This PDF file contains the front matter associated with SPIE Proceedings Volume 11216, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

There has been little headway against Alzheimer’s disease despite decades of research into causes, conditions, and cures. Much of this effort has focused on the amyloid beta protein; however, it remains unclear whether it plays a causative or correlative role. Amyloid beta is notoriously difficult to study, and many of the current tools to monitor oligomerization and fibril formation are inadequate and are incapable of measuring the subtle details of plaque formation and deformation. Our preliminary data suggests that multispectral nanoparticle analysis can monitor the sigmoidal self-assembly (R2 = 0.94) of physiologically relevant populations of amyloid beta aggregates in real-time with single fibril resolution.

Ulcerative Colitis (UC) is an idiopathic autoimmune inflammatory disorder of the colon mucosa characterized by leukocyte infiltration into the submucosa and ulcer formation in the epithelium, followed by epithelial cell proliferation and restitution of the mucosal barrier. Optical methods sensitive to tissue oxygen demand and epithelial cell metabolism are ideally suited for clinical guidance of disease progression and remission in UC. Here we present endoscopic reporters of submucosal oxygen demand (i.e. inflammatory-related changes) and epithelial cell metabolism (i.e. mucosal re-epithelialization) within the ulcer bed and surrounding microenvironment in a murine model of UC as putative biomarkers of mucosal healing.

Photoacoustic tomography (PAT), in the forms of computed tomography or microscopy, provides in vivo functional, metabolic, molecular, and histologic imaging. PAT has the unique strength of high-resolution multiscale imaging across organelles, cells, tissues, organs in both animals and humans as well as small-animal organisms with consistent optical absorption contrast. The wavelength of the excitation laser can be broadly tuned to target various endogenous or exogenous molecules. PAT has the potential to empower omniscale biology and accelerate trans-scale clinical translation. Potential medical applications include imaging of the breast, brain, thyroid, muscle, joint, skin, vascular system, lymphatic system, prostate, esophagus, colon, cervix, bladder, and uterus.

In recent years we have developed open-top light-sheet microscopes for a variety of clinical applications. Here we present a new directions and next-generation clinical applications of the technology.

Innate differences among breast cancer phenotypes are often studied from the perspective of single protein expressions or by fluorescent imaging using molecular tags. While these techniques can offer useful insights into breast cancer taxonomy, they do not easily translate to clinical care. Nonlinear optical microscopy has revolutionized our ability to study biochemical processes, as it offers a label-free approach to study differences in cancer phenotypes that may provide insight into factors affecting prognosis and treatment strategies. The endogenous chemical specificity offered by nonlinear imaging modalities like Stimulated Raman Scattering (SRS) and Multiphoton Fluorescence (MPF) are attractive alternatives to fluorescent imaging to study intracellular biochemistry. Here we utilize a multimodal imaging platform to characterize lipogenesis in HER2+ cell lines through SRS and metabolic activity by MPF of NADH/FAD to investigate unique signals related to HER2 status.

Bladder cancer is the 6th most common cancer in the United States.[1] While cystoscopy is routinely performed for patients under surveillance, there is a critical need to improve its ability to accurately characterize bladder lesions with high specificity. Previously we developed a real-time and low-cost (<$5,000) confocal high-resolution microendoscope (confocal HRME) and demonstrated that it can resolve subcellular and clinically relevant features in highly scattering tissue samples.[2] In this study, we evaluate the utility of the confocal HRME in conjunction with conventional cystoscopy to characterize bladder precancer and cancer lesions at different scales. Patients scheduled for cystoscopy at Lyndon B. Johnson Hospital in Houston, Texas were first examined with a standard cystoscope, followed by intravesical instillation of proflavine (0.01% w/v in sterile PBS) for fluorescence imaging. In vivo HRME images of normal and suspicious lesions were then acquired and compared to histopathology as the gold standard. Our preliminary results showed that confocal HRME images of normal bladder epithelium was characterized by an intact lining of umbrella cells. As disease progressed in urothelial carcinoma, the regular layered structure became disrupted, accompanied with nuclear crowding and enlargement of urothelial cells. We also compared the imaging performance of the confocal HRME with the non-confocal mode, and confocal images revealed improved imaging contrast of nuclear morphology, especially in lesions with crowded nuclei. In summary, the confocal HRME, when combined with widefield cystoscopy, can resolve disease associated features at the subcellular level and has the potential to improve early detection of bladder cancer. References: 1. SEER/NIC. Seer Stats Fact Sheets: Bladder Cancer. Retrieved July 22, 2019 from https://seer.cancer.gov/statfacts/html/urinb.html. 2. Y. Tang, A. Kortum, I. Vohra, M. Othman, S. Dhingra, N. Mansour, J. Carns, S. Anandasabapathy, and R. Richards-Kortum, "Improving nuclear morphometry imaging with real-time and low-cost line-scanning confocal microendoscope," Opt. Lett. 44, 654 (2019).

Photomultiplier tubes are the standard for high sensitivity detection of fluorescence signals, but are costly, fragile, have limited QE and worse sensitivity at far-red wavelengths. Silicon photomultipliers (SIPMs) combine the higher shot-noise limited sensitivity of silicon sensors with the single photon sensitivity of PMTs. We evaluate the new generation of SIPMs for high speed fluorescent imaging applications and find that for a range of operating conditions, SIPMs offer higher SNR and dynamic range than GaAsP or GaAs PMTs. We show how to design low cost electronics that enable extremely high sensitivity, dynamic range, and bandwidth, discuss how to customize detectors for high speed fluorescent imaging, and show the results of human tissue imaging comparing PMTs and SIPMs.

Cerebral blood flow regulation is impaired in many diseases and treatment based on optimizing blood flow regulation is promising for patient outcome, but may require intracranial pressure (ICP) to be known. We have recently developed a non-invasive alternative based on a combination of near-infrared spectroscopy and diffuse correlation spectroscopy. This talk will summarize the optical imaging setup, experimental procedures, and data analysis. Data from non-human primates as well as clinical data from pediatric critical care patients will be presented. We will further present data on neurovascular coupling during ICP changes, suggesting a link between blood flow regulation and neuronal activity.

The change in the optical properties of tissue during thermal treatment can be potentially used to monitor procedures like Radiofrequency Ablation (RFA). We present key features in the optical absorption and scattering of tissue during the RFA procedure and during post-ablation cooling down to room temperature. We have used time-resolved diffuse optical spectroscopy for the measurement of the optical properties of tissue for the wavelengths from 650 to 1100 nm. Ex vivo experiments were conducted using a clinical RFA system on bovine liver tissue. Measurements were performed for two temperatures (70°C and 105°C). The following features were observed in the optical properties. First, there was a decrease in optical absorption and an increase in scattering during the treatment. With overtreatment, the absorption increased for initial part of the spectrum (until 910 nm) and scattering decreased in comparison to normal treatment. Secondly, a redshift of the hemoglobin peak and blue shift around water peak was observed in the optical absorption. Finally, a new peak around 840 nm and a valley around 920 nm appeared with heating. When the tissue was allowed to cool down, most of the changes in the absorption around the water peak partially reversed including the blue shift and the valley around 920 nm. Additionally scattering decreased with cooling. Results show key features in the optical properties of tissue during RFA, the effect of overtreatment and post-treatment cooling in ex vivo tissue. Insights from this study will help in advancing optical methods in monitoring thermal treatment.

We developed combined diffuse reflectance spectroscopy – near-infrared diffuse correlation spectroscopy system. Cuff occlusion test, blood phantom test and test with anesthetic agents in a rat model were conducted for system verification. When the result of system verification follows the expected change in hemoglobin concentration change and the result of test with anesthetic agents shows effect of agents, then we can say that this combined system can be used for monitoring depth of anesthesia. This verified system can be used as the system for establishment of brain disease biomarker under anesthesia state.

Functional near-infrared spectroscopy (fNIRS) has been gaining much attention in biophotonics fields because it provides brain activity based on monitoring of hemodynamic changes. Even though fNIRS has shown significant results in brain research, the question has been raised about the origin of hemodynamic changes, due to the uncertainty of the light path in the brain structure. The goal of this study is to separate the scalp and brain layer hemodynamic by developing diffuse reflectance spectroscopy based on two-layered photon diffusion reflectance equation. In order to validate our approach, the simulation experiments were carried out. During the experiment, various tissue reflectance spectra corresponding to various hemodynamic conditions of the superficial and brain layers were generated by simulation. The results show the potential of our approach that separating brain hemodynamics from tissue reflectance spectrums.

We have used Raman spectroscopy to assess biomolecular changes of two human head and neck cancer xenografts in response to radiation therapy. We injected radiation-sensitive UM-SCC-22B and –resistant UM-SCC-47 cell lines into the flanks of 80 mice to grow tumor xenografts. Animals were distributed into control and radiation groups where the latter group received a single dose of 2 Gy. In vivo Raman spectroscopy was conducted before, 1, 24, and 48 hours after radiation and data were decomposed using principal component analysis and multi variate curve resolution. We found statistically significant differences in biomolecular changes among radiation-sensitive and –resistant groups.

Cancer cell growth has been shown to affect receptor expression and organelle morphology. Therefore, differences in mitochondrial morphology and function in cancer cells are of key importance for the successful implementation of targeted drug therapy in the clinic. To improve our understanding of the heterogeneity of mitochondrial morphology in breast cancer cells, we have analyzed the morphology of mitochondria in a comparative manner across 2D-culture breast cancer cell systems. Moreover, we used machine learning methods to develop a classification method that can assign each 3D rendered mitochondrial object to their respective breast cancer cell type with good accuracy.

Irradiation of tissue causes delivery of dose to the volume, which causes widespread damage, the most important being production of radical oxygen species and the associated DNA damage. Part of the dose deposition is from soft electron collisions, which result in Cherenkov light emission from the relativistic dielectric interaction. This visible light emission is produced at the level of hundreds of photons per x-ray photon, and is significant enough to be detected with single photon imaging cameras. Radiation dosimetry with these cameras is now possible, providing a non-contact way to detect radiation dose deposition in cancer therapy. The demonstration of this imaging methodology and the theoretical underpinnings will be illustrated.

Angiogenesis inhibiting cancer therapy has become a standard treatment for many cancer types. The ability to examine the effects of these drugs in tumors noninvasively could help assess efficacy early in the treatment course or identify optimal times to introduce other combinatorial treatments. Herein, we examine whether a paired agent MRI-coupled fluorescence tomography approach can be used to monitor the effects of anti-angiogenesis therapy. Using small animal models bearing orthotopic glioma xenografts, we demonstrate noninvasive quantification of paired-agent uptake in response to anti-angiogenesis therapy in vivo. The result provides insights on receptor targeted drug delivery in altered vasculature, a potential important development for treatment monitoring and combinatorial strategies.

Recently we have demonstrated that spatial frequency modulation imaging can use extended excitation sources in linear and nonlinear image modalities, is compatible with single element detection, and results in enhanced lateral resolution across the excitation beam. In this paper, we will present new methods where the SPIFI platform goes from one-dimensional to two-dimensional imaging while still exhibiting the enhanced resolution across the added dimension. Significantly, we present the physical mechanism responsible for the resolution enhancement for all imaging modalities, we provide computational models that support the physical model for the increased resolution, and finally, present experimental verification of the resolution enhancement.

Positive outcomes for colorectal cancer treatment have been linked to early detection. The difficulty in detecting early lesions is the limited contrast with surrounding mucosa and minimal definitive markers to distinguish between hyperplastic and carcinoma lesions. Colorectal cancer is the 3rd leading cancer for incidence and mortality rates which is potentially linked to missed early lesions which allow for increased growth and metastatic potential. One potential technology for early-stage lesion detection is hyperspectral imaging. Traditionally, hyperspectral imaging uses reflectance spectroscopic data to provide a component analysis, per pixel, of an image in fields such as remote sensing, agriculture, food processing and archaeology. This work aims to acquire higher signal-to-noise fluorescence spectroscopic data, harnessing the autofluorescence of tissue, adding a hyperspectral contrast to colorectal cancer detection while maintaining spatial resolution at video-rate speeds. We have previously designed a multi-furcated LED-based spectral light source to prove this concept. Our results demonstrated that the technique is feasible, but the initial prototype has a high light transmission loss (~98%) minimizing spatial resolution and slowing video acquisition. Here, we present updated results in developing an optical ray-tracing model of light source geometries to maximize irradiance throughput for excitation-scanning hyperspectral imaging. Results show combining solid light guide branches have a compounding light loss effect, however, there is potential to minimize light loss through the use of optical claddings. This simulation data will provide the necessary metrics to verify and validate future physical optical components within the hyperspectral endoscopic system for detecting colorectal cancer.

Time-domain near-infrared spectroscopy (TD-NIRs) and Time-Domain Diffuse Correlation Spectroscopy (TD-DCS) are emerging imaging techniques that use a near-infrared, long coherence, pulsed laser to characterize oxygenation levels and blood flow. TD-DCS is a promising tool for bedside monitoring of brain activity due to its high time-resolution and portability. One potential new application area for TD-DCS is for detecting non-compressible torso hemorrhages (NCTH). NCTH is a serious traumatic injury that requires surgical intervention and is a leading cause of death in the military due to the lack of a rapid and portable imaging system sensitive enough to detect injury. Applying long wavelengths (1064 nm and 1120 nm) and time gating, TD-DCS can penetrate the superficial tissue layers and potentially detect bleeding deep within an organ. One limitation of current time-gating system is its reliance on full knowledge of the target tissue layers and properties in order to apply gating effectively. An automatic gating scheme that can adjust the time gate to quickly recalibrate itself to different imaging conditions, such as a different body area, can eliminate this limitation. Here, we use modeling and Monte Carlo simulations to search for characteristics in return signal profiles, specifically the time-of-flight and intensity profiles, as first step toward an automatic time-gating algorithm. We detail the simulation setups, parameter sweeps, and preliminary results in this report. These results show promise for TD-DCS as a tool for rapid and continuous monitoring of injuries in the field.

Various contrast mechanisms in imaging applications in biological and material sciences are of great importance for multimodal sample visualization. Nonlinear-optical interactions in the sample provide multitude of possibilities to imaging with different contrasts, including sensitivity to chemically-specific vibrational signatures. Cubic order nonlinearity is present in all materials since it does not require broken inversion symmetry. Cubic non- linearity offers several useful interaction modalities, including vibrationally resonant ones, such as third-order sum-frequency generation (TSFG) and four-wave mixing (FWM), which we explore in this work using femtosecond lasers in a laser-scanning all-reflective microscope. We observe strong dependence of image contrast on delay between interacting pulses and the frequency of the mid-IR laser relative to the CH vibrational mode of the sample. Images of oil-water interfaces demonstrate striking visual contrast and impressive signal-to-noise ratio in our system. Pathways to expand TSFG and FWM imaging onto biological samples are explored.

Diffuse Optical Breast Scanner (DOB-Scan) probe utilizes a linear charge-coupled device (CCD) array as the detector, measuring back-scattered light intensity above the breast tissue to create cross-sectional concentration images for different constituents. The response received at each pixel of the sensor is determined by optical properties of the scattering medium, source-detector distance, integration time of CCD array, and intensity of the light source. However, the performance of the probe is limited by the inherent electronic properties of CCD array, which cause saturation at high response region and high noise level at low response region. In this paper, an algorithm to enhance the dynamic range of the CCD array is presented. The objective is to maximize the dynamic range of the CCD array without any data loss while minimizing the noise-to-signal ratio where the response of the CCD array is relatively low. The desired output can be achieved by capturing multiple sets of data with different integration time and light intensity settings, ensuring the best CCD performance in each pixel range of interest. The profile of CCD array’s linearity and optical power measurement of the light source allow different sets of data to be translated into the same scale and joined accordingly into one. A series of phantom studies are conducted and confirm the feasibility of the probe’s dynamic range intensification.

Blood is a crucial body fluid which contains erythrocytes and leukocytes and platelets, the number and status of which directly indicate the physiological state of the body. The first response to the infection is mediated by the number of leukocytes in the blood. The number and type of immune cells change vary in the disease state and identification of the type of immune cell provides information about the healthy state of body. Determining the identities of cells of the immune system usually involves destructive fixation and chemical staining, or labeling with fluorescently labeled antibodies. Raman microscopy is ideal for live cell studies or real-time diagnosis of disease, because it does not require the use of labels that may harm cells. It has potential to be carried out in vivo conditions. Raman spectroscopy has been used to investigate the differences between the leukocytes subtypes. The complex chemical composition of cells leads to complex Raman spectra, it is difficult to distinguish the categary of five subtypes white blood cells. We propose a partition principal component analysis (PPCA) method based on Raman spectroscopy using wavelet anlysis at the single cell level to separate Raman spectra of five subtypes of leukocytes, which are respectively lymphocytes, nuetrophils, monocytes, eosinophils and basophils. We achieved the identification and differentiation of five subtypes with a minimum discrimination efficiency of 85%. Systematic studies of five leukocyte subtypes have important guiding significance for the study of various leukocyte-associated cancers.