Fischell Department of Bioengineering
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Item TUNABLE ATOMIC LINE MONOCHROMATORS FOR BRILLOUIN SPECTROSCOPY(2024) Hutchins, Romanus Joshua; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Brillouin microscopy, a non-contact, spatially-resolved imaging method, provides insights into the mechanical information of samples. The first generation of Brillouin microscopes combined confocal microscopes and etalon-based spectrometers. In this setup, a confocal microscope scans a laser across the sample pixel-by-pixel, while the etalon spectrometer measures the Brillouin shift frequency at each pixel. Despite the extended image acquisition times in biological samples (>20 ms/pixel), advancements have been made in the field to enhance the overall speed of Brillouin imaging. For example, line-scan Brillouin spectrometers use orthogonal detection to measure the Brillouin scattering at a row of pixels in a single shot. The pixel multiplexing in one-dimension (1D) improved the Brillouin imaging speeds 20-fold. Further multiplexing to two dimensions, or full-field spectroscopy, where the frequency domain is sequentially acquired but all the pixels in the field of view are simultaneously measured at each frequency, can further improve the average image acquisition time. However, there are currently no solutions for sub-picometer (sub-GHz) spectral resolution, two-dimensional (2D) multiplexing of Brillouin images. Here, I use the laser induced circular dichroism (LICD) effect in atomic vapors to create monochromators for 2D multiplexing at high spectral resolutions. These atomic line monochromators possess spectral resolutions dependent on the linewidth of the atomic resonance (~MHz), and they are ideal for pixel multiplexing because they have spectral analysis capabilities that do not depend on the spatial separation of spectral components. First, I present a full characterization of a tunable atomic line monochromator. I measure the transmission, spectral resolution, and spectral tunability of the device, as well as demonstrate whole-image transmission through the atomic line monochromator. Next, for practical implementations of the device to Brillouin spectroscopy, I created an atomic line monochromator based on a ladder-type atomic transition. This iteration of the device suffers from less noise than the previous version, leading to the first Brillouin measurements with this device. Finally, I present the first full-field Brillouin microscope by demonstrating whole Brillouin imaging with orthogonal detection with an atomic line monochromator.Item MECHANICAL MAPPING OF NEURAL TUBE CLOSURE IN LIVE EMBRYOS USING BRILLOUIN MICROSCOPY(2022) Handler, Chenchen; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neurulation is a process that serves as the precursor to the spinal cord in vertebrates. Neural tube closure (NTC), part of primary neurulation, involves the extensive coordination of cellular, molecular, and mechanical events to transform the flat neural epithelium to a luminated epithelial tube. Neural tube defects (NTD) are the result of mechanical failures that arise during neurulation. Recent research has focused on understanding the molecular mechanisms underlying neurulation but has difficulty correlating them to physical mechanisms. To better understand how physical mechanisms are integrated and responsible for neurulation, several techniques have been applied to study NTC in a range of in vitro environments. However, many of these techniques have been limited due requiring the specimen to be fixed and/ or being invasive and requiring physical contact with the specimen to extract the modulus. As such, there is limited resolution and only the superficial layer of the sample is measured making assessing 2D/3D tissue mechanics inside a growing organism is highly challenging. In this dissertation, we aim to quantify the mechanical state of the neural tube without disruption to development. To do this, we adapted Brillouin microscopy, a non-invasive, label- and contact-free imaging technique, to allows us to probe thelongitudinal modulus of the neural plate at every step of NTC with cellular resolution. This quantification is performed as the embryo develops in real time using time-lapse Brillouin and an improved ex-ovo culture method. We observed an increase in the Brillouin modulus of the neural plate as the embryo develops from Hamburger-Hamilton stage (HH)-6 to HH-12. This increase in modulus is consistent with previous data from other vertebrates such as Xenopus and Mouse embryos and demonstrates the process of neurulation is driven by mechanical forces. Time-lapse Brillouin imaging depicted stiffening and thickening of the neural plate during NTC, suggesting these are coordinated events for NTC. Here, we show that tissue stiffness plays an integral role in NTC and directly quantifying tissue mechanics during neurulation should allow us to better determine the biomechanical nature of NTD.Item Development of Fluorescent Imaging Methods and Systems to Determine Photodynamic Potential and Inform Cancer Treatment Efficacy(2022) Gaitan, Brandon; Huang, Huang-Chiao; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photodynamic therapy (PDT) is a treatment modality that has gained rapid popularity in both research and clinical settings over the past 20 years. PDT involves harmless red/near-infrared light excitation of non-toxic photosensitizers to generate reactive molecular species (RMS) that can induce tissue damage and/or cell death. In addition, the fluorescence signal generated from the photosensitizer can also be used for optical imaging. These effects have been harnessed for image-guided treatment of cancer and other diseases. As PDT gains popularity, it is crucial to understand and monitor different factors that could impact overall treatment efficacy. These factors include, but are not limited to, the RMS yield of photosensitizers, the distribution of photosensitizers in tissue, and the PDT activation depth in tissues. Our work focused on developing methodologies and devices to characterize and improve PDT treatment. In collaboration with the FDA, we developed a cell-free assay to rapidly and more quantitatively determine the potential phototoxicity of fluorescent probes through the measurement of singlet oxygen. We also developed a method to compare the maximal PDT activation depth of FDA-approved photosensitizers (BPD and PpIX) in the brain. We found that BPD can be activated 50% deeper into brain tissues compared to PpIX at the same radiant exposure. Next, we tested the ability of a 3D imaging system, Fluorescence Laminar Optical Tomography (FLOT), to image the distribution of photosensitizers in the rodent brain. We demonstrated that FLOT could accurately map the photosensitizer distribution up to 0.5 mm in tissues. Lastly, we developed an autofluorescent-based endoscopic imaging system to measure the metabolic impact of PDT on cancer and normal tissues, finding that PDT leads to significant changes in tissue metabolism immediately after treatment. In summary, we have developed a series of systems that can aid in PDT treatment optimization in three major ways:1) rapidly quantifying the singlet oxygen production of photosensitizers, 2) more accurately measuring a photosensitizers localization and activatable depth, and 3) developing the ability to measure a tissues response to PDT in real-time.Item Brillouin confocal microscopy in off-axis configuration(2021) Fiore, Antonio; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three-dimensional Brillouin confocal microscopy is an imaging modality that correlates with mechanical properties in biological media from subcellular to tissue level. Over the years we developed new approaches to this technique that improve the spectral performance and can measure directly the local refractive index as well as the complex modulus of the sample; to achieve this goal, we probed two co-localized Brillouin scattering geometries. The confocal microscopy setting ensures three-dimensional mapping with high resolution, while the back scattering configuration allows access to the sample from the same side. For these reasons, such an instrument constitutes a new approach in investigating biological phenomena providing both local index of refraction and mechanical information with a single measurement. This technique has been improved in speed and spatial resolution in order to be applied to some specific challenging material characterization such as liquid-liquid phase separation.Item Controlling light Propagation in complex media for Imaging, focusing and Brillouin measurements(2018) Edrei, Eitan Y; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Imaging and focusing light through turbid media are two fundamental challenges of optical sciences that have attracted significant attention in recent years. Traditional optical systems such as confocal microscopy, optical coherence tomography and multi-photon microscopy utilize ballistic photons traveling in straight trajectories to generate an image; however, with increasing depth, the signal to noise ratio (SNR) decreases as the number of ballistic photons decays exponentially. In the first part of this thesis I present two novel techniques for imaging through scattering medium by decoding seemingly random scattered light patterns and demonstrate the highest resolution and acquisition speed to date. For point scanning applications I also study methods to focus light through scattering materials and report on a fundamental trade-off between the focal point intensity and the focal plane in which it is generated. In the second part of the thesis I investigate how the ability to control light propagation within turbid media can be used to enhance point scanning measurements such as Brillouin scattering spectroscopy, a technology recently developed in our lab to characterize material stiffness without contact. To do this, I first present a novel optical system (“spectral coronagraph”) which yields an improved extinction ratio when inserted into Brillouin spectrometers to enable the spectral separation in the presence of scattering or close to interfaces. Additionally, to enhance the Brillouin signal, I apply adaptive optics techniques, first developed for astronomy applications, where the incident wave front is shaped to circumvent for optical phase aberrations. Using adaptive optics, I show signal enhancement in artificial and biological samples, an important feature in the context of Brillouin microscopy to promote high SNR imaging in practical scenarios.Item NOVEL TECHNOLOGIES AND APPLICATIONS FOR FLUORESCENT LAMINAR OPTICAL TOMOGRAPHY(2017) Tang, Qinggong Tang; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Laminar optical tomography (LOT) is a mesoscopic three-dimensional (3D) optical imaging technique that can achieve both a resolution of 100-200 µm and a penetration depth of 2-3 mm based either on absorption or fluorescence contrast. Fluorescence laminar optical tomography (FLOT) can also provide large field-of-view (FOV) and high acquisition speed. All of these advantages make FLOT suitable for 3D depth-resolved imaging in tissue engineering, neuroscience, and oncology. In this study, by incorporating the high-dynamic-range (HDR) method widely used in digital cameras, we presented the HDR-FLOT. HDR-FLOT can moderate the limited dynamic range of the charge-coupled device-based system in FLOT and thus increase penetration depth and improve the ability to image fluorescent samples with a large concentration difference. For functional mapping of brain activities, we applied FLOT to record 3D neural activities evoked in the whisker system of mice by deflection of a single whisker in vivo. We utilized FLOT to investigate the cell viability, migration, and bone mineralization within bone tissue engineering scaffolds in situ, which allows depth-resolved molecular characterization of engineered tissues in 3D. Moreover, we investigated the feasibility of the multi-modal optical imaging approach including high-resolution optical coherence tomography (OCT) and high-sensitivity FLOT for structural and molecular imaging of colon tumors, which has demonstrated more accurate diagnosis with 88.23% (82.35%) for sensitivity (specificity) compared to either modality alone. We further applied the multi-modal imaging system to monitor the drug distribution and therapeutic effects during and after Photo-immunotherapy (PIT) in situ and in vivo, which is a novel low-side-effect targeted cancer therapy. A minimally-invasive two-channel fluorescence fiber bundle imaging system and a two-photon microscopy system combined with a micro-prism were also developed to verify the results.Item OPTICAL COHERENCE TOMOGRAPHY FOR NEUROSURGEY AND CANCER RESEARCH(2014) Liang, Chia-Pin; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Optical Coherence Tomography (OCT) provides non-labeling, real-time and high resolution images, which has the potential to transform the paradigm of surgical guidance and preclinical animal studies. The design and development of OCT devices for neurosurgery guidance and novel imaging algorithms for monitoring anti-cancer therapy have been pursued in this work. A forward-imaging needle-type OCT probe was developed which can fit into minimally invasive tools (I.D. ~ 1mm), detect the at-risk blood vessels, and identify tissue micro-landmarks. This promising guidance tool improves the safety and the accuracy of needle-based procedures, which are currently performed without imaging feedback. Despite the great imaging capability, OCT is limited by the shallow imaging depth (1-2 mm). In order to address this issue, the first MRI compatible OCT system has been developed. The multi-scale and multi-contrast MRI/OCT imaging combination significantly improves the accuracy of intra-operative MRI by two orders (from 1mm to 0.01 mm). In contrast to imaging systems, a thin (0.125 mm), low-cost (1/10 cost of OCT system) and simple fiber sensor technology called coherence gated Doppler (CGD) was developed which can be integrated with many surgical tools and aid in the avoidance of intracranial hemorrhage. Furthermore, intra-vital OCT is a powerful tool to study the mechanism of anti-cancer therapy. Photo-immunotherapy (PIT) is a low-side-effect cancer therapy based on an armed antibody conjugate that induces highly selective cancer cell necrosis after exposure to near infrared light both in vitro and in vivo. With novel algorithms that remove the bulk motion and track the vessel lumen automatically, OCT reveals dramatic hemodynamic changes during PIT and helps to elucidate the mechanisms behind the PIT treatment. The transformative guidance tools and the novel image processing algorithms pave a new avenue to better clinical outcomes and preclinical animal studies.