Label-free Multiphoton Microscopy Research Articles

Quantification of age-related changes in the structure and mechanical function of skin with multiscale imaging.

The mechanical properties of skin change during aging but the relationships between structure and mechanical function remain poorly understood. Previous work has shown that young skin exhibits a substantial decrease in tissue volume, a large macro-scale Poisson's ratio, and an increase in micro-scale collagen fiber alignment during mechanical stretch. In this study, label-free multiphoton microscopy was used to quantify how the microstructure and fiber kinematics of aged mouse skin affect its mechanical function. In an unloaded state, aged skin was found to have less collagen alignment and more non-enzymatic collagen fiber crosslinks. Skin samples were then loaded in uniaxial tension and aged skin exhibited a lower mechanical stiffness compared to young skin. Aged tissue also demonstrated less volume reduction and a lower macro-scale Poisson's ratio at 10% uniaxial strain, but not at 20% strain. The magnitude of 3D fiber realignment in the direction of loading was not different between age groups, and the amount of realignment in young and aged skin was less than expected based on theoretical fiber kinematics affine to the local deformation. These findings provide key insights on how the collagen fiber microstructure changes with age, and how those changes affect the mechanical function of skin, findings which may help guide wound healing or anti-aging treatments.

Rapid label-free detection of early-stage lung adenocarcinoma and tumor boundary via multiphoton microscopy.

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. This article is protected by copyright. All rights reserved.

Label-free multiphoton microscopy enables histopathological assessment of colorectal liver metastases and supports automated classification of neoplastic tissue

As the state of resection margins is an important prognostic factor after extirpation of colorectal liver metastases, surgeons aim to obtain negative margins, sometimes elaborated by resections of the positive resection plane after intraoperative frozen sections. However, this is time consuming and results sometimes remain unclear during surgery. Label-free multimodal multiphoton microscopy (MPM) is an optical technique that retrieves morpho-chemical information avoiding all staining and that can potentially be performed in real-time. Here, we investigated colorectal liver metastases and hepatic tissue using a combination of three endogenous nonlinear signals, namely: coherent anti-Stokes Raman scattering (CARS) to visualize lipids, two-photon excited fluorescence (TPEF) to visualize cellular patterns, and second harmonic generation (SHG) to visualize collagen fibers. We acquired and analyzed over forty thousand MPM images of metastatic and normal liver tissue of 106 patients. The morphological information with biochemical specificity produced by MPM allowed discriminating normal liver from metastatic tissue and discerning the tumor borders on cryosections as well as formalin-fixed bulk tissue. Furthermore, automated tissue type classification with a correct rate close to 95% was possible using a simple approach based on discriminant analysis of texture parameters. Therefore, MPM has the potential to increase the precision of resection margins in hepatic surgery of metastases without prolonging surgical intervention.

Mapping the 3D remodeling of the extracellular matrix in human hypertrophic scar by multi-parametric multiphoton imaging using endogenous contrast

The hypertrophic scar is an aberrant form of wound healing process, whose clinical efficacy is limited by a lack of understanding of its pathophysiology. Remodeling of collagen and elastin fibers in the extracellular matrix (ECM) is closely associated with scar progression. Herein, we perform label-free multiphoton microscopy (MPM) of both fiber components from human skin specimens and propose a multi-fiber metrics (MFM) analysis model for mapping the structural remodeling of the ECM in hypertrophic scars in a highly-sensitive, three-dimensional (3D) manner. We find that both fiber components become wavier and more disorganized in scar tissues, while content accumulation is observed from elastin fibers only. The 3D MFM analysis can effectively distinguish normal and scar tissues with better than 95% in accuracy and 0.999 in the area under the curve value of the receiver operating characteristic curve. Further, unique organizational features with orderly alignment of both fibers are observed in scar-normal adjacent regions, and an optimized combination of features from 3D MFM analysis enables successful identification of all the boundaries. This imaging and analysis system uncovers the 3D architecture of the ECM in hypertrophic scars and exhibits great translational potential for evaluating scars in vivo and identifying individualized treatment targets.

Automated classification of breast cancer histologic grade using multiphoton microscopy and generative adversarial networks

Histological grade is one of the most powerful prognostic factors for breast cancer and impacts treatment decisions. However, a label-free and automated classification system for histological grading of breast tumors has not yet been developed. In this study, we employed label-free multiphoton microscopy (MPM) to acquire subcellular-resolution images of unstained breast cancer tissues. Subsequently, a deep-learning algorithm based on the generative adversarial network (GAN) was introduced to learn a representation using only MPM images without the histological grade information. Furthermore, to obtain abundant image information and determine the detailed differences between MPM images of different grades, a multiple-feature discriminator network based on the GAN was leveraged to learn the multi-scale spatial features of MPM images through unlabeled data. The experimental results showed that the classification accuracies for tumors of grades 1, 2, and 3 were 92.4%, 88.6%, and 89.0%, respectively. Our results suggest that the fusion of multiphoton microscopy and the GAN-based deep learning algorithm can be used as a fast and powerful clinical tool for the computer-aided intelligent pathological diagnosis of breast cancer.

Label free deep penetration single photon microscopic imaging with ultralong anti-diffracting beam

Label free single photon microscopic imaging has natural advantages in noninvasive in vivo tissue imaging such as high resolution and rapid imaging speed. Although label free multi-photon microscopy can be used for imaging thick tissue samples, it requires high excitation light power and is phototoxic to the samples. Conventional label free single photon microscopy requires lower excitation light power, but it has limited imaging depth. Observing some highly scattering thick tissue samples with single photon microscopy is a great challenge. To solve the problem, we developed a label free deep penetration single photon microscopic imaging technique with an ultralong anti-diffracting (UAD) beam. The penetrating ability of the UAD beam was verified by passing through turbid media and performed with autofluorescence of chloroplasts in fresh Epipremnum aureum leaves. Benefiting from the anti-diffracting properties and the elongated focal depth of the UAD beam, single photon UAD microscopy has deeper penetration depth and better anti-scattering ability and is one of the ideal methods to observe the deep structure of biological samples.

Abstract 2472: Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy

Abstract We have developed a live tumor fragment (LTF) platform for predicting clinical response to cancer drugs. The success of this approach depends on screening tissue fragments derived from biopsies and excisions before drug treatment to select those that have acceptable levels of viability and tumor content. To enable this screening, we are developing label-free methods for integrated assessment of LTF histology, viability, and metabolic status using intrinsic multiphoton-excited fluorescence lifetime microscopy (MP-FLIM). We cut live EMT6 tumors and mammary fat pad into rectangular fragments that were then sorted and cultured in glass-bottomed multi-well plates. To induce cancer cell death, we treated one group with a therapeutic agent, e.g., the multi-kinase inhibitor, staurosporine, or the alkylating agent, cisplatin. Before and after treatment, we imaged LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide dinucleotides (NAD(P)H) and flavin adenine dinucleotides (FAD) fluorescence intensity and lifetime using dual-excitation multiphoton microscopy. As a ground truth viability reference, we then stained and re-imaged the fragments using nuclear and cell viability probes such as Hoechst, SYTOX, TMRE and/or propidium iodide (PI). We analyzed the data by fitting fluorescence decay curves with dual or triple exponents to generate images of lifetime parameters, including the mean and individual component lifetimes and amplitudes. Finally, we co-registered the extrinsically labeled and autofluorescent lifetime images for 3D spatial analysis using commercial and custom software. We verified the methods using monolayer cell cultures and ATP luminescence and flow cytometry viability assays. Image spatial heterogeneity was quantified utilizing entropy metrics. Intrinsic contrast from multiphoton imaging revealed cellular and tissue structures. The median entropy was higher and its distribution negatively skewed for EMT6 (p<0.01), allowing us to distinguish tumor from its corresponding normal tissue of origin. In viable cells, nuclei were distinguishable from the cytoplasm via higher cytoplasmic NADH signal. Upon loss of cell viability, nuclear NADH signals increased, with characteristically short lifetimes, and the overall NADH intensity decreased, reducing the contrast between nuclei and their surrounding cytoplasm. We detected an increase in the whole fragment mean lifetime (τm p<0.002) and concomitant decrease in the short lifetime component (α1 p< 0.002) within 24 hours of staurosporine exposure. We have demonstrated that MP-FLIM can distinguish tumor from normal and live from dead in LTFs without labels. This approach will facilitate selection of fragments with acceptable viability and tumor content for subsequent drug treatment as part of a platform built for personalizing cancer therapy. Citation Format: Jonathan Ouellette, John Rafter, Kelsey Tweed, Leung Kau Tang, Christina Scribano, Pichet Adstamongkonkul, Mikaela Schultz, Justine Coburn, Maged Aldeeb, Neil Anthony, Christin Johnson, Kevin Eliceiri, Jonathan Oliner, Tomasz Zal. Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2472.

In vivo simultaneous nonlinear absorption Raman and fluorescence (SNARF) imaging of mouse brain cortical structures

Label-free multiphoton microscopy is a powerful platform for biomedical imaging. Recent advancements have demonstrated the capabilities of transient absorption microscopy (TAM) for label-free quantification of hemoglobin and stimulated Raman scattering (SRS) microscopy for pathological assessment of label-free virtual histochemical staining. We propose the combination of TAM and SRS with two-photon excited fluorescence (TPEF) to characterize, quantify, and compare hemodynamics, vessel structure, cell density, and cell identity in vivo between age groups. In this study, we construct a simultaneous nonlinear absorption, Raman, and fluorescence (SNARF) microscope with the highest reported in vivo imaging depth for SRS and TAM at 250–280 μm to enable these multimodal measurements. Using machine learning, we predict capillary-lining cell identities with 90% accuracy based on nuclear morphology and capillary relationship. The microscope and methodology outlined herein provides an exciting route to study several research topics, including neurovascular coupling, blood-brain barrier, and neurodegenerative diseases.

Imaging Arm Regeneration: Label-Free Multiphoton Microscopy to Dissect the Process in Octopus vulgaris.

Cephalopod mollusks are endowed with an impressive range of features that have captured the attention of scientists from different fields, the imaginations of artists, and the interests of the public. The ability to spontaneously regrow lost or damaged structures quickly and functionally is among one of the most notable peculiarities that cephalopods possess. Microscopical imaging techniques represent useful tools for investigating the regenerative processes in several species, from invertebrates to mammals. However, these techniques have had limited use in cephalopods mainly due to the paucity of specific and commercially available markers. In addition, the commonly used immunohistochemical staining methods provide data that are specific to the antigens studied. New microscopical methods were recently applied to vertebrates to investigate regenerative events. Among them, multiphoton microscopy appears promising. For instance, it does not depend on species-related epitopes, taking advantage of the specific characteristics of tissues and allowing for its use in a species-independent way. Here, we illustrate the results obtained by applying this label-free imaging technique to the injured arm of Octopus vulgaris, a complex structure often subject to injury in the wild. This approach allowed for the characterization of the entire tissue arm architecture (muscular layers, nerve component, connective tissues, etc.) and elements usually hardly detectable (such as vessels, hemocytes, and chromatophores). More importantly, it also provided morpho-chemical information which helped decipher the regenerative phases after damage, from healing to complete arm regrowth, thereby appearing promising for regenerative studies in cephalopods and other non-model species.

Multiphoton Microscopy for Identifying Collagen Signatures Associated with Biochemical Recurrence in Prostate Cancer Patients.

Prostate cancer is a heterogeneous disease that remains dormant for long periods or acts aggressively with poor clinical outcomes. Identifying aggressive prostate tumor behavior using current glandular-focused histopathological criteria is challenging. Recent evidence has implicated the stroma in modulating prostate tumor behavior and in predicting post-surgical outcomes. However, the emergence of stromal signatures has been limited, due in part to the lack of adoption of imaging modalities for stromal-specific profiling. Herein, label-free multiphoton microscopy (MPM), with its ability to image tissue with stromal-specific contrast, is used to identify prostate stromal features associated with aggressive tumor behavior and clinical outcome. MPM was performed on unstained prostatectomy specimens from 59 patients and on biopsy specimens from 17 patients with known post-surgery recurrence status. MPM-identified collagen content, organization, and morphological tumor signatures were extracted for each patient and screened for association with recurrent disease. Compared to tumors from patients whose disease did not recur, tumors from patients with recurrent disease exhibited higher MPM-identified collagen amount and collagen fiber intensity signal and width. Our study shows an association between MPM-identified stromal collagen features of prostate tumors and post-surgical disease recurrence, suggesting their potential for prostate cancer risk assessment.

Innovative Diagnostic Endoscopy in Inflammatory Bowel Diseases: From High-Definition to Molecular Endoscopy.

High-definition endoscopy is one essential step in the initial diagnosis of inflammatory bowel disease (IBD) characterizing the extent and severity of inflammation, as well as discriminating ulcerative colitis (UC) from Crohn's disease (CD). Following general recommendations and national guidelines, individual risk stratification should define the appropriate surveillance strategy, biopsy protocol and frequency of endoscopies. Beside high-definition videoendoscopy the application of dyes applied via a spraying catheter is of additional diagnostic value with a higher detection rate of intraepithelial neoplasia (IEN). Virtual chromoendoscopy techniques (NBI, FICE, I-scan, BLI) should not be recommended as a single surveillance strategy in IBD, although newer data suggest a higher comparability to dye-based chromoendoscopy than previously assumed. First results of oral methylene blue formulation are promising for improving the acceptance rate of classical chromoendoscopy. Confocal laser endomicroscopy (CLE) is still an experimental but highly innovative endoscopic procedure with the potential to contribute to the detection of dysplastic lesions. Molecular endoscopy in IBD has taken application of CLE to a higher level and allows topical application of labeled probes, mainly antibodies, against specific target structures expressed in the tissue to predict response or failure to biological therapies. First pre-clinical and in vivo data from label-free multiphoton microscopy (MPM) are now available to characterize mucosal and submucosal inflammation on endoscopy in more detail. These new techniques now have opened the door to individualized and highly specific molecular imaging in IBD in the future and pave the path to personalized medicine approaches. The quality of evidence was stated according to the Oxford Center of evidence-based medicine (March 2009). For this review a Medline search up to January 2021 was performed using the words “inflammatory bowel disease,” “ulcerative colitis,” “crohn's disease,” “chromoendoscopy,” “high-definition endoscopy,” “confocal laser endomicroscopy,” “confocal laser microscopy,” “molecular imaging,” “multiphoton microscopy.”

Effect of valproic acid on functional bleb morphology in a rabbit model of minimally invasive surgery

PurposeTo determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS).MethodsNine New Zealand white rabbits were subjected...

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD) is the most common valvular heart disease. CAVD results in a considerable socio-economic burden, especially considering the aging population in Europe and North America. The only treatment standard is surgical valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. Novel diagnostic techniques and biomarkers for early detection and monitoring of CAVD progression are thus a pressing need. Additionally, non-destructive tools are required for longitudinal in vitro and in vivo assessment of CAVD initiation and progression that can be translated into clinical practice in the future. Multiphoton microscopy (MPM) facilitates label-free and non-destructive imaging to obtain quantitative, optical biomarkers that have been shown to correlate with key events during CAVD progression. MPM can also be used to obtain spatiotemporal readouts of metabolic changes that occur in the cells. While cellular metabolism has been extensively explored for various cardiovascular disorders like atherosclerosis, hypertension, and heart failure, and has shown potential in elucidating key pathophysiological processes in heart valve diseases, it has yet to gain traction in the study of CAVD. Furthermore, MPM also provides structural, functional, and metabolic readouts that have the potential to correlate with key pathophysiological events in CAVD progression. This review outlines the applicability of MPM and its derived quantitative metrics for the detection and monitoring of early CAVD progression. The review will further focus on the MPM-detectable metabolic biomarkers that correlate with key biological events during valve pathogenesis and their potential role in assessing CAVD pathophysiology.

Label-Free Multiphoton Microscopy: Much More Than Fancy Images.

Multiphoton microscopy has recently passed the milestone of its first 30 years of activity in biomedical research. The growing interest around this approach has led to a variety of applications from basic research to clinical practice. Moreover, this technique offers the advantage of label-free multiphoton imaging to analyze samples without staining processes and the need for a dedicated system. Here, we review the state of the art of label-free techniques; then, we focus on two-photon autofluorescence as well as second and third harmonic generation, describing physical and technical characteristics. We summarize some successful applications to a plethora of biomedical research fields and samples, underlying the versatility of this technique. A paragraph is dedicated to an overview of sample preparation, which is a crucial step in every microscopy experiment. Afterwards, we provide a detailed review analysis of the main quantitative methods to extract important information and parameters from acquired images using second harmonic generation. Lastly, we discuss advantages, limitations, and future perspectives in label-free multiphoton microscopy.

Widely-tunable synchronisation-free picosecond laser source for multimodal CARS, SHG, and two-photon microscopy

We demonstrate a continuous wave (CW) seeded synchronization-free optical parametric amplifier (OPA) pumped by a picosecond, 1 µm laser and show its performance when used as a simple yet powerful source for label-free coherent anti-Stokes Raman scattering (CARS), concurrent second harmonic generation (SHG), and two-photon fluorescence microscopy in an epi-detection geometry. The average power level of above 175 mW, spectral resolution of 8 cm−1, and 2 ps pulse duration are well optimized for CARS microscopy in bio-science and bio-medical imaging systems. Our OPA is a much simpler setup than either the “gold-standard” laser and optical parametric oscillator (OPO) combination traditionally used for CARS imaging, or the more recently developed OPA systems pumped with femtosecond pulses [1]. Rapid and accurate tuning between resonances was achieved by changing the poled channels and temperature of the periodically-poled lithium niobate (PPLN) OPA crystal together with the OPA seed wavelength. The Pump-Stokes frequency detuning range fully covered the C-H stretching band used for the imaging of lipids. By enabling three multiphoton techniques using a compact, synchronization free laser source, our work paves the way for the translation of label-free multi-photon microscopy imaging from biomedical research to an imaging based diagnostic tool for use in the healthcare arena.

Label-free multiphoton microscopy as a tool to investigate alterations of cerebral aneurysms

Cerebral aneurysms are abnormal focal dilatations of arterial vessel walls with pathological vessel structure alterations. Sudden rupture can lead to a subarachnoid hemorrhage, which is associated with a high mortality. Therefore, the origin of cerebral aneurysms as well as the progression to the point of rupture needs to be further investigated. Label-free multimodal multiphoton microscopy (MPM) was performed on resected human aneurysm domes and integrated three modalities: coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence and second harmonic generation. We showed that MPM is a completely label-free and real-time powerful tool to detect pathognomonic histopathological changes in aneurysms, e.g. thickening and thinning of vessel walls, intimal hyperplasia, intra-wall haemorrhage, calcification as well as atherosclerotic changes. In particular, the loss or fragmentation of elastin as well as fibromatous wall remodelling appeared very distinct. Remarkably, cholesterol and lipid deposits were clearly visible in the multiphoton images. MPM provides morphological and biochemical information that are crucial for understanding the mechanisms of aneurysm formation and progression.

High-speed, multi-modal, label-free imaging of pathological slices with a Bessel beam

Optical imaging of stained pathological slices has become the gold standard for disease diagnosis. However, the procedure of sample preparation is complex and time-consuming. Multiphoton microscopy (MPM) is promising for label-free imaging, but the imaging speed is limited, especially for whole slice imaging. Here we propose a high-speed, multi-modal, label-free MPM by Bessel scan-based strip mosaicking. With a Bessel beam for excitation, the extended depth-of-focus not only enables full axial information acquisition at once, but also alleviates the demanding requirement of sample alignment. With the strip mosaicking protocol, we can save the time of frequent sample transferring. Besides, we add a closely-attached reflection mirror under the sample for enhancing epi-detection signals, and employ circularly polarized beams for recording comprehensive information. We demonstrate its application in multi-modal, label-free imaging of human gastric cancer slices and liver cancer slices, and show its potential in rapid disease diagnosis.

Quantifying Age-Related Changes in Skin Wound Metabolism Using In Vivo Multiphoton Microscopy.

Objective: The elderly are at high risk for developing chronic skin wounds, but the effects of intrinsic aging on skin healing are difficult to isolate due to common comorbidities like diabetes. Our objective is to use multiphoton microscopy (MPM) to find endogenous, noninvasive biomarkers to differentiate changes in skin wound healing metabolism between young and aged mice in vivo. Approach: We utilized MPM to monitor skin metabolism at the edge of full-thickness, excisional wounds in 24- and 4-month-old mice of both sexes for 10 days. MPM can assess quantitative biomarkers of cellular metabolism in vivo by utilizing autofluorescence from the cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). Results: An optical redox ratio of FAD/(NADH+FAD) autofluorescence and NADH fluorescence lifetime imaging revealed dynamic changes in keratinocyte function during healing. Aged female mice demonstrated an attenuation of keratinocyte proliferation during wound healing detectable optically through a higher redox ratio and longer NADH fluorescence lifetime. By measuring the correlation between NADH lifetime and the optical redox ratio at each day, we also demonstrate sensitivity to the proliferative phase of wound healing. Innovation: Label-free MPM was used to longitudinally monitor individual wounds in vivo, which revealed age-dependent differences in wound metabolism. Conclusion: These results indicate in vivo MPM can provide quantitative biomarkers of age-related delays in healing, which can be used in the future to provide patient-specific wound care.

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label-free multiphoton microscopic images.

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.

Label-free multiphoton imaging of β-amyloid plaques in Alzheimer's disease mouse models.

.-Amyloid () plaque, representing the progressive accumulation of the protein that mainly consists of , is one of the prominent pathological hallmarks of Alzheimer’s disease (AD). Label-free imaging of plaques holds the potential to be a histological examination tool for diagnosing AD. We applied label-free multiphoton microscopy to identify extracellular plaque as well as intracellular accumulation for the first time from AD mouse models. We showed that a two-photon-excited fluorescence signal is a sensitive optical marker for revealing the spatial–temporal progression and the surrounding morphological changes of deposition, which demonstrated that both extracellular and intracellular accumulations play an important role in the progression of AD. Moreover, combined with a custom-developed image-processing program, we established a rapid method to visualize different degrees of deposition by color coding. These results provide an approach for investigating pathophysiology of AD that can complement traditional biomedical procedures.