Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

2-Photon Characterization of Optical Proteolytic Beacons for Imaging Changes in Matrix-Metalloprotease Activity in a Mouse Model of Aneurysm.

Abdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p<0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease.

Large-scale time series microscopy of neovessel growth during angiogenesis.

During angiogenesis, growing neovessels must effectively navigate through the tissue space as they elongate and subsequently integrate into a microvascular network. While time series microscopy has provided insight into the cell activities within single growing neovessel sprouts, less is known concerning neovascular dynamics within a large angiogenic tissue bed. Here, we developed a time-lapse imaging technique that allowed visualization and quantification of sprouting neovessels as they form and grow away from adult parent microvessels in three dimensions over cubic millimeters of matrix volume during the course of up to 5 days on the microscope. Using a new image acquisition procedure and novel morphometric analysis tools, we quantified the elongation dynamics of growing neovessels and found an episodic growth pattern accompanied by fluctuations in neovessel diameter. Average elongation rate was 5 µm/h for individual vessels, but we also observed considerable dynamic variability in growth character including retraction and complete regression of entire neovessels. We observed neovessel-to-neovessel directed growth over tens to hundreds of microns preceding tip-to-tip inosculation. As we have previously described via static 3D imaging at discrete time points, we identified different collagen fibril structures associated with the growing neovessel tip and stalk, and observed the coordinated alignment of growing neovessels in a deforming matrix. Overall analysis of the entire image volumes demonstrated that although individual neovessels exhibited episodic growth and regression, there was a monotonic increase in parameters associated with the entire vascular bed such as total network length and number of branch points. This new time-lapse imaging approach corroborated morphometric changes in individual neovessels described by us and others, as well as captured dynamic neovessel behaviors unique to days-long angiogenesis within the forming neovascular network.

Formation of microvascular networks: role of stromal interactions directing angiogenic growth.

In the adult, angiogenesis leads to an expanded microvascular network as new vessel segments are added to an existing microcirculation. Necessarily, growing neovessels must navigate through tissue stroma as they locate and grow toward other vessel elements. We have a growing body of evidence demonstrating that angiogenic neovessels reciprocally interact with the interstitial matrix of the stroma resulting in directed neovascular growth during angiogenesis. Given the compliance and the viscoelastic properties of collagen, neovessel guidance by the stroma is likely due to compressive strain transverse to the direction of primary tensile forces present during active tissue deformation. Similar stromal strains control the final network topology of the new microcirculation, including the distribution of arterioles, capillaries, and venules. In this case, stromal-derived stimuli must be present during the post-angiogenesis remodeling and maturation phases of neovascularization to have this effect. Interestingly, the preexisting organization of vessels prior to the start of angiogenesis has no lasting influence on the final, new network architecture. Combined, the evidence describes interplay between angiogenic neovessels and stroma that is important in directed neovessel growth and invasion. This dynamic is also likely a mechanism by which global tissue forces influence vascular form and function.

Differences in the microstructure and biomechanical properties of the recurrent laryngeal nerve as a function of age and location.

Idiopathic onset of unilateral vocal fold paralysis (UVP) is caused by damage to the recurrent laryngeal nerve (RLN) and results in difficulty speaking, breathing, and swallowing. This damage may occur in this nerve as it loops around the aortic arch, which is in a dynamic biomechanical environment. The goal of this study is to determine if the location-dependent biomechanical and microstructural properties of the RLN are different in piglets versus adolescent pigs. The neck/distal and thoracic/proximal (near the aortic arch) regions of the RLN from eight adolescent pigs and six piglets were isolated and mechanically assessed in uni-axial tension. Two-photon imaging (second harmonic) data were collected at 5%, 10%, and 15% strain during the mechanical test. The tangential modulus (TM) and the strain energy density (W) were determined at each level of strain. The mean mode of the preferred fiber angle and the full width at half maximum (FWHM, a measure of fiber splay) were calculated from the imaging data. We found significantly larger values of TM, W, and FWHM in the proximal segments of the left RLN when compared to the distal segments (18.51 MPa ± 1.22 versus 10.78 MPa ± 1.22, p < 0.001 for TM, 0.046 MPa ± 0.01 versus 0.026 MPa ± 0.01, p < 0.003 for W, 15.52 deg ± 1.00 versus 12.98 deg ± 1.00, p < 0.001 for FWHM). TM and W were larger in the left segments than the right (15.32 MPa ± 1.20 versus 11.80 MPa ± 1.20, p < 0.002 for TM, 0.038 MPa ± 0.01 versus 0.028 MPa ± 0.01, p < 0.0001 for W). W was larger in piglets when compared to adolescent pigs (0.042 MPa ± 0.01 versus 0.025 MPa ± 0.01, p < 0.04). The proximal region of the left porcine RLN is more stiff than the distal region and has a higher degree of fiber splay. The left RLN of the adolescent pigs also displayed a higher degree of strain stiffening than the right. These differences may develop as a result of the more dynamic environment the left RLN is in as it loops around the aortic arch.

Mechanical interaction of angiogenic microvessels with the extracellular matrix.

Angiogenesis is the process by which new blood vessels sprout from existing blood vessels, enabling new vascular elements to be added to an existing vasculature. This review discusses our investigations into the role of cell-matrix mechanics in the mechanical regulation of angiogenesis. The experimental aspects of the research are based on in vitro experiments using an organ culture model of sprouting angiogenesis with the goal of developing new treatments and techniques to either promote or inhibit angiogenic outgrowth, depending on the application. Computational simulations were performed to simulate angiogenic growth coupled to matrix deformation, and live two-photon microscopy was used to obtain insight into the dynamic mechanical interaction between angiogenic neovessels and the extracellular matrix. In these studies, we characterized how angiogenic neovessels remodel the extracellular matrix (ECM) and how properties of the matrix such as density and boundary conditions influence vascular growth and alignment. Angiogenic neovessels extensively deform and remodel the matrix through a combination of applied traction, proteolytic activity, and generation of new cell-matrix adhesions. The angiogenic phenotype within endothelial cells is promoted by ECM deformation and remodeling. Sensitivity analysis using our finite element model of angiogenesis suggests that cell-generated traction during growth is the most important parameter controlling the deformation of the matrix and, therefore, angiogenic growth and remodeling. Live two-photon imaging has also revealed numerous neovessel behaviors during angiogenesis that are poorly understood such as episodic growth/regression, neovessel colocation, and anastomosis. Our research demonstrates that the topology of a resulting vascular network can be manipulated directly by modifying the mechanical interaction between angiogenic neovessels and the matrix.

Two-photon excited fluorescence imaging of endogenous contrast in a mouse model of ovarian cancer.

BACKGROUND AND OBJECTIVE: Ovarian cancer has an extremely high mortality rate resulting from poor understanding of the disease. In order to aid understanding of disease etiology and progression, we identify the endogenous fluorophores present in a mouse model of ovarian cancer and describe changes in fluorophore abundance and distribution with age and disease. STUDY DESIGN/MATERIALS AND METHODS: A mouse model of ovarian cancer was created by dosing with 4-vinylcyclohexene diepoxide, which induces follicular apoptosis (simulating menopause), and 7,12-dimethylbenz[a]anthracene, a known carcinogen. Imaging of ovarian tissue was completed ex vivo with a multiphoton microscope using excitation wavelength of 780 nm and emission collection from 405 to 505 nm. Two-photon excited fluorescence images and corresponding histologic sections with selective stains were used to identify endogenous fluorophores. RESULTS: The majority of collected fluorescence emission was attributed to NADH and lipofuscin, with additional contributions from collagen and elastin. Dim cellular fluorescence from NADH did not show observable changes with age. Changes in ovarian morphology with disease development frequently caused increased fluorescence contributions from collagen and adipose tissue-associated NADH. Lipofuscin fluorescence was much brighter than NADH fluorescence and increased as a function of both age and disease. CONCLUSIONS: Our finding of NADH fluorescence patterns similar to that seen previously in human ovary, combined with the observation of lipofuscin accumulation with age and disease also seen in human organs, suggests that the findings from this model may be relevant to human ovarian disease. Increased lipofuscin fluorescence might be used as an indicator of disease in the ovary and this finding warrants further study.

Enhanced visibility of colonic neoplasms using formulaic ratio imaging of native fluorescence.

BACKGROUND AND OBJECTIVES: Colonoscopy is the preferred method for colon cancer screening, but can miss polyps and flat neoplasms with low color contrast. The objective was to develop a new autofluorescence method that improves image contrast of colonic neoplasms. STUDY DESIGN/MATERIALS AND METHODS: We selected the three strongest native fluorescence signals and developed a novel method where fluorescence images are processed in a ratiometric formula to represent the likely cellular and structural changes associated with neoplasia. Native fluorescence images of fresh surgical specimens of the colon containing normal mucosa, polypoid and flat adenomas as well as adenocarcinoma were recorded using a prototype multi-spectral imager. Sixteen patients, with a mean age of 62 years (range 28-81) undergoing elective resection for colonic neoplasms were enrolled. High contrast images were seen with fluorescence from tryptophan (Tryp), flavin adenine dinucleotide (FAD) and collagen. RESULTS: When the image intensity of Tryp was divided pixel by pixel, by the intensities of FAD and collagen, the resulting formulaic ratio (FR) images were of exceptionally high contrast. The FR images of adenomas and adenocarcinomas had increased Weber contrast. CONCLUSIONS: FR imaging is a novel imaging process that represents the likely metabolic and structural changes in colonic neoplasia that produces images with remarkably high contrast.

Intravital confocal and two-photon imaging of dual-color cells and extracellular matrix mimics.

We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

Altered tissue behavior of a non-aneurysmal descending thoracic aorta in the mouse model of Marfan syndrome.

Aortic aneurysm is predominantly found in the ascending aorta in patients with Marfan syndrome (MFS). However, descending aortic disease has emerged as a problem since people are living longer because of improved medical and surgical management of the ascending aorta. Diagnostic procedures before disease onset and the mechanisms involved in the transition of normal aortic tissue to aneurysm remain unclear. We determined signs of descending aortic disease before disease onset in mice with a mutation in the fibrillin 1 gene (Fbn1(+/C1039G)), a validated mouse model of disease susceptibility and progression of aortic aneurysm of MFS. We analyzed a tubular unfixed non-aneurysmal descending thoracic aorta from 8-month-old wild-type and Fbn1(+/C1039G) mice by a tubular biaxial tester that works in conjunction with a two-photon nonlinear microscope. Fbn1(+/C1039G) mouse aorta was more compliant in the circumferential direction. Two-photon imaging showed defective organization of adventitial collagen fibers in the pressurized aortas of Fbn1(+/C1039G) mice. Moreover, disruption in the elastic lamina was noted in the absence of aneurysms in pressurized aortas but not unpressurized aortas of Fbn1(+/C1039G) mice. At the molecular level, this altered tissue behavior in non-aneurysmal descending aortas of Fbn1(+/C1039G) mice was accompanied by an increasing trend of canonical but not noncanonical, transforming growth factor-ß (TGFß) signaling. Finally, assays of in vitro collagen lattice formation in mouse wild-type and TGFß1-deficient embryonic fibroblasts indicate that TGFß1 can regulate collagen organization. The ability to reveal the presence of altered biomechanics and microstructure coupled with subtle changes in TGFß signaling provides a novel surrogate measure of tissue susceptibility to aneurysm before disease onset.

Parallel factor analysis of ovarian autofluorescence as a cancer diagnostic.

BACKGROUND AND OBJECTIVES: Endogenous fluorescence from certain amino acids, structural proteins, and enzymatic co-factors in tissue is altered by carcinogenesis. We evaluate the potential of these changes in fluorescence to predict a diagnosis of malignancy and to estimate the risk of developing ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Ovarian biopsies were interrogated over 270-550 nm excitation and fluorescence was collected from 290 to 700 nm. Two hundred forty-nine measurements were performed on 49 IRB-consented patients undergoing oophorectomy. Data are analyzed using parallel factor analysis to determine excitation and emission spectra of the underlying fluorophores that contribute to the total detected fluorescence intensity. RESULTS: Using multivariate normal distribution fits and cross-validation techniques, sensitivity and specificity of 88% and 93%, respectively, are achieved when classifying malignant samples versus others, while 88% and 80%, respectively, are achieved when classifying normal post-menopausal patients as being either at high- or low-risk of developing ovarian cancer based on their personal and family history of cancer. Performance of classifying cancer increases when the normal group does not include benign neoplasm and endometriosis samples. Performance of high- versus low-risk classification decreases when normal samples include both pre- and post-menopausal women. Excitation over 270-400 and 380-560 nm, respectively, have the best diagnostic performance for cancer detection and risk-status assessment. CONCLUSIONS: Assessing the endogenous fluorescence could be useful in screening women at increased risk of developing ovarian cancer.

Ultra-slim plastic endomicroscope objective for non-linear microscopy.

Non-linear microscopy has the potential to provide clinically useful information on the structure of biological tissue in vivo via an endomicroscope. The ability to use plastic as the optical material in a multiphoton objective was evaluated based on several criteria including autofluorescence, injection molding induced birefringence, and pulse broadening due to group velocity dispersion. An all-plastic, refractive ultra-slim endoscope objective was built with design specifications of NA=0.4, FOV=250 µm, 1.27 mm outer diameter, and 0.8 mm clear aperture. Initial images of second-harmonic generation signal (illumination at 780 nm) in collagen fibers and two-photon excited fluorescence (illumination at 920 nm) of Convallaria rhizome are reported.

Adaptation of a planar microbiaxial optomechanical device for the tubular biaxial microstructural and macroscopic characterization of small vascular tissues.

Murine models of disease are a powerful tool for researchers to gain insight into disease formation, progression, and therapies. The biomechanical indicators of diseased tissue provide a unique insight into some of these murine models, since the biomechanical properties in scenarios such as aneurysm and Marfan syndrome can dictate tissue failure and mortality. Understanding the properties of the tissue on the macroscopic scale has been shown to be important, as one can then understand the tissue's ability to withstand the high stresses seen in the cardiac pulsatile cycle. Alterations in the biomechanical response can foreshadow prospective mechanical failure of the tissue. These alterations are often seen on the microstructural level, and obtaining detailed information on such changes can offer a better understanding of the phenomena seen on the macroscopic level. Unfortunately, mouse models present problems due to the size and delicate features in the mechanical testing of such tissues. In addition, some smaller arteries in large-animal studies (e.g., coronary and cerebral arteries) can present the same issues, and are sometimes unsuitable for planar biaxial testing. The purpose of this paper is to present a robust method for the investigation of the mechanical properties of small arteries and the classification of the microstructural orientation and degree of fiber alignment. This occurs through the cost-efficient modification of a planar biaxial tester that works in conjunction with a two-photon nonlinear microscope. This system provides a means to further investigate how microstructure and mechanical properties are modified in diseased transgenic animals where the tissue is in small tube form. Several other hard-to-test tubular specimens such as cerebral aneurysm arteries and atherosclerotic coronary arteries can also be tested using the described modular device.

A gimbal-mounted pressurization chamber for macroscopic and microscopic assessment of ocular tissues.

The biomechanical model of glaucoma considers intraocular pressure-related stress and resultant strain on load bearing connective tissues of the optic nerve and surrounding peripapillary sclera as one major causative influence that effects cellular, vascular, and axonal components of the optic nerve. By this reasoning, the quantification of variations in the microstructural architecture and macromechanical response of scleral shells in glaucomatous compared to healthy populations provides an insight into any variations that exist between patient populations. While scleral shells have been tested mechanically in planar and pressure-inflation scenarios the link between the macroscopic biomechanical response and the underlying microstructure has not been determined to date. A potential roadblock to determining how the microstructure changes based on pressure is the ability to mount the spherical scleral shells in a method that does not induce unwanted stresses to the samples (for instance, in the flattening of the spherical specimens), and then capturing macroscopic and microscopic changes under pressure. Often what is done is a macroscopic test followed by sample fixation and then imaging to determine microstructural organization. We introduce a novel device and method, which allows spherical samples to be pressurized and macroscopic and microstructural behavior quantified on fully hydrated ocular specimens. The samples are pressurized and a series of markers on the surface of the sclera imaged from several different perspectives and reconstructed between pressure points to allow for mapping of nonhomogenous strain. Pictures are taken from different perspectives through the use of mounting the pressurization scheme in a gimbal that allows for positioning the sample in several different spherical coordinate system configurations. This ability to move the sclera in space about the center of the globe, coupled with an upright multiphoton microscope, allows for collecting collagen, and elastin signal in a rapid automated fashion so the entire globe can be imaged.

Design and demonstration of a microbiaxial optomechanical device for multiscale characterization of soft biological tissues with two-photon microscopy.

The biomechanical response of tissues serves as a valuable marker in the prediction of disease and in understanding the related behavior of the body under various disease and age states. Alterations in the macroscopic biomechanical response of diseased tissues are well documented; however, a thorough understanding of the microstructural events that lead to these changes is poorly understood. In this article we introduce a novel microbiaxial optomechanical device that allows two-photon imaging techniques to be coupled with macromechanical stimulation in hydrated planar tissue specimens. This allows that the mechanical response of the microstructure can be quantified and related to the macroscopic response of the same tissue sample. This occurs without the need to fix tissue in strain states that could introduce a change in the microstructural configuration. We demonstrate the passive realignment of fibrous proteins under various types of loading, which demonstrates the ability of tissue microstructure to reinforce itself in periods of high stress. In addition, the collagen and elastin response of tissue during viscoelastic behavior is reported showing interstitial fluid movement and fiber realignment potentially responsible for the temporal behavior. We also demonstrate that nonhomogeneities in fiber strain exist over biaxial regions of assumed homogeneity.

Moving from Pedagogy to Andragogy in Biomedical Engineering Design: Strategies for Lab-at-Home and Distance Learning.

Engineering design courses are particularly challenging to deliver in online or distance modalities because of the hands-on, collaborative nature of the design process and the need for physical resources and work spaces. In this work, we describe how we rapidly transformed two design courses in the middle two years of the biomedical engineering (BME) program to an online format during the 2019 coronavirus pandemic. In addition to time and safety constraints, we identified access to design spaces with biochemistry, computing, electronic, computing, and manufacturing tools, and team-based learning as major challenges to distance learning in BME design courses. To this end, we mapped and translated various course and design activities to an online environment using a combination of customized at-home laboratory kits and distributed team structures. Drawing upon our pilot experience as well as principles from online and adult learning theories, we offer an overview of strategies to retain hands-on and team-based activities and rapidly implement BME design courses in online or distance modalities.

Using FDA-approved drugs as off-label fluorescent dyes for optical biopsies: from in silico design toex vivoproof-of-concept.

Optical biopsies bring the microscope to the patient rather than the tissue to the microscope, and may complement or replace the tissue-harvesting component of the traditional biopsy process with its associated risks. In general, optical biopsies are limited by the lack of endogenous tissue contrast and the small number of clinically approvedin vivodyes. This study tests multiple FDA-approved drugs that have structural similarity to research dyes as off-labelin situfluorescent alternatives to standardex vivohematoxylin & eosin tissue stain. Numerous drug-dye combinations shown here may facilitate relatively safe and fastin situor possiblyin vivostaining of tissue, enabling real-time optical biopsies and other advanced microscopy technologies, which have implications for the speed and performance of tissue- and cellular-level diagnostics.

Racioethnic differences in the biomechanical response of the lamina cribrosa.

Glaucoma is the second leading cause of irreversible blindness in the world with a higher prevalence in those of African Descent (AD) and Hispanic Ethnicity (HE) than in those of European Descent (ED). The objective of this study was to investigate the pressure dependent biomechanical response of the lamina cribrosa (LC) in normal human donor tissues from these racioethnic backgrounds. Pressure inflation tests were performed on 24 human LCs (n = 9 AD, n = 6 ED, and n = 9 HE) capturing the second harmonic generation (SHG) signal of collagen at 5, 15, 30, and 45 mmHg from an anterior view. A non-rigid image registration technique was utilized to determine the 3D displacement field in each LC from which 3D Green strains were calculated. The peak shear strain in the superior quadrant of the LC in those of ED was significantly higher than in those of AD and HE (p-value = 0.005 & 0.034, respectively) where ED = 0.017 [IQR = 0.012-0.027], AD = 0.0002 [IQR = -0.001-0.007], HE = 0.0016 [IQR = -0.002-0.012]). There were also significant differences in the regional strain heterogeneity in those of AD and HE that were absent in those of ED. This work represents, to our knowledge, the first ex-vivo study identifying significant differences in the biomechanical response of the LC in populations at increased risk of glaucoma. Future work will be necessary to assess if and how these differences play a role in predisposing those of Hispanic Ethnicity and African Descent to the onset and/or progression of primary open angle glaucoma. STATEMENT OF SIGNIFICANCE: Glaucoma is the second leading cause of irreversible blindness in the world and occurs more frequently in those of African Descent and Hispanic Ethnicity than in those of European Descent. To date, there has been no ex-vivo study quantifying differences in the biomechanical response of the non-glaucomatous lamina cribrosa (LC) across these racioethnic backgrounds. In this work we report, for the first time, differences in the pressure dependent biomechanical response of LC across different racioethnic groups as quantified using nonlinear optical microscopy. This study lays the foundation for future work investigating if and how these differences may play a role in predisposing those at increased risk to the onset and/or progression of primary open angle glaucoma.

Spectral background and transmission characteristics of fiber optic imaging bundles.

The emission and transmission properties of three commercially produced coherent fiber optic imaging bundles were evaluated. Full fluorescence excitation versus emission data were collected from 250 to 650 nm excitation for high-resolution Sumitomo, Fujikura, and Schott fiber bundles. The results generated show regions of autofluorescence and inelastic Raman scattering in the imaging bundles that represent a wavelength-dependent background signal when these fibers are used for imaging applications. The high-resolution fiber bundles also exhibit significant variation in transmission with the angle of illumination, which affects the overall coupling and transmission efficiency. Knowledge of these properties allows users of high-resolution imaging bundles to optimally design systems that utilize such bundles.

Endogenous optical biomarkers of ovarian cancer evaluated with multiphoton microscopy.

PURPOSE: Among gynecologic cancers, ovarian cancer is the second most common and has the highest mortality. Currently, there is no accurate early diagnostic technique for ovarian cancer. Furthermore, little is understood regarding the early progression of this disease. We have imaged multiphoton interactions of endogenous tissue constituents from normal and abnormal ovarian biopsies that were kept viable during transport from the operating room and microscopy. EXPERIMENTAL DESIGN: The ovarian surface and underlying stroma were assessed with two-photon excited fluorescence (2PEF) and second harmonic generation (SHG). High-resolution, optically sectioned images were analyzed for epithelial morphology based on 2PEF and collagen density and structural integrity based on SHG. Additionally, multiwavelength 2PEF provided an estimation of the cellular redox ratio of epithelial cells. RESULTS: Normal tissue exhibited a uniform epithelial layer with highly structured collagen in the stroma, whereas abnormal tissue exhibited varied epithelium with large cells and substantial quantitative changes to the collagen structure. Samples from patients at high risk for developing ovarian cancer (based on their personal/family history of cancer) exhibited highly variable cellular redox ratios and changes in collagen structure that trended toward cancer samples. CONCLUSION: This study highlights differences in endogenous signals in viable ovarian biopsies based on quantitative collagen structural changes and redox ratio estimates that may lead to improved detection and further insights in ovarian cancer, particularly in the early stages of the disease.