Computer-Aided Multiphoton Microscopy Diagnosis of 5 Different Primary Architecture Subtypes of Meningiomas.

Meningiomas rank among the most common intracranial tumors, and surgery stands as the primary treatment modality for meningiomas. The precise subtyping and diagnosis of meningiomas, both before and during surgery, play a pivotal role in enabling neurosurgeons choose the optimal surgical program. In this study, we utilized multiphoton microscopy (MPM) based on 2-photon excited fluorescence and second-harmonic generation to identify 5 common meningioma subtypes. The morphological features of these subtypes were depicted using the MPM multichannel mode. Additionally, we developed 2 distinct programs to quantify collagen content and blood vessel density. Furthermore, the lambda mode of the MPM characterized architectural and spectral features, from which 3 quantitative indicators were extracted. Moreover, we employed machine learning to differentiate meningioma subtypes automatically, achieving high classification accuracy. These findings demonstrate the potential of MPM as a noninvasive diagnostic tool for meningioma subtyping and diagnosis, offering improved accuracy and resolution compared with traditional methods.

Improving the diagnosis of ductal carcinoma in situ with microinvasion without immunohistochemistry: An innovative method with H&E-stained and multiphoton microscopy images.

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).

Prognostic significance of collagen signatures in pancreatic ductal adenocarcinoma obtained from second-harmonic generation imaging.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) ranks among the deadliest types of cancer, and it will be meaningful to search for new biomarkers with prognostic value to help clinicians tailor therapeutic strategies. METHODS: Here we tried to use an advanced optical imaging technique, multiphoton microscopy (MPM) combining second-harmonic generation (SHG) and two-photon excited fluorescence (TPEF) imaging, for the label-free detection of PDAC tissues from a cohort of 149 patients. An automated image processing method was used to extract collagen features from SHG images and the Kaplan-Meier survival analysis and Cox proportional hazards regression were used to assess the prognostic value of collagen signatures. RESULTS: SHG images clearly show the different characteristics of collagen fibers in tumor microenvironment. We gained eight collagen morphological features, and a Feature-score was derived for each patient by the combination of these features using ridge regression. Statistical analyses reveal that Feature-score is an independent factor, and can predict the overall survival of PDAC patients as well as provide well risk stratification. CONCLUSIONS: SHG imaging technique can potentially be a tool for the accurate diagnosis of PDAC, and this optical biomarker (Feature-score) may help clinicians make more approximate treatment decisions.

Exploration of the relationship between tumor-infiltrating lymphocyte score and histological grade in breast cancer.

BACKGROUND: The histological grade is an important factor in the prognosis of invasive breast cancer and is vital to accurately identify the histological grade and reclassify of Grade2 status in breast cancer patients. METHODS: In this study, data were collected from 556 invasive breast cancer patients, and then randomly divided into training cohort (n = 335) and validation cohort (n = 221). All patients were divided into actual low risk group (Grade1) and high risk group (Grade2/3) based on traditional histological grade, and tumor-infiltrating lymphocyte score (TILs-score) obtained from multiphoton images, and the TILs assessment method proposed by International Immuno-Oncology Biomarker Working Group (TILs-WG) were also used to differentiate between high risk group and low risk group of histological grade in patients with invasive breast cancer. Furthermore, TILs-score was used to reclassify Grade2 (G2) into G2 /Low risk and G2/High risk. The coefficients for each TILs in the training cohort were retrieved using ridge regression and TILs-score was created based on the coefficients of the three kinds of TILs. RESULTS: Statistical analysis shows that TILs-score is significantly correlated with histological grade, and is an independent predictor of histological grade (odds ratio [OR], 2.548; 95%CI, 1.648-3.941; P < 0.0001), but TILs-WG is not an independent predictive factor for grade (P > 0.05 in the univariate analysis). Moreover, the risk of G2/High risk group is higher than that of G2/Low risk group, and the survival rate of patients with G2/Low risk is similar to that of Grade1, while the survival rate of patients with G2/High risk is even worse than that of patients with G3. CONCLUSION: Our results suggest that TILs-score can be used to predict the histological grade of breast cancer and potentially to guide the therapeutic management of breast cancer patients.

Quantitative Assessment of Hepatic Steatosis Using Label-Free Multiphoton Imaging and Customized Image Processing Program.

Nonalcoholic fatty liver disease is rapidly becoming one of the most common causes of chronic liver disease worldwide and is the leading cause of liver-related morbidity and mortality. A quantitative assessment of the degree of steatosis would be more advantageous for diagnostic evaluation and exploring the patterns of disease progression. Here, multiphoton microscopy, based on the second harmonic generation and 2-photon excited fluorescence, was used to label-free image the samples of nonalcoholic fatty liver. Imaging results confirm that multiphoton microscopy is capable of directly visualizing important pathologic features such as normal hepatocytes, hepatic steatosis, Mallory bodies, necrosis, inflammation, collagen deposition, microvessel, and so on and is a reliable auxiliary tool for the diagnosis of nonalcoholic fatty liver disease. Furthermore, we developed an image segmentation algorithm to simultaneously assess hepatic steatosis and fibrotic changes, and quantitative results reveal that there is a correlation between the degree of steatosis and collagen content. We also developed a feature extraction program to precisely display the spatial distribution of hepatocyte steatosis in tissues. These studies may be beneficial for a better clinical understanding of the process of steatosis as well as for exploring possible therapeutic targets.

Rapid and label-free detection of gastrointestinal stromal tumor via a combination of two-photon microscopy and imaging analysis.

BACKGROUND: Gastrointestinal stromal tumor (GIST) is currently regarded as a potentially malignant tumor, and early diagnosis is the best way to improve its prognosis. Therefore, it will be meaningful to develop a new method for auxiliary diagnosis of this disease. METHODS: Here we try out a new means to detect GIST by combining two-photon imaging with automatic image processing strategy. RESULTS: Experimental results show that two-photon microscopy has the ability to label-freely identify the structural characteristics of GIST such as tumor cells, desmoplastic reaction, which are entirely different from those from gastric adenocarcinoma. Moreover, an image processing approach is used to extract eight collagen morphological features from tumor microenvironment and normal muscularis, and statistical analysis demonstrates that there are significant differences in three features-fiber area, density and cross-link density. The three morphological characteristics may be considered as optical imaging biomarkers to differentiate between normal and abnormal tissues. CONCLUSION: With continued improvement and refinement of this technology, we believe that two-photon microscopy will be an efficient surveillance tool for GIST and lead to better management of this disease.

Prognostic value of tumor necrosis based on the evaluation of frequency in invasive breast cancer.

BACKGROUND: Tumor necrosis (TN) was associated with poor prognosis. However, the traditional classification of TN ignored spatial intratumor heterogeneity, which may be associated with important prognosis. The purpose of this study was to propose a new method to reveal the hidden prognostic value of spatial heterogeneity of TN in invasive breast cancer (IBC). METHODS: Multiphoton microscopy (MPM) was used to obtain multiphoton images from 471 patients. According to the relative spatial positions of TN, tumor cells, collagen fibers and myoepithelium, four spatial heterogeneities of TN (TN1-4) were defined. Based on the frequency of individual TN, TN-score was obtained to investigate the prognostic value of TN. RESULTS: Patients with high-risk TN had worse 5-year disease-free survival (DFS) than patients with no necrosis (32.5% vs. 64.7%; P < 0.0001 in training set; 45.8% vs. 70.8%; P = 0.017 in validation set), while patients with low-risk TN had a 5-year DFS comparable to patients with no necrosis (60.0% vs. 64.7%; P = 0.497 in training set; 59.8% vs. 70.8%; P = 0.121 in validation set). Furthermore, high-risk TN "up-staged" the patients with IBC. Patients with high-risk TN and stage I tumors had a 5-year DFS comparable to patients with stage II tumors (55.6% vs. 62.0%; P = 0.565 in training set; 62.5% vs. 66.3%; P = 0.856 in validation set), as well as patients with high-risk TN and stage II tumors had a 5-year DFS comparable to patients with stage III tumors (33.3% vs. 24.6%; P = 0.271 in training set; 44.4% vs. 39.3%; P = 0.519 in validation set). CONCLUSIONS: TN-score was an independent prognostic factor for 5-year DFS. Only high-risk TN was associated with poor prognosis. High-risk TN "up-staged" the patients with IBC. Incorporating TN-score into staging category could improve its performance to stratify patients.

Computer-assisted quantification of tumor-associated collagen signatures to improve the prognosis prediction of breast cancer.

BACKGROUND: Collagen fibers play an important role in tumor initiation, progression, and invasion. Our previous research has already shown that large-scale tumor-associated collagen signatures (TACS) are powerful prognostic biomarkers independent of clinicopathological factors in invasive breast cancer. However, they are observed on a macroscale and are more suitable for identifying high-risk patients. It is necessary to investigate the effect of the corresponding microscopic features of TACS so as to more accurately and comprehensively predict the prognosis of breast cancer patients. METHODS: In this retrospective and multicenter study, we included 942 invasive breast cancer patients in both a training cohort (n = 355) and an internal validation cohort (n = 334) from one clinical center and in an external validation cohort (n = 253) from a different clinical center. TACS corresponding microscopic features (TCMFs) were firstly extracted from multiphoton images for each patient, and then least absolute shrinkage and selection operator (LASSO) regression was applied to select the most robust features to build a TCMF-score. Finally, the Cox proportional hazard regression analysis was used to evaluate the association of TCMF-score with disease-free survival (DFS). RESULTS: TCMF-score is significantly associated with DFS in univariate Cox proportional hazard regression analysis. After adjusting for clinical variables by multivariate Cox regression analysis, the TCMF-score remains an independent prognostic indicator. Remarkably, the TCMF model performs better than the clinical (CLI) model in the three cohorts and is particularly outstanding in the ER-positive and lower-risk subgroups. By contrast, the TACS model is more suitable for the ER-negative and higher-risk subgroups. When the TACS and TCMF are combined, they could complement each other and perform well in all patients. As expected, the full model (CLI+TCMF+TACS) achieves the best performance (AUC 0.905, [0.873-0.938]; 0.896, [0.860-0.931]; 0.882, [0.840-0.925] in the three cohorts). CONCLUSION: These results demonstrate that the TCMF-score is an independent prognostic factor for breast cancer, and the increased prognostic performance (TCMF+TACS-score) may help us develop more appropriate treatment protocols.

Visualization of lymphatic vascular invasion in breast cancer by multiphoton microscopy.

Lymphatic vascular invasion (LVI) is regarded as one of the independent factors which affect the prognosis of breast cancer. Once LVI is formed, it indicates the tumor has metastasized or has the possibility of metastasis. In this work, multiphoton microscopy (MPM), which relies on the two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was applied to identify the typical morphology of LVI and also visualize the histological features of LVI. Furthermore, the pixel density of collagen fibers was extracted as a quantitative parameter to differentiate LVI from the ductal carcinoma in situ (DCIS). By comparing with the corresponding H&E-stained images, it was confirmed that MPM can be used as an auxiliary tool for pathologists to diagnose LVI, and has a possibility for the application in clinical examination.

Multimodal multiphoton imaging for label-free monitoring of early gastric cancer.

BACKGROUND: Early gastric cancer is associated with a much better prognosis than advanced disease, and strategies to improve prognosis is strictly dependent on earlier detection and accurate diagnosis. Therefore, a label-free, non-invasive imaging technique that allows the precise identification of morphologic changes in early gastric cancer would be of considerable clinical interest. METHODS: In this study, multiphoton microscopy (MPM) using two-photon excited fluorescence combined with second-harmonic generation was used for the identification of early gastric cancer. RESULTS: This microscope was able to directly reveal improved cellular detail and stromal changes during the development of early gastric cancer. Furthermore, two features were quantified from MPM images to assess the cell change in size and stromal collagen change as gastric lesion developed from normal to early cancer. CONCLUSIONS: These results clearly show that multiphoton microscopy can be used to examine early gastric cancer at the cellular level without the need for exogenous contrast agents. This study would be helpful for early diagnosis and treatment of gastric cancer, and may provide the groundwork for further exploration into the application of multiphoton microscopy in clinical practice.

Label-free detection of residual breast cancer after neoadjuvant chemotherapy using biomedical multiphoton microscopy.

Neoadjuvant chemotherapy has become a standard treatment for breast cancer as it has been shown to increase the rate of breast preservation and to improve outcome in patients. However, how to accurately detect residual tumors is still a challenge. In this work, we tried to use multiphoton imaging to look for residual breast tumors after preoperative therapy. Imaging results demonstrate that multiphoton microscopy can identify remaining tumor tissues and can even detect rarely residual tumor cells, which would be helpful for surgeons to accurately assess the surgical margin in real time to confirm negative margins during operation. We also performed a quantification analysis of the nuclear area of tumor cells before and after treatment with neoadjuvant chemotherapy. The measurement data show that the tumor cell nuclei after chemotherapy are significantly larger than those without treatment, and there is a statistically significant difference in the nuclear areas between the pre-treatment and post-treatment mammary carcinoma. Our pilot study indicates the potential utility of multiphoton imaging for detecting residual breast carcinoma tissues in fresh, ex vivo specimens without the use of exogenous contrast agents. We foresee real-time intraoperative applications of multiphoton microscopy in evaluating therapy response, and thereby helping clinicians develop individualized treatment plans.

Diagnosing pituitary adenoma in unstained sections based on multiphoton microscopy.

PURPOSE: If we can find a new method that can achieve rapid diagnosis of adenoma during operation, it will help surgeon shorten the operation time and enhance the treatment efficacy. This study discusses the feasibility of multiphoton microscopy (MPM) in diagnosing pituitary adenoma. METHOD: MPM, based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) is performed for the diagnosis of pituitary adenoma in unstained sections. RESULTS: Our results show that MPM can reveal the variation of reticulin fiber by SHG signals of collagen, combined with the measurement of area of acinus, thickness of collagen fiber and collagen percentage. MPM can further reflect the change of meshwork in normal pituitary and hyperplasia quantitatively. And the characteristics of typical growth patterns of pituitary adenoma are demonstrated by the overlay of SHG and TPEF images. What's more, we can identify the boundary of normal pituitary, hyperplasia and adenoma from MPM images. And the experiment also results verify the feasibility of this method in frozen sections. CONCLUSION: These results indicated that MPM can make a diagnosis of pituitary adenoma by the morphological changes without routine pathological processing including hematoxylin-eosin (H&E) staining and other special staining. Therefore, this technique is expected to help diagnosis of pituitary adenoma during operation.

Nonlinear optical microscopy for label-free detection of gastrointestinal neuroendocrine tumors.

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.

Detection of morphologic alterations in rectal carcinoma following preoperative radiochemotherapy based on multiphoton microscopy imaging.

BACKGROUND: Preoperative radiochemotherapy improves outcomes in patients with locally advanced rectal carcinoma, and has been used increasingly in patient management. However, there is a strong clinical need to assess tumor response to neoadjuvant treatment, and a non-invasive technique that allows the precise identification of morphologic changes in tumors would be of considerable clinical interest. METHODS: In this study, we used multiphoton microscopy (MPM) to detect morphologic alterations in rectal adenocarcinomas in patients treated with preoperative radiochemotherapy. RESULTS: MPM was able to identify histopathologic alterations in rectal cancer following preoperative radiochemotherapy, and allowed the qualitative assessment of treatment efficacy and feasibility in relation to dose or strategy. CONCLUSION: These findings may provide the groundwork for evaluating tumor response to neoadjuvant treatment, thus allowing the tailoring of effective treatment doses and strategies.

Layer-resolved colorectal tissues using nonlinear microscopy.

In this work, multiphoton microscopy (MPM), based on the nonlinear optical processes two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), was extended to evaluate the feasibility of using MPM to distinguish layers of the bowel wall. It was found that MPM has the ability to identify the four-layer microstructures of colorectal tissues including mucosa, submucosa, muscularis propria, and serosa as there are many intrinsic signal sources in each layer. Our results also showed the capability of using the quantitative analyses of MPM images for quantifying some feature parameters including the nuclear area, nuclear-to-cytoplasmic ratio, and optical redox ratio. This work demonstrates that MPM has the potential in noninvasively monitoring the development and progression of colorectal diseases and then guiding effective treatment.

Label-free assessment of pathological changes in pancreatic intraepithelial neoplasia by biomedical multiphoton microscopy.

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.

Prognostic significance of collagen signatures at breast tumor boundary obtained by combining multiphoton imaging and imaging analysis.

PURPOSE: Collagen features in breast tumor microenvironment is closely associated with the prognosis of patients. We aim to explore the prognostic significance of collagen features at breast tumor border by combining multiphoton imaging and imaging analysis. METHODS: We used multiphoton microscopy (MPM) to label-freely image human breast tumor samples and then constructed an automatic classification model based on deep learning to identify collagen signatures from multiphoton images. We recognized three kinds of collagen signatures at tumor boundary (CSTB I-III) in a small-scale, and furthermore obtained a CSTB score for each patient based on the combined CSTB I-III by using the ridge regression analysis. The prognostic performance of CSTB score is assessed by the area under the receiver operating characteristic curve (AUC), Cox proportional hazard regression analysis, as well as Kaplan-Meier survival analysis. RESULTS: As an independent prognostic factor, statistical results reveal that the prognostic performance of CSTB score is better than that of the clinical model combining three independent prognostic indicators, molecular subtype, tumor size, and lymph nodal metastasis (AUC, Training dataset: 0.773 vs. 0.749; External validation: 0.753 vs. 0.724; HR, Training dataset: 4.18 vs. 3.92; External validation: 4.98 vs. 4.16), and as an auxiliary indicator, it can greatly improve the accuracy of prognostic prediction. And furthermore, a nomogram combining the CSTB score with the clinical model is established for prognosis prediction and clinical decision making. CONCLUSION: This standardized and automated imaging prognosticator may convince pathologists to adopt it as a prognostic factor, thereby customizing more effective treatment plans for patients.

Supercontinuum intrinsic fluorescence imaging heralds free view of living systems.

Optimal imaging strategies remain underdeveloped to maximize information for fluorescence microscopy while minimizing the harm to fragile living systems. Taking hint from the supercontinuum generation in ultrafast laser physics, we generated supercontinuum fluorescence from untreated unlabeled live samples before nonlinear photodamage onset. Our imaging achieved high-content cell phenotyping and tissue histology, identified bovine embryo polarization, quantified aging-related stress across cell types and species, demystified embryogenesis before and after implantation, sensed drug cytotoxicity in real-time, scanned brain area for targeted patching, optimized machine learning to track small moving organisms, induced two-photon phototropism of leaf chloroplasts under two-photon photosynthesis, unraveled microscopic origin of autumn colors, and interrogated intestinal microbiome. The results enable a facility-type microscope to freely explore vital molecular biology across life sciences.

Gentle label-free nonlinear optical imaging relaxes linear-absorption-mediated triplet.

Sample health is critical for live-cell fluorescence microscopy and has promoted light-sheet microscopy that restricts its ultraviolet-visible excitation to one plane inside a three-dimensional sample. It is thus intriguing that laser-scanning nonlinear optical microscopy, which similarly restricts its near-infrared excitation, has not broadly enabled gentle label-free molecular imaging. We hypothesize that intense near-infrared excitation induces phototoxicity via linear absorption of intrinsic biomolecules with subsequent triplet buildup, rather than the commonly assumed mechanism of nonlinear absorption. Using a reproducible phototoxicity assay based on the time-lapse elevation of auto-fluorescence (hyper-fluorescence) from a homogeneous tissue model (chicken breast), we provide strong evidence supporting this hypothesis. Our study justifies a simple imaging technique, e.g., rapidly scanned sub-80-fs excitation with full triplet-relaxation, to mitigate this ubiquitous linear-absorption-mediated phototoxicity independent of sample types. The corresponding label-free imaging can track freely moving C. elegans in real-time at an irradiance up to one-half of water optical breakdown.

Two-photon imaging reveals histopathological changes in the gastric tumor microenvironment induced by neoadjuvant treatment.

There is a close association between tumor response and survival in gastric cancer patients after receiving neoadjuvant treatment. An accurate and rapid assessment of therapeutic efficacy would be helpful for subsequent treatments and individual prognosis. At present, pathological examination is the gold standard for evaluating treatment response, however, it requires additional staining and the process is tedious, labor-intensive, as well as time-consuming. Here, we introduce a label-free imaging technique, two-photon imaging, to evaluate histopathological changes induced by pre-operative therapy, with a focus on assessing tumor regression as well as stromal response. Imaging data show that two-photon imaging allows label-free, rapid visualization of various aspects of pathological alterations in tumor microenvironment such as fibrotic reaction, inflammatory cell infiltration, mucinous response, isolated residual tumor cells. Moreover, a semi-automatic image processing approach is developed to extract the collagen morphological features, and statistical results show that there are significant differences in collagen area, length, width, cross-link space between the gastric cancer tissues with and without treatment. With the advent of a portable, miniaturized two-photon imaging device, we have enough reason to believe that this technique will become as an important auxiliary diagnostic tool in assessing neoadjuvant treatment response and thereby tailoring the most appropriate therapy strategies for the patients.