Optical and X-Ray Technology Synergies Enabling Diagnostic and Therapeutic Applications in Medicine

X-ray and optical technologies are the two central pillars for human imaging and therapy. The strengths of x-rays are deep tissue penetration, effective cytotoxicity, and the ability to image with robust projection and computed-tomography methods. The major limitations of x-ray use are the lack of molecular specificity and the carcinogenic risk. In comparison, optical interactions with tissue are strongly scatter dominated, leading to limited tissue penetration, making imaging and therapy largely restricted to superficial or endoscopically directed tissues. However, optical photon energies are comparable with molecular energy levels, thereby providing the strength of intrinsic molecular specificity. Additionally, optical technologies are highly advanced and diversified, being ubiquitously used throughout medicine as the single largest technology sector. Both have dominant spatial localization value, achieved with optical surface scanning or x-ray internal visualization, where one often is used with the other. Therapeutic delivery can also be enhanced by their synergy, where radio-optical and optical-radio interactions can inform about dose or amplify the clinical therapeutic value. An emerging trend is the integration of nanoparticles to serve as molecular intermediates or energy transducers for imaging and therapy, requiring careful design for the interaction either by scintillation or Cherenkov light, and the nanoscale design is impacted by the choices of optical interaction mechanism. The enhancement of optical molecular sensing or sensitization of tissue using x-rays as the energy source is an important emerging field combining x-ray tissue penetration in radiation oncology with the molecular specificity and packaging of optical probes or molecular localization. The ways in which x-rays can enable optical procedures, or optics can enable x-ray procedures, provide a range of new opportunities in both diagnostic and therapeutic medicine. Taken together, these two technologies form the basis for the vast majority of diagnostics and therapeutics in use in clinical medicine

Fast Two-Photon Excited Fluorescence Imaging for the Human Retina

In der vorliegenden Doktorarbeit wird ein neuartiges Zwei-Photonen-Mikrosokop, basierend auf einem schnellen ophthalmoskopischen Scanner, zur hochauflösenden strukturellen und funktionellen Abbildung der menschlichen Netzhaut entwickelt und aufgebaut. An menschlichen Spenderaugen werden die Autofluoreszenzeigenschaften der Netzhaut erprobt und auf ihren diagnostischen Wert hin analysiert. Die sich auf dem retinalen Pigmentepithel (RPE) mit Alter und Krankheit ansammelnde Lipofuszingranula können dabei hochauflösend mit Hilfe der Zwei-Photonen-Anregung abgebildet werden. Exzessive Lipofuszinmengen im RPE werden in der Ophthalmologie als mögliche Ursache für die Pathogenese u.a. der altersabhängigen Makulardegeneration (AMD) vermutet und dessen hochauflösende, selektive Anregung mittels Zwei-Photonen-Absorption erscheint besonders interessant. Zudem ist es erstmals möglich, die feinen Photorezeptor Zapfen und Stäbchen sowie die darüberliegenden Ganglionzellen und Nervenfaserschicht der neurosensorischen Netzhaut mittels Zwei-Photonen Autofluoreszenz darzustellen, allerdings sind dazu höhere Anregungsenergien nötig. Desweiteren wird die Abhängigkeit von kurzen Pixelverweilzeiten auf die Fluoreszenzausbeute untersucht. Am RPE Lipofuszin wird experimentell nachgewiesen, dass vermutlich aufgrund von unterdrückter Tripletzustand Ansammlung, durch schnelles Scannen eine Fluoreszenzsignalzunahme erzielt werden kann. Zuletzt wird die potenzielle Anwendung am lebenden menschlichen Auge zur diagnostischen Abbildung der RPE Zellschicht simuliert und diskutiert. Die Anwendung der Zwei-Photonen Scanning-Laser-Ophthalmoskopie erscheint unseren Berechnungen, nach neuesten Lasersicherheitsbestimmungen, zufolge als nichtinvasiv

High-Resolution Label Free Imaging of Endogenous Chromophore via Non-Linear Photoacoustic Microscopy

Molecular specific subcellular imaging of biological tissues is vital for understanding the mechanisms of various pathologies. Current technologies for subcellular absorption contrast imaging, such as fluorescence confocal microscopy, require exogenous contrast agents to gain access to relevant biomolecules. All non-fluorescing biomolecules must therefore be tagged by a fluorescent marker to be visible in fluorescence confocal images. While these markers are effective, they can change the local environments, and any exogenous contrast agent must first achieve FDA approval for wide-spread use in humans. Photoacoustic microscopy (PAM) is a hybrid imaging modality combining optical absorption imaging with ultrasonic detection capable of endogenous absorption contrast. Unfortunately, traditional photoacoustic microscopy suffers from poor axial resolution, precluding it from three-dimensional subcellular imaging. High axial resolution may be lent to PAM through the addition of a pump-probe spectroscopy technique known as transient absorption. This high resolution PAM technique, known as transient absorption ultrasonic microscopy (TAUM) enables three-dimensional subcellular imaging of endogenous biomolecules. The pump-probe spectroscopy properties inherent to TAUM provide optically resolved point spread functions, access to ground state recovery time, and access to transient absorption spectrum measurements. This manuscript describes the author’s efforts to improve the processing capabilities of both PAM and TAUM. In this manuscript various TAUM systems are designed and characterized in detail. A second generation TAUM system improves the processing speed of TAUM to enable processing in parallel with data acquisition. Following the improvements to processing, a novel optical schematic of TAUM is developed, greatly simplifying the design requirements of TAUM images. This system is validated by collecting volumetric images of erythrocytes in blood smears. This work enables any PAM system to be converted to a TAUM system through the addition of an optical modulator. The culmination of this work is a multispectral TAUM system hybridized with a confocal microscope to enable high resolution imaging with both scattering and absorption contrast of biological tissues. The capabilities of this PAM and TAUM are demonstrated by obtaining high resolution images of the endogenous chromophores: hemoglobin, melanin, and cytochrome C

Optical Studies of Oxidative Stress in Lung Tissue: Rodent Models

Objectives: There currently exists a need for reliable measurements of tissue metabolic state at cellular levels. The objective of this research was to study tools capable of evaluating cellular redox states in intact tissue. To meet this goal, three different instruments (cryoimager, fluorometer, and fluorescent microscope) were used to study the metabolism and functions of the mitochondria at different levels and regimes (cryo, ex vivo, in vivo and in vitro). Introduction: Through optical monitoring of autofluorescent mitochondrial metabolic coenzymes, as well as exogenous fluorophores, the state of mitochondria can be probed in real time in many intact organs and in vitro. Autofluorescent mitochondrial metabolic coenzymes, studied here, include NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), and the ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of the tissue. Exogenous fluorophores include but are not limited to tetramethylrhodamine (TMRM) and Mito-SOX, which are used to evaluate the mitochondrial membrane potential and level of reactive oxygen species (ROS) in the mitochondria, respectively. Methods: Different optical imaging and acquisition techniques were studied to evaluate oxidative stress in lung tissue and cells in cryogenic temperatures, in vivo, ex vivo, and in vitro. Though in essence the underlying technological and biological principles appear to be the same, imaging in each of these regimes imposed unique challenges requiring significantly different approaches to system design, data acquisition, and processing. A brief description of each technique is provided here and each is described in detail in the following chapters. The first device utilized is a cryoimager, which sequentially slices tissue and acquires fluorescence images of up to five fluorophores in cryogenic temperatures (-40oC). Rapid freezing of organs preserves the tissue\u27s metabolic state and subsequent low temperature fluorescence imaging (cryoimaging) provides high fluorescence quantum yield as compared with room temperature. Sequential slicing of the tissue provides 3D spatial distribution of NADH and FAD fluorescence intensities throughout the tissue. These studies were conducted using the cryoimager in the Biophotonics Lab on different models of lung injuries including ischemia, hyperoxia, and BPD (bronchopulmonary dysplasia). The second device is a fluorometer, which was designed and implemented in the Biophotonics Lab. It is capable of monitoring the dynamics of the metabolism of the tissue through the use of optical surface fluorescence measurements of NADH and FAD. The ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. Surface fluorescence signals from NADH and FAD were acquired in the absence (baseline) and presence of metabolic perturbers (e.g. pentachlorophenol, rotenone, or potassium cyanide), in the presence of blood, and eventually in vivo. The third instrument, a fluorescent microscope, is used to image slides and dishes containing stained cells (e.g. endothelial cells, perycites, or fibroblasts) from lungs, hearts, and retinas to study their structure and dynamics at cellular level. Images of retinas were classified as normal or injured using developed cytometry tools and morphologic parameters. For heart and lung, the dynamics of concentration of reactive oxygen species (mainly superoxide) and calcium is monitored over time in cultured live cells. Results: In the cryogenic temperatures, lung treatment with KCN (inhibitor of Complex IV), resulted in an increase in RR and sets the upper limit of the NADH signal level while injured lungs (BPD model, hyperoxia and IR) showed a more oxidized chain compared with control lungs, and as a result more oxidative stress. In ex vivo fluorometric studies, an increase in RR from chain inhibitors (including KCN and rotenone), and a decrease in the same due to an uncoupler (PCP), all from baseline was observed which was consistent with the cryoimaging results. The same experiments in isolated perfused lungs previously treated with hyperoxia showed the same direction but different levels indicating the impairment in different complexes due to hyperoxia. Segmentation algorithm developed here showed 90% accuracy comparing to manual counting, and studying the cells in retina slides confirms apoptosis and oxidative stress in retinas from injured mice. In live cells, studying the dynamics of calcium concentration in the presence of different perturbations enabled us to study the behavior of mitochondrial regulated calcium channels. Also, changes in the Mito-SOX channel gave us the dynamics of mitochondrial ROS in the presence of chain perturbers (chemicals and gas). Conclusion: Optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue due to oxidative stress

Multispectral three-dimensional optical coherence tomography

A spectral-domain OCT system operating at 1300 nm wavelength region, capable of acquiring 47,000 A-lines/s, was designed and developed. Its axial and transverse resolutions were 6 micro and 15 &micro respectively. OCT images of human skin were obtained in vivo using three OCT systems, in order to find the optimal wavelength region for dermal imaging. 800 nm OCT system provided better image contrast over other two wavelength regions. Meanwhile, 1300 nm wavelength region was needed to obtain information from deeper dermal layers. To determine the effect of melanin pigmentation on OCT, images were taken from subjects with different ethnic origins. Interestingly, melanin pigmentation was found to have little effect on penetration depth in OCT. In vitro tumour samples, comprising samples with different degrees of dysplasia, were imaged at 800 nm, 1060 nm and 1300 nm wavelength regions to find the capability of OCT to diagnose microstructural changes occurring during tumour progression. 800 nm OCT system was capable to detect the malignant changes with higher contrast than other wavelength regions. However, higher wavelength regions were required to penetrate deeper in densely scattering tumour samples at advanced stages. OCT system operating at 1060 nm was combined with a photoacoustic imaging (PAT) system to obtain complementary information from biological tissues. This multimodal OCT/PAT system demonstrated its potential to deliver microstructural information based on optical scattering and vascular information based on optical absorption in living mice and human skin. The results indicate OCT as a promising imaging modality that can have profound applications in several areas of clinical diagnostic imaging