neoadjuvant therapy; especially normal tissue, pre- and post-therapeutic cancerous tissues were investigated in order to present more detailed information and make comparison. It was found that the MPM has ability not only to directly visualize histopathologic changes in rectal carcinoma, including stromal fibrosis, colloid response, residual tumors, blood vessel hyperplasia, and inflammatory reaction, which had been proven to have important influence on estimation of the prognosis and the effect of neoadjuvant treatment, but also to provide quantitative optical biomarkers including the intensity ratio of SHG over TPEF and collagen orientation index. These results show that the MPM will become a useful tool for clinicians to determine whether neoadjuvant therapy is effective or treatment strategy is approximate, and this study may provide the groundwork for further exploration into the application of MPM in a clinical setting. [ABSTRACT FROM PUBLISHER]

Rabbits are often preferred to be experimental animals during the skin research. The visualizing and understanding the full-thickness structure of rabbit skin has significance in biology, medicine, and animal husbandry. In this study, multiphoton microscopy (MPM) was employed to examine the rabbit skin on the back, which was based on second harmonic generation and two-photon excited fluorescence. High-resolution images were achieved from the fresh, unfixed, and unstained tissues, showing detailed microstructure of the skin without the administration of exogenous contrast agents. The morphology and distribution of the main components of epidermis and dermis, such as keratin, collagen fibers, elastic fibers, and hair follicles, can be distinctly identified in MPM images. Since the changes in these components are tightly related to skin diseases and wound healing, the noninvasive nature of MPM enables it become a valuable tool in skin research for detecting and monitoring. SCANNING 37:95-100, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Recently, targeting tumor microenvironment has become a novel approach for cancer therapy. Collagen is one of important components of tissue microenvironment, and has been considered as a new visible target for cancer therapy. In this work, multiphoton microscopy (MPM) was used to monitor the changes of collagen in tumor microenvironment during tumor progression. It was found that MPM facilitates imaging of tumor cells and collagen. MPM images in different tumor microenvironment during tumor progression shows obvious increase in cell number and collagen degration. In addition, the quantitative analysis of collagen content and orientation index in tumor microenvironment shows significant alteration during tumor progression. These results suggest that MPM has the ability to monitor the changes of collagen morphology in tumor microenvironment and quantify content and orientation index of collagen during tumor progression. Therefore this technique is a powerful imaging tool for the investigation of targeting tumor microenvironment for cancer therapy. SCANNING 37:17-22, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM) was applied for imaging and quantifying the elastic cartilage regeneration tissue in a rabbit ear model without using labeling agents. Morphology of cells and collagen matrix were analysis, showing significant difference between regenerated and intact cartilage in cellular size and collagen distribution. The results demonstrate that high resolution images provide by MPM are consistent with the histological results, and show additional biological behavior which is not visible in standard histology. Advantages in instrumentation may lead to the application of MPM for intravital detection and treatment. SCANNING 36:540-546, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Elastic cartilage in the rabbit external ear is an important animal model with attractive potential value for researching the physiological and pathological states of cartilages especially during wound healing. In this work, nonlinear optical microscopy based on two-photon excited fluorescence and second harmonic generation were employed for imaging and quantifying the intact elastic cartilage. The morphology and distribution of main components in elastic cartilage including cartilage cells, collagen and elastic fibers were clearly observed from the high-resolution two-dimensional nonlinear optical images. The areas of cell nuclei, a parameter related to the pathological changes of normal or abnormal elastic cartilage, can be easily quantified. Moreover, the three-dimensional structure of chondrocytes and matrix were displayed by constructing three-dimensional image of cartilage tissue. At last, the emission spectra from cartilage were obtained and analyzed. We found that the different ratio of collagen over elastic fibers can be used to locate the observed position in the elastic cartilage. The redox ratio based on the ratio of nicotinamide adenine dinucleotide (NADH) over flavin adenine dinucleotide (FAD) fluorescence can also be calculated to analyze the metabolic state of chondrocytes in different regions. Our results demonstrated that this technique has the potential to provide more accurate and comprehensive information for the physiological states of elastic cartilage. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy (MPM), based on two-photon excited fluorescence and second harmonic generation, enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents. In this paper, we used MPM to image the microstructures of the mucosa in fresh, unfixed, and unstained intestinal tissue of mouse. The morphology and distribution of the main components in mucosa layer such as columnar cells, goblet cells, intestinal glands, and a little collagen fibers were clearly observed in MPM images, and then compared with standard H&E images from paired specimens. Our results indicate that MPM combined with endoscopy and miniaturization probes has the potential application in the clinical diagnosis and in vivo monitoring of early intestinal cancer. [ABSTRACT FROM AUTHOR]

Papulonodular mucinosis ( PM) is a cutaneous clue to the presence and activity of silent lupus erythematosus ( LE), but the exact pathogenesis is still under secret. Moreover, the currently available treatments for PM are not satisfactory. To demonstrate the possibility of multiphoton microscopy ( MPM) to trace the pathological state of PM and evaluate the treatment efficacy, epidermal and dermal alteration in skin lesion with PM before and after treatment was examined using MPM. Microstructure of epidermis as well as content and distribution of collagen and elastin in dermis were quantified to characterize the pathological states of PM. The results showed significant morphological difference between skin lesion before and after treatment, indicating the possibility of MPM to assess the therapeutic efficacy. With the advancement on MPM miniaturization and enhancement of contrast and depth of imaging, the MPM technique can be applied in in vivo tracking PM formation and progression, and leading the better understanding the PM pathogenesis and mechanism of response to any treatment, helping to establish novel effective therapies for PM. SCANNING 35:22-27, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Liver or lung biopsy for suspicious lesions has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. The ability to directly observe cellular and subcellular details and then perform 'optical biopsy' is a major goal in the development of new interventional techniques. Multiphoton microscopy ( MPM) enables real-time noninvasive visualization of tissue architecture and cell morphology in live tissue. We performed a study to evaluate whether MPMcan make real-time optical diagnosis for liver cancer and lung metastasis using an orthotopic rat model with Morris hepatoma. We found that real-time high-resolution MPMimaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, MPMimaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, MPMimaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPMimages were comparable to golden standard hematoxylin-eosin staining images. Moreover, MPMimaging had deep penetration with the capability of optical sectioning. In short, MPMcan make real-time optical diagnosis for liver cancer and lung metastasis. This study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive 'optical biopsy' for liver cancer and lung metastasis in the near future. SCANNING 34: 271-277, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In contrast to colonoscopy biopsy, which contains several disadvantages such as bleeding, sampling error, crush artifact, and time-consuming pathological procedure, multiphoton microscopy (MPM) enables direct noninvasive visualization of tissue architecture and cell morphology in live tissues without the administration of exogenous contrast agents. We performed a proof-of-principle study to evaluate the feasibility of using MPM to make real-time noninvasive optical diagnosis of colorectal cancer by investigating 30 fresh, unfixed, and unstained full-thickness colorectal specimens. We found that MPM images demonstrated irregular tubular structures, reduced stroma, and cellular and nuclear pleomorphism in the cancerous tissues. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were clearly observed in MPM images, which were comparable to golden standard hematoxylin-eosin staining images. Our findings showed that MPM had the potential to make real-time noninvasive optical diagnosis of colorectal cancer. With miniaturization and integration of colonoscopy, MPM has a promising future in real-time noninvasive 'optical biopsy' for colorectal cancer. SCANNING 34: 181-185, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We demonstrate the application of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation signals in imaging two-layered structures of cervical tissue in mice. It was found that MPM is effective for visualizing two-layered cervical microstructures, monitoring the metabolic activity and the density (nuclear-to-cytoplasmic) of epithelial cells, measuring the size of epithelial cell nuclei and the thickness of the epithelial layer, and quantifying collagen in the stroma. These results suggest that, with the advent of clinical portability of typical multiphoton endoscopes, the MPM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of cervical cancer. [ABSTRACT FROM AUTHOR]

In the current clinical care practice, Gleason grading system is one of the most powerful prognostic predictors for prostate cancer (PCa). The grading system is based on the architectural pattern of cancerous epithelium in histological images. However, the standard procedure of histological examination often involves complicated tissue fixation and staining, which are time‐consuming and may delay the diagnosis and surgery. In this study, label‐free multiphoton microscopy (MPM) was used to acquire subcellular‐resolution images of unstained prostate tissues. Then, a deep learning architecture (U‐net) was introduced for epithelium segmentation of prostate tissues in MPM images. The obtained segmentation results were then merged with the original MPM images to train a classification network (AlexNet) for automated Gleason grading. The developed method achieved an overall pixel accuracy of 92.3% with a mean F1 score of 0.839 for epithelium segmentation. By merging the segmentation results with the MPM images, the accuracy of Gleason grading was improved from 72.42% to 81.13% in hold‐out test set. Our results suggest that MPM in combination with deep learning holds the potential to be used as a fast and powerful clinical tool for PCa diagnosis. [ABSTRACT FROM AUTHOR]

Tumor microenvironment and metabolic activity in gliomas are the important biomarkers to evaluate the progression of gliomas. Many evidences have suggested that the targeting of metabolic activity and tumor microenvironment simultaneously can be more effective to take the tumor therapy. Therefore, the noninvasive, accurate assessment of tumor microenvironment and metabolic activity is quite important in clinical practice. Multiphoton microscopy (MPM), based on two‐photon‐excited fluorescence and second harmonic generation was performed on unstained glioma tissues. With our combined image analysis approaches, our research findings indicate that MPM is able to qualitatively and quantitatively describe the microenvironment characteristics in gliomas, such as collage deposition in extracellular matrix, lymphocyte infiltration and tumor angiogenesis, etc. Meanwhile, the metabolic activity can also be quantitatively evaluated by optical redox ratio, NADH and FAD intensity. With the microendoscope and fiberscope are portable, MPM technique can be used to perform in‐vivo studies and clinical examinations in gliomas. [ABSTRACT FROM AUTHOR]