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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., (© 2024 Wiley‐VCH GmbH.)

Neuroendocrine tumors (NETs), which are rare and slow-growing neoplasms, pose a diagnostic challenge as they are clinically silent at the time of presentation. Here, gastrointestinal neuroendocrine tumors were researched by nonlinear microscopy, and results demonstrate that this technique has the capability to identify neuroendocrine tumors in the absence of labels and can, in particular, detect rare neuroendocrine tumor cells, vascular invasion, desmoplastic reaction, and fibroelastosis induced by neuroendocrine tumors. These conclusions highlight the possibility of nonlinear optical microscopy as a diagnostic tool for label-freely differentiating neuroendocrine tumors by these histopathologic features.

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., (© Wiley Periodicals, Inc.)

Stroma plays an important role during epithelial tumor progression. Probing stroma alteration may become an intrinsic indicator for evaluating epithelial tumor progression. In this review, we summarize our recent works on stromal alterations as quantitative optical biomarkers of epithelial tumor progression by use of nonlinear optical microscopy., (© 2013 Wiley Periodicals, Inc.)

Esophageal cancer is one of the most common cancer and leading cause of cancer death worldwide. Multiphoton microscopy (MPM) has become a novel optical tool of choice for imaging tissue architecture and cellular morphology based on two-photon excited fluorescence and second harmonic generation. In this study, we used MPM to image microstructure of human normal esophagus, carcinoma in situ, and early invasive carcinoma in order to investigate the morphological change of tissue structure during the early phase of tumor progression. The diagnostic features such as the appearance of cancerous cells, the absence of the basement membrane were extracted to distinguish between normal and cancerous esophagus tissue. The infiltration depth during tumor progression was determined by the appearance of cancerous cells. The significant change of layer structure between cancerous tissue and normal esophagus was described. We also quantitatively described the differences of morphology between normal and cancerous cells. These results correlated well with the corresponding histological findings. With the advancement of clinically miniaturized MPM and the multi-photon probe, combining MPM with standard endoscopy will therefore allow us to make a real-time in vivo diagnosis of early esophageal cancer at the cellular level., (© Wiley Periodicals, Inc.)

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., (© Wiley Periodicals, Inc.)

Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast.

Nonlinear optical microscopy (NLOM) was applied for monitoring dermal wound healing after mesenchymal stem cell (MSC) transplantation. Our results showed that NLOM can reveal different regeneration processes of collagen in nontreated and MSC-treated wound dermis. Specifically, the temporal increases in the intensity of second-harmonic-generation signals can quantify kinetic properties of collagen regeneration. Orientation analysis of collagen fiber bundles can monitor the formation of new normal collagen fiber bundles, which is an indicator for evaluating the therapy response. It was also found that NLOM can track MSCs' location and recruitment. These findings suggested that NLOM is ideal for monitoring the progress of dermal wound healing.

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]

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]

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]

Glioblastoma (GBM) is a highly angiogenesis malignant tumor with a significant heterogeneity of vascular morphology. The vascular patterns could be served as an indicator of vascular morphological complexity that associated with postoperative prognosis of patients with GBM. Here, multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), is applied for detecting the architectural feature of blood vessels from ex vivo, unfixed, and unstained human GBM specimens. Combined with the image processing algorithms, the morphology, location, orientation, alignment, and density of vascular collagen are qualitatively and quantitatively visualized, revealing the presented method not only contribute to differentiate GBM from normal brain tissue, but also provide important indicators of different vascular patterns, which may serve as a reference to the prognosis more accurately. Therefore, these results can provide prognosis-related histopathological diagnosis of GBM, holding a translational potential to be used as a label-free, quick, and on-site imaging tool for clinicians to determine the extent of excision involving brain functional areas. [ABSTRACT FROM AUTHOR]

Breast cancer can be cured by early diagnosis. Appropriate and effective clinical treatment benefits from accurate pathological diagnosis. However, due to the lack of effective screening and diagnostic imaging methods, early stages of breast cancer often progress to malignant breast cancer. In this study, multiphoton microscopy (MPM) via two-photon excited fluorescence combined with second-harmonic generation was used for identifying the early stages of breast ductal carcinoma. The results showed differences in both cytological features and collagen distribution among normal breast tissue, atypical ductal hyperplasia, low-grade ductal carcinoma in situ, and high-grade ductal carcinoma in situ with microinvasion. Furthermore, three features extracted from the MPM images were used to describe differences in cytological features, collagen density, and basement membrane circumference in the early stages of breast ductal carcinoma. They revealed that MPM has the ability to identify early stages of breast ductal carcinoma label-free, which would contribute to the early diagnosis and treatment of breast cancer. This study may provide the groundwork for the further application of MPM in the clinic. [ABSTRACT FROM AUTHOR]

Background and Aims: Endoscopic submucosal dissection (ESD) has become the primary option for the treatment of early gastric cancer (EGC). Thus, it is necessary to diagnose whether residual cancer cells exist in the ESD specimen margins, which can affect tumor recurrence and survival rates in the future. Multiphoton microscopy (MPM) can be suitably used for nondestructive imaging of biological tissue on a cellular level to enable real-time guidance during endoscopic therapy. Considering this, the objective of this study is to explore the practicality of MPM for the diagnosis of ESD specimen margins in the case of EGC.Methods: First, a total of 20 surgical samples was imaged using the proposed MPM technique to obtain two-photo excited fluorescence signal from the intrinsic fluorescent substances within cells and second-harmonic generation signal from collagen; these signals were used to determine MPM pathological features for margin diagnosis. Then, a double-blind study of 50 samples was conducted to evaluate the diagnosis results based on the obtained MPM pathological features.Results: Multiphoton microscopy can accurately identify the cytological and morphological differences between tissue in the negative and positive margin. The sensitivity, specificity, accuracy, negative predictive, and positive predictive values of MPM in the diagnosis of ESD specimen margins were 97.62, 75.00, 94.00, 95.35, and 85.71%, respectively.Conclusion: These results indicate that MPM can be used as an effective, real-time, and label-free novel method to determine intraoperative resection margins. [ABSTRACT FROM AUTHOR]

Without sophisticated data inversion algorithms, nonlinear optical microscopy can acquire images at subcellular resolution and relatively large depth, with plausible endogenous contrasts indicative of authentic biological and pathological states. Although independent contrasts have been derived by sequentially imaging the same sample plane or volume under different and often optimized excitation conditions, new laser source engineering with inputs from key biomolecules surprisingly enable real-time simultaneous acquisition of multiple endogenous molecular contrasts to segment a rich set of cellular and extracellular components. Since this development allows simple single-beam single-shot excitation and simultaneous multicontrast epidirected signal detection, the resulting platform avoids perturbative sample pretreatments such as fluorescent labeling, mechanical sectioning, scarce or interdependent contrast generation, constraints to the sample or imaging geometry, and intraimaging motion artifacts that have limited in vivo nonlinear optical molecular imaging. [ABSTRACT FROM AUTHOR]

The development of imaging technique to visualize and quantify the structural alteration of the spinal cord injury (SCI) may lead to better understanding and treatments of the injuries. In this work, multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) was tentatively applied to quantitatively visualize the cellular microstructures of SCI to demonstrate the feasibility and superiority of MPM in SCI imaging. High-contrast MPM images of normal and injured spinal cord tissue were obtained for comparison. Moreover, the changes of injured spinal cord were characterized by the quantitative analysis of the MPM images. These results showed that MPM combined with quantitative method has the ability to identify the characteristics of spinal cord injury including the changes in the contents of nerve fibers and extracellular matrix. With the advancement of MPM, we believe that this technique has great potential to provide the histological diagnosis for the monitoring and evaluation of SCI. [ABSTRACT FROM AUTHOR]

Cortical structures in the central nervous system exhibit an ordered laminar organization. Defined cell layers are significant to our understanding of brain structure and function. In this work, multiphoton microscopy (MPM), based on second harmonic generation and two-photon excited fluorescence, is applied for visualizing the mouse and human cerebral cortex from fresh, unfixed, and unstained specimen. We show that label-free MPM is able to effectively visualize microscopic scale of cortical neurons, six-layer cortical lamination, and functional cortex that correlates with the corresponding stained histological images. Moreover, we further demonstrate that MPM is feasible for identifying focal cortical dysplasia (FCD) in the human cerebral cortex. The border between gray and white matter, abnormal radial cortical lamination, immature small diameter neurons, pia mater, and blood vessels are also clearly visualized as the typical histopathologic hallmark of FCD. In addition, to diagnose FCD more accurately and rapidly, an image processing method is employed to extract MPM image features for auxiliary identification of FCD specimen. These results offer additional metrics to pathologists and surgeons, which hold the translational potential to be used as a diagnostic tool into routine clinical use. [ABSTRACT FROM AUTHOR]

In this paper, a nonlinear optical microscopy employing two-photon excited fluorescence and second-harmonic generation was used for the detection of premalignant gastric lesions. It was found that gland morphology and collagen structure in mucosa will change with the progression of gastric diseases from normal to intestinal metaplasia, to low-grade intraepithelial neoplasia, and to high-grade intraepithelial neoplasia, and this microscopy was able to directly distinguish these warning symptoms. Furthermore, two features were quantified from nonlinear optical images to demonstrate the changes of gland size and collagen content during the development process of preneoplastic lesions. These results clearly show that nonlinear optical microscopy can effectively differentiate normal and precancerous gastric tissues without contrast agents, which would be helpful for early diagnosis and treatment of gastric diseases. This study may provide the groundwork for further application of nonlinear optical microscopy in clinical practice. [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]

Cartilage is one of the most important tissues in mammalian growth and is classified into three types: hyaline, fibro, and elastic. To illustrate its potential as a clinical diagnostic tool, nonlinear optical microscopy (NLOM) was applied to simultaneously investigate images and spectra from three different types of rat cartilage. High-resolution images were obtained using the two-photon excited fluorescence and second-harmonic generation (SHG) of structures within fresh specimens. Since samples were imaged without hematoxylin and eosin (H&E) staining, detailed observations of tissue morphology and microstructural distribution were obtained without shrinkage or distortion. Nonlinear optical spectra confirmed that elastic cartilage is rich in elastic fibers, of which there are few in both hyaline and fibro cartilages. Structural dimensions were also measured with this technique, revealing the average area of the lacuna (398.36 \pm 52.95\ \mu\textm^2), or the average thickness of the territorial matrix (0.83 \pm 0.26\ \mu\textm), within elastic cartilage. Fourier analysis of SHG images was performed to further confirm that collagen in fibrocartilage showed significantly greater organization and directionality compared with collagen in hyaline cartilage $(p\ <\ 0.01)$. Our results show that each type of cartilage has different structural features and proportion of matrix, which may significantly contribute to pathology when damaged. Our findings demonstrate that NLOM holds the potential as a clinically relevant diagnostic tool for imaging degenerative tissues or assessing wound repair following cartilage injury. [ABSTRACT FROM PUBLISHER]

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]

Scar margins dynamic with time were quantitatively characterized using multiphoton microscopy (MPM). 2D large-area and 3D focused images of elastin and collagen at scar margins were obtained to extract quantitative parameters. An obvious boundary was observed at the scar margin, showing altered morphological patterns of elastin and collagen on both sides. Content alteration of elastin and collagen between the two sides of boundary were defined to characterize scar margins from different individuals. The statistical results from 15 normal scar samples strongly demonstrated that content alteration degree of elastin and collagen had decreasing tendency with the increase of patient age or scar duration, consistent with the fact of normal scars regressing spontaneously over time. It indicated that alteration degree can potentially serve as quantitative indicators to examine wound healing and scar progression over time. With the advent of clinical portable multiphoton endoscopes, the MPM technique can be applied in tracking scar formation and progression in vivo by examination of scar margin. [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]

We investigate the prognostic value of tumour-infiltrating lymphocytes (TILs) based on the evaluation of the present frequency in patients with breast cancer rather than that of the density proposed in previous research. Multiphoton microscopy (MPM) was introduced to label-freely obtain TIL images from a total of 564 patients, and then TILs were redefined as TILs-1 to TILs-3 from MPM images according to the relative positions between TILs, tumour cells and collagen fibres. More seminally, a new method, which was based on the present frequency of TILs-1 to TILs-3, was presented for assessing the predictive ability of TILs, and then a tumour-infiltrating lymphocytes score (TILs-score) for each patient was obtained by ridge regression analysis. Data results from Cox proportional hazards regression analysis showed that the TILs-score was an independent prognostic factor for both disease-free survival (DFS) and overall survival (OS) in the complete cohort (n = 564), oestrogen receptor (ER)–positive subgroup (n = 352) and ER-negative subgroup (n = 212), but was more suitable for the ER-positive subgroup. Furthermore, the nomogram model combining the TILs-score with independent clinical factors further improved the predictive ability for the ER-positive subgroup: area under the curve (AUC) at 5-year DFS (OS) and hazard ratio (HR) for DFS (OS) in the training cohort increase from 0.735 (0.785) to 0.814 (0.830) and from 3.156 (5.845) to 4.643 (7.006), respectively, and in the validation cohort from 0.749 (0.748) to 0.804 (0.830) and from 3.104 (3.701) to 3.729 (5.132), respectively. The TILs-score is an independent prognostic factor and displays a strong prognostic value for ER-positive breast cancer. To our knowledge, this is the first time to use MPM for studying the prognostic value of TILs in breast cancer. • A new method is presented for tumour-infiltrating lymphocytes score (TILs-score). • The TILs-score is an independent prognostic factor. • The TILs-score is more suitable for oestrogen receptor-positive breast cancer. [ABSTRACT FROM AUTHOR]