Ovarian cancer remains the most deadly gynecological cancer with a poor aggregate survival rate. To improve upon this situation, we utilized collagen-specific Second Harmonic Generation (SHG) imaging microscopy and optical scattering measurements to probe structural differences in the extracellular matrix of normal stroma, benign tumors, endometrioid tumors, and low and high-grade serous (LGS and HGS) tumors. The SHG signatures of the emission directionality and conversion efficiency as well as the optical scattering are related to the organization of collagen on the sub-micron size. The wavelength dependence of these readouts adds additional characterization of the size and distribution of collagen fibrils/fibers relative to the interrogating wavelengths. We found strong wavelength dependent dependencies of these metrics that were different between the different tumors that are related to respective structural attributes in the collagen organization. These sub-resolution determinations are consistent with the dualistic classification of type I and II serous tumors. However, type I endometrioid tumors have strongly differing ECM architecture than the serous malignancies. Moreover, our analyses are further consistent with LGS and benign tumors having similar etiology. We identified optimal wavelengths for the SHG metrics as well as optical scattering measurements. The SHG metrics and optical scattering measurements were then used to form a linear discriminant model to classify the tissues, and we obtained high accuracy (~90%) between the tissue types. This delineation is superior to current clinical performance and has potential applicability in supplementing histological analysis, understanding the etiology, as well as development of an in vivo screening tool.

Preterm birth (PTB) presents a serious medical heath concern in both economically developed and developing nations, with incidence rate from 15%-11% respectively. Changes in cervical collagen bundle orientation and distribution may prove to be a predictor of PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent biological tissue such as those rich in collagen. Non-invasive, full-field Mueller Matrix polarimetry (MMP) imaging methodologies, optical coherence tomography (OCT), and second harmonic generation (SHG) microscopy were used to assess cervical collagen content and structure in non-pregnant cervices. In vivo studies using a Mueller Matrix colposcope are underway. Further studies of cervical collagen orientation throughout pregnancy are needed to understand if Mueller matrix polarimetry can effectively identify at-risk conditions for PTB.

The cervix is primarily composed of two types of epithelium: stratified squamous ectocervix and simple columnar endocervix. In between these two layers lies a metaplastic squamocolumnar junction commonly referred to as the transformation zone (T-zone). During puberty, the cervical epithelium undergoes dynamic changes including cervical ectropion and increased area and rates of metaplasia. Although these metaplastic changes have been linked to higher incidence of cervical cancer among young women, research in this field has been limited to surface analysis using computerized planimetry of colopophotographs. Here, we present a novel multiplexed low coherence interferometry (mLCI) system for interrogating the cervical epithelium. The system is comprised of 6 parallel Mach-Zehnder interferometers in a time-multiplexed configuration that increases throughput by 6-fold to realize a combined 36-channel acquisition. A custom designed endoscopic handheld probe is used to collect sparsely sampled, depth-resolved scattering intensity profiles (A-scans) from a large field of view (25 x 25 mm) on the cervical epithelium in vivo. The instrument incorporates white light imaging through a plastic fiber bundle to co-register the mLCI A-scans to colpophotographs which are analyzed by a clinician to manually segment the cervical epithelium. Our preliminary data shows significant differences in characteristic A-scans from endocervical and ectocervical epithelium. These results demonstrate the feasibility of using mLCI as both a research tool for studying the relationship between cervical ectopy and cancer as well as a clinical instrument for identifying the at-risk T-zone on the cervix in vivo as a means to improve biopsy targeting. Further analysis will be performed to develop an algorithm for distinguishing the mLCI A-scans of endocervical, ectocervical, and metaplastic epithelium in real time.

Label-free multi-photon imaging has been a powerful tool for studying tissue microstructures and biochemical distributions, particularly for investigating tumors and their microenvironments. However, it remains challenging for traditional bench-top multi-photon microscope systems to conduct ex vivo tumor tissue imaging in the operating room due to their bulky setups and laser sources. In this study, we designed, built, and clinically demonstrated a portable multi-modal nonlinear label-free microscope system that combined four modalities, including two- and three- photon fluorescence for studying the distributions of FAD and NADH, and second and third harmonic generation, respectively, for collagen fiber structures and the distribution of micro-vesicles found in tumors and the microenvironment. Optical realignments and switching between modalities were motorized for more rapid and efficient imaging and for a light-tight enclosure, reducing ambient light noise to only 5% within the brightly lit operating room. Using up to 20 mW of laser power after a 20x objective, this system can acquire multi-modal sets of images over 600 μm × 600 μm at an acquisition rate of 60 seconds using galvo-mirror scanning. This portable microscope system was demonstrated in the operating room for imaging fresh, resected, unstained breast tissue specimens, and for assessing tumor margins and the tumor microenvironment. This real-time label-free nonlinear imaging system has the potential to uniquely characterize breast cancer margins and the microenvironment of tumors to intraoperatively identify structural, functional, and molecular changes that could indicate the aggressiveness of the tumor.

Previous work has shown that cellular-level Optical Metabolic Imaging (OMI) of organoids derived from human breast cancer cell-line xenografts accurately and rapidly predicts in vivo response to therapy. To validate OMI as a predictive measure of treatment response in an immune-competent model, we used the polyomavirus middle-T (PyVmT) transgenic mouse breast cancer model. The PyVmT model includes intra-tumoral heterogeneity and a complex tumor microenvironment that can influence treatment responses. Three-dimensional organoids generated from primary PyVmT tumor tissue were treated with a chemotherapy (paclitaxel) and a PI3K inhibitor (XL147), each alone or in combination. Cellular subpopulations of response were measured using the OMI Index, a composite endpoint of metabolic response comprised of the optical redox ratio (ratio of the fluorescence intensities of metabolic co-enzymes NAD(P)H to FAD) as well as the fluorescence lifetimes of NAD(P)H and FAD. Combination treatment significantly decreased the OMI Index of PyVmT tumor organoids (p<0.0001) and in vivo tumors (p<0.0001) versus controls. Subpopulation analyses revealed a homogeneous response to combined therapy in both cultured organoids and in vivo tumors, while single agent treatment with XL147 alone or paclitaxel alone elicited heterogeneous responses in organoids. Tumor volume decreased with combination treatment through treatment day 30. These results indicate that OMI of organoids generated from PyVmT tumors can accurately reflect drug response in heterogeneous allografts with both innate and adaptive immunity. Thus, this method is promising for use in humans to predict long-term treatment responses accurately and rapidly, and could aid in clinical treatment planning.

Confocal microscopy is in clinical use to diagnose skin cancers in the United States and in Europe. Potentially, this technology may provide bed-side pathology in breast cancer surgery during tumor removal. Initial studies have described major findings of invasive breast cancers as seen on fluorescence confocal microscopy. In many of these studies the region of interest (ROI) used in the analysis was user-selected and small (typically 15 square-mm). Although these important findings open exploration into rapid pathology, further development and implementation in a surgical setting will require examination of large specimens in a blinded fashion that will address the needs of typical surgical settings. In post surgery pathology viewing, pathologists inspect the entire pathology section with a low (2X) magnification objective lens initially and then zoomed in to ROIs with higher magnification lenses (10X to 40X) magnifications to further investigate suspected regions. In this study we explore the possibility of implementation in a typical surgical setting with a new microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 400 square-mm (2 cm x 2 cm) of tissue with cellular level resolution in 10 minutes. CSM images of 34 human breast tissue specimens from 18 patients were blindly analyzed by a board-certified pathologist and correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM images. Thirty specimens were concordant for images-to-histopathology correlation while four were discordant. Preliminary results from on-going work to molecularly target tumor margin will also be presented.

The current standard of care for early stages of breast cancer is breast-conserving surgery (BCS). BCS involves a lumpectomy procedure, during which the tumor is removed with a rim of normal tissue-if cancer cells found in that rim of tissue, it is called a positive margin and means part of the tumor remains in the breast. Currently there is no method to determine if cancer cells exist at the margins of lumpectomy specimens aside from time-intensive histology methods that result in reoperations in up to 38% of cases. We used fluorescence lifetime imaging (FLIm) to measure time-resolved autofluorescence from N=13 ex vivo human breast cancer specimens (N=10 patients undergoing lumpectomy or mastectomy) and compared our results to histology. Tumor (both invasive and ductal carcinoma in situ), fibrous tissue, fat and fat necrosis have unique fluorescence signatures. For instance, between 500-580 nm, fluorescence lifetime of tumor was shortest (4.7 ± 0.4 ns) compared to fibrous tissue (5.5 ± 0.7 ns) and fat (7.0 ± 0.1 ns), P<0.05 (ANOVA). These differences are due to the biochemical properties of lipid, nicotineamide adenine dinucleotide (NADH) and collagen fibers in the fat, tumor and fibrous tissue, respectively. Additionally, the FLIm data is augmented to video of the breast tissue with image processing algorithms that track a blue (450 nm) aiming beam used in parallel with the 355 nm excitation beam. This allows for accurate histologic co-registration and in the future will allow for three-dimensional lumpectomy surfaces to be imaged for cancer margin delineation.

We have developed ultrasound (US)-guided diffuse optical tomography (DOT) technique to assist US diagnosis of breast cancer and to predict neoadjuvant chemotherapy response of breast cancer patients. The technique was implemented using a hand-held hybrid probe consisting co-registered US transducer and optical source and detector fibers which couple the light illumination from laser diodes and photon detection to PMT detectors. With the US guidance, diffused light measurements were made at the breast lesion site and the normal contralateral reference site which was used to estimate the background tissue optical properties for imaging reconstruction. However, background optical properties were affected by the chest wall underneath the breast tissue. In this study, we have analyzed data from 297 female patients and results have shown statistical significant correlation between fitted optical properties (μa and μs’) and the chest wall depth detected by a boundary detection algorithm applied to co-registered US images (r < 0.27, p < 1.0 x 10^-4). After subtracting the background total hemoglobin (tHb) computed with μa at each wavelength, the difference between malignant and benign lesion groups has improved. The Area-under-the- ROC curve (AUC) has improved from 88.5% to 91.5% (sensitivity improved from 85.0% to 87.5% and specificity from 90.2% to 92.6%). Statistical test has revealed significant difference of the AUC improvements after subtracting background tHb values.

Breast cancer is the third leading cause of death in women in the United States. In human breast tissue, adipose cells are infiltrated or replaced by cancer cells during the development of breast tumor. Therefore, an adipose map can be an indicator of identifying cancerous region. We developed an automated classification method to generate adipose map within human breast. To facilitate the automated classification, we first mask the B-scans from OCT volumes by comparing the signal noise ratio with a threshold. Then, the image was divided into multiple blocks with a size of 30 pixels by 30 pixels. In each block, we extracted texture features such as local standard deviation, entropy, homogeneity, and coarseness. The features of each block were input to a probabilistic model, relevance vector machine (RVM), which was trained prior to the experiment, to classify tissue types. For each block within the B-scan, RVM identified the region with adipose tissue. We calculated the adipose ratio as the number of blocks identified as adipose over the total number of blocks within the B-scan. We obtained OCT images from patients (n = 19) in Columbia medical center. We automatically generated the adipose maps from 24 B-scans including normal samples (n = 16) and cancerous samples (n = 8). We found the adipose regions show an isolated pattern that in cancerous tissue while a clustered pattern in normal tissue. Moreover, the adipose ratio (52.30 ± 29.42%) in normal tissue was higher than the that in cancerous tissue (12.41 ± 10.07%).

Polarized light has many applications in biomedical imaging. The interaction of a biological sample with polarized light reveals information about its biological composition, both structural and functional. The most comprehensive type of polarimetry analysis is to measure the Mueller matrix, a polarization transfer function that completely describes how a sample interacts with polarized light. However, determination of the Mueller matrix requires tissue analysis under many different states of polarized light; a time consuming and measurement intensive process. Here we address this limitation with a new rapid polarimetry system, and use this polarimetry platform to investigate a variety of tissue changes associated with breast cancer. We have recently developed a rapid polarimetry imaging platform based on four photoelastic modulators (PEMs). The PEMs generate fast polarization modulations that allow the complete sample Mueller matrix to be imaged over a large field of view, with no moving parts. This polarimetry system is then demonstrated to be sensitive to a variety of tissue changes that are relevant to breast cancer. Specifically, we show that changes in depolarization can reveal tumor margins, and can differentiate between viable and necrotic breast cancer metastasized to the lymph nodes. Furthermore, the polarimetric property of linear retardance (related to birefringence) is dependent on collagen organization in the extracellular matrix. These findings indicate that our polarimetry platform may have future applications in fields such as breast cancer diagnosis, improving the speed and efficacy of intraoperative pathology, and providing prognostic information that may be beneficial for guiding treatment.

Determination of ovarian status and follicle monitoring are common methods of diagnosing female infertility. We evaluated the suitability of selective plane illumination microscopy (SPIM) for the study of ovarian follicles. Owing to the large field of view and fast acquisition speed of our newly developed SPIM system, volumetric image stacks from entire intact samples of pig ovaries have been rendered demonstrating clearly discernible follicular features like follicle diameters (70 μm - 2.5 mm), size of developing Cumulus oophorus complexes (COC ) (40 μm - 110 μm), and follicular wall thicknesses (90 μm-120 μm). The observation of clearly distinguishable COCs protruding into the follicular antrum was also shown possible, and correlation with the developmental stage of the follicles was determined. Follicles of all developmental stages were identified, and even the small primordial follicle clusters forming the egg nest could be observed. The ability of the system to non-destructively generate sub-cellular resolution 3D images of developing follicles, with excellent image contrast and high throughput capacity compared to conventional histology, suggests that it can be used to monitor follicular development and identify structural abnormalities indicative of ovarian ailments. Accurate folliculometric measurements provided by SPIM images can immensely help the understanding of ovarian physiology and provide important information for the proper management of ovarian diseases.

Both optical coherence tomography (OCT) and selective plane illumination microscopy (SPIM) are frequently used in mouse embryonic research for high-resolution three-dimensional imaging. However, each of these imaging methods provide a unique and independent advantage: SPIM provides morpho-functional information through immunofluorescence and OCT provides a method for whole-embryo 3D imaging. In this study, we have combined rotational imaging OCT and SPIM into a single, dual-modality device to image E9.5 mouse embryos. The results demonstrate that the dual-modality setup is able to provide both anatomical and functional information simultaneously for more comprehensive tissue characterization.

Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

Over 500,000 women per year in the United States drink during pregnancy, and 1 in 5 of this population also binge drink. Up to 40% of live-born children with prenatal alcohol exposure (PAE) present with congenital heart defects (CHDs) including life-threatening outflow and valvuloseptal anomalies. Previously we established a PAE model in the avian embryo and used optical coherence tomography (OCT) imaging to assay looping-stage (early) cardiac function/structure and septation-stage (late) cardiac defects. Early-stage ethanol-exposed embryos had smaller cardiac cushions (valve precursors) and increased retrograde flow, while late-stage embryos presented with gross head/body defects, and exhibited smaller atrio-ventricular (AV) valves, interventricular septae, and aortic vessels. However, supplementation with the methyl donor betaine reduced gross defects, prevented cardiac defects such as ventricular septal defects and abnormal AV valves, and normalized cardiac parameters. Immunofluorescent staining for 5-methylcytosine in transverse embryo sections also revealed that DNA methylation levels were reduced by ethanol but normalized by co-administration of betaine. Furthermore, supplementation with folate, another methyl donor, in the PAE model appeared to normalize retrograde flow levels which are typically elevated by ethanol exposure. Studies are underway to correlate retrograde flow numbers for folate with associated cushion volumes. Finally, preliminary findings have revealed that glutathione, a key endogenous antioxidant which also regulates methyl group donation, is particularly effective in improving alcohol-impacted survival and gross defect rates. Current investigations will determine whether glutathione has any positive effect on PAE-related CHDs. Our studies could have significant implications for public health, especially related to prenatal nutrition recommendations.

Premature infants are at a high risk for respiratory diseases owing to an underdeveloped respiratory system that is very susceptible to infection and inflammation. One aspect of respiratory health is the state of the ciliated respiratory epithelium which lines the trachea and bronchi. The ciliated epithelium is responsible for trapping and removing pathogens and pollutants from the lungs and an impairment of ciliary functionality can lead to recurring respiratory infections and subsequent lung damage. Mechanisms of cilia-driven fluid flow itself but also factors influenced by development like ciliary density and flow generation are incompletely understood. Furthermore, medical interventions like intubation and accidental aspiration can lead to focal or diffuse loss of cilia and disruption of flow. In this study we use two animal models, Xenopus embryo and ex vivo mouse trachea, to analyze flow defects in the injured ciliated epithelium. Injury is generated either mechanically with a scalpel or chemically by calcium chloride (CaCl2) shock, which efficiently but reversibly deciliates the embryo skin. In this study we used optical coherence tomography (OCT) and particle tracking velocimetry (PTV) to quantify cilia driven fluid flow over the surface of the Xenopus embryo. We additionally visualized damage to the ciliated epithelium by capturing 3D speckle variance images that highlight beating cilia. Mechanical injury disrupted cilia-driven fluid flow over the injured site, which led to a reduction in cilia-driven fluid flow over the whole surface of the embryo (n=7). The calcium chloride shock protocol proved to be highly effective in deciliating embryos (n=6). 3D speckle variance images visualized a loss of cilia and cilia-driven flow was halted immediately after application. We also applied CaCl2-shock to cultured ex vivo mouse trachea (n=8) and found, similarly to effects in Xenopus embryo, an extensive loss of cilia with resulting cessation of flow. We investigated the regeneration of the ciliated epithelium after an 8 day incubation period, and found that cilia had regrown and flow was completely restored. In conclusion, OCT is a valuable tool to visualize injury of the ciliated epithelium and to quantify reduction of generated flow. This method allows for systematic investigation of focal and diffuse injury of the ciliated epithelium and the assessment of mechanisms to compensate for loss of flow.

The Drosophila melanogaster shares many similarities with vertebrates in heart development. Comparison of heart structural and functional characteristic between male and female Drosophila melanogaster at different developmental stages is helpful to understand heart morphogenesis and function for different genders. And also, it opens up the possibility to uncover the role of sex-related genes in heart development. In this longitudinal study, we cultured and tracked dozens of individually labeled flies throughout their lifecycle. The heart characteristic was measured at different developmental stages during culturing. The gender of each individual fly was determined by adult stage so that the collected data of early stages could be classified to male or female group. We adapted a high-speed optical coherence microscopy (OCM) system with axial and transverse resolution of 2um and 4um, respectively, to perform non-invasive M-mode imaging at a frame rate of 132Hz in Drosophila heart at third instar larva, early pupa and adult stage. Based on those GPU processed M-mode OCM images, we segmented the fly heart region and then quantified the cardiac structural and functional parameters such as heart rate, heart chamber size and so on. Despite large variances of wild type Drosophila in terms of some cardiac characteristic, our results suggest that the heart rate is lower for male flies than for female flies, especially at third instar larva stage. The end diastolic area (EDA) and end systolic area (ESA) of the heart are both slightly larger in female flies than in male flies at larva and adult stage. In summary, we showed gender differences of wild type drosophila in heart functional and structural characteristic.

The study of the developing cardiovascular system in mice is important for understanding human cardiogenesis and congenital heart defects. Our research focuses on imaging early development in the mouse embryo to specifically understand cardiovascular development under the regulation of dynamic factors like contractile force and blood flow using optical coherence tomography (OCT). We have previously developed an OCT based approach that combines static embryo culture and advanced image processing with computational modeling to live-image mouse embryos and obtain 4D (3D+time) cardiodynamic datasets. Here we present live 4D dynamic blood flow imaging of the early embryonic mouse heart in correlation with heart wall movement. We are using this approach to understand how specific mutations impact heart wall dynamics, and how this influences flow patterns and cardiogenesis. We perform studies in mutant embryos with cardiac phenotypes such as myosin regulatory light chain 2, atrial isoform (Mlc2a). This work is brings us closer to understanding the connections between dynamic mechanical factors and gene programs responsible for early cardiovascular development.

Abnormal cell proliferation and migration during heart development can lead to severe congenital heart defects (CHDs). Studying the spatial distribution of cells during embryonic development helps our understanding of how the heart develops and the etiology of certain CHDs. However, imaging large groups of single cells in intact tissue volumes is challenging. No current technique can accomplish this task in both a time-efficient and cost-effective manner. OCT has potential with its large field of view and micron-scale resolution, but even the highest resolution OCT systems have poor contrast for counting cells and have a small field of view compared to conventional OCT. We propose using a conventional OCT system and processing the sample to enhance cellular contrast. Inspired by the recently developed Expansion Microscopy, we permeated whole-mount embryonic tissue with a superabsorbent monomer solution and polymerized into a hydrogel. When hydrated in DI water, the tissue-hydrogel complex was uniformly enlarged (~5X in all dimensions) without distorting the microscopic structure. This had a twofold effect: it increased the resolution by a factor of 5 and decreased scattering, which allowed us to resolve cellular level features deep in the tissue with high contrast using conventional OCT. We noted that cell nuclei caused significantly more backscattering than the other subcellular structures after expansion. Based on this property, we were able to distinguish individual cell nuclei, and thus count cells, in expanded OCT images with simple intensity thresholding. We demonstrate the technique with embryonic quail hearts at various developmental stages.

Altered hemodynamics in developing embryonic hearts lead to congenital heart diseases, motivating close monitoring of blood flow over several stages of development. Doppler OCT can assess blood flow in tubular hearts, but the maximum velocity increases drastically during the period of cardiac cushion (valve precursors) formation. Therefore, the limited dynamic range of Doppler OCT velocity measurement makes it difficult to conduct longitudinal studies without phase wrapping at high velocities or loss of sensitivity to slow velocities. We have built a high-speed OCT system using an FDML laser (Optores GmbH, Germany) at a sweep rate of 1.68 MHz (axial resolution - 12 μm, sensitivity - 105 dB, phase stability - 17 mrad). The speed of this OCT system allows us to acquire high-density B-scans to obtain an extended velocity dynamic range without sacrificing the frame rate. The extended dynamic range within a frame is achieved by varying the A-scan interval at which the phase difference is found, enabling detection of velocities ranging from tens of microns per second to hundreds of mm per second. The extra lines in a frame can also be utilized to improve the structural and Doppler images via complex averaging. In structural images where presence of blood causes additional scattering, complex averaging helps retrieve features located deeper in the tissue. Moreover, high-density frames can be registered to 4D volumes to determine the orthogonal direction of flow and calculate shear stress. In conclusion, our high-speed OCT system will enable automated Doppler imaging of embryonic hearts in cohort studies.

Hemodynamic force is vital to cardiovascular remodeling in the early post-implantation mouse embryo. Here, we present work using microCT and lightsheet microscopy to establish the critical sequence of developmental events required for forming functional vasculature and circulation in the embryo, yolk sac, and placenta in the context of normal and impaired flow. A flow impaired model, Mlc2a+/- will be used to determine how hemodynamic force affects the specific events during embryonic development and vascular remodeling between the 4 and 29-somite stage using microCT. We have recently established high-resolution methods for the generation of 3D image volumes from the whole embryo within the deciduum (Hsu et al., in revision). This method enables the careful characterization of 3D images of vitelline and umbilical vessel remodeling to define how poor blood flow impacts both vitelline and umbilical vessel remodeling. Novel lightsheet live imaging techniques will be used to determine the consequence of impaired blood flow on yolk sac vasculature remodeling and formation of umbilical vessels using transgenic reporters: Flk-myr::mCherry, Flk1-H2B::YFP, or εGlobin-GFP. High-resolution 3D imaging of fixed and ScaleA2-cleared whole mount embryos labeled with Ki67 and Caspase3 will also be performed using lightsheet microscopy to quantify the proliferation and apoptotic indexes of early post-implanted embryos and yolk sac. This multi-modality approach is aimed at revealing further information about the cellular mechanisms required for proper vessel remodeling and the initial stages in placentation during early post-implantation development.

Preterm birth (PTB) presents a serious medical heath concern throughout the world. There is a high incidence of PTB in both developed and developing countries ranging from 11%-15%, respectively. Studies have shown there may be numerous precursors to PTB including infections, genetic predisposition, nutrition and various other morbidities which all lead to a premature disorganization in the cervical collagen resulting in the weakening of the structure designed to keep the fetus in utero. The changes in cervical collagen orientation and distribution may prove to be a predictor of PTB. Polarization imaging is an effective means to measure optical anisotropy in birefringent materials such as those rich in collagen as the cervix is. Non-invasive, full-field Mueller Matrix polarimetry (MMP) imaging methodologies and ex-vivo second harmonic generation (SHG) imaging were used to assess cervical collagen content and structure in non-pregnant porcine cervices. The SHG microscopy was used to verify the efficacy of the MMP in assessing changes in collagen orientation.

Autofluorescence microscopy of NAD(P)H and FAD provides functional metabolic measurements at the single-cell level. Here, density-based clustering algorithms were applied to metabolic autofluorescence measurements to identify cell-level heterogeneity in tumor cell cultures. The performance of the density-based clustering algorithm, DENCLUE, was tested in samples with known heterogeneity (co-cultures of breast carcinoma lines). DENCLUE was found to better represent the distribution of cell clusters compared to Gaussian mixture modeling. Overall, DENCLUE is a promising approach to quantify cell-level heterogeneity, and could be used to understand single cell population dynamics in cancer progression and treatment.

We are investigating the ability of targeted rare earth (RE) doped nanocomposites to detect and track micrometastatic breast cancer lesions to distant sites in pre-clinical in vivo models. Functionalizing RE nanocomposites with AMD3100 promotes targeting to CXCR4, a recognized marker for highly metastatic disease. Mice were inoculated with SCP-28 (CXCR4 positive) and 4175 (CXCR4 negative) cell lines. Whole animal in vivo SWIR fluorescence imaging was performed after bioluminescence imaging confirmed tumor burden in the lungs. Line-scanning confocal fluorescence microscopy provided high-resolution imaging of RE nanocomposite uptake and native tissue autofluorescence in ex vivo lung specimens. Co-registered optical coherence tomography imaging allowed assessment of tissue microarchitecture. In conclusion, multiscale optical molecular imaging can be performed in pre-clinical models of metastatic breast cancer, using targeted RE-doped nanocomposites.

Optical coherence microscopy (OCM) has unique advantages of high-resolution volumetric imaging without relying on exogenous labels or dyes. It combines the coherence-gated depth discrimination of optical coherence tomography (OCT) with the high lateral resolution of confocal microscopy, offering an excellent balance between the resolutions and imaging depth. However, as the lateral resolution becomes higher, the imaging depth of OCM decreases and its three-dimensional imaging capability is greatly degraded. To overcome this limitation, we used amplitude apodization to create quasi-Bessel beam illumination in order to extend the depth of focus. The lateral and axial resolutions of our OCM system were measured to be 1.6 μm and 2.9 μm in tissue. The imaging depth was extended by ~3.0X (~100 μm) beyond that of the standard Gaussian beam OCM. Using zebrafish embryos as a test system, we demonstrate extendedfocus OCM for structural imaging studies, which revealed the detailed anatomy deep in embryos.

Changes in mechanical properties represent one of the driving factors behind cell differentiation during embryonic development. However, measuring these changes without disrupting the normal progression of morphogenesis or destroying the developing organism is not trivial. Brillouin microspectroscopy has been shown to be capable of nocontact, non-destructive and non-disruptive assessment of elastic properties in developing zebrafish embryos. The present study builds upon the previous work, and observes the changes in elasticity during the development of heart and brain in zebrafish embryos from 8 to 28 hpf (hours post-fertilization) at regular intervals. Brillouin microspectroscopy has proved to be a suitable technique to continuously monitor tissue differentiation and the development of individual organs with high spatial resolution without harming the developing organism.

Early detection of breast carcinoma is vital for effective treatment option and to enhance the survival rate. Existing breast imaging systems such as ultrasound, mammography, and magnetic resonance imaging (MRI) have been utilized for early detection of breast carcinoma which requires contact with the breast surface. However, these existing methods require contact to the breast surface, which causes discomfort to the test subject. Hence, there is a need for alternative modality, which exhibits a total non-contact nature. Structured light profilometry has developed into a vital system with its application in diverse fields of surface metrology analysis. Therefore, in this work structured light profilometry based on phase shift technique is setup to analyze the surface variation of the breast due to the presence of a lesion in the context of surface tension. The sinusoidal fringe pattern is projected through three step phase shift onto the surface of the breast, and a resulting phase map is produced. Pixel tracing was performed to evaluate the variation of surface changes on the breast based on surface marker coordinates. The comparison was made between breast with lump and breast without a lump. Maiden results have established that the structured light profilometry is capable of detecting breast surface changes at various locations on the breast.

Optical sensing technique has inherited non-contact nature for generating 3D surface mapping where its application ranges from MEMS component characterization, corrosion analysis, and vibration analysis. In particular, the digital fringe projection is utilized for 3D mapping of objects through the illumination of structured light for medical application extending from oral dental measurements, lower back deformation analysis, monitoring of scoliosis and 3D face reconstruction for biometric identification. However, the usage of digital fringe projection for 3D mapping of human breast is very minimal. Thus, this paper addresses the application of digital fringe projection for 3D mapping of breast surface based on total non-contact nature. In this work, phase shift method is utilized to perform the 3D mapping. The phase shifted fringe pattern are displayed through a digital projector onto the breast surface, and the distorted fringe patterns are captured by a CCD camera. A phase map is produced, and phase unwrapping was executed to obtain the 3D surface mapping of the breast. The surface height profile from 3D fringe projection was compared with the surface height measured by a direct method using electronic digital vernier caliper. Preliminary results showed the feasibility of digital fringe projection in providing a 3D mapping of breast and its application could be further extended for breast carcinoma detection.