Technological advances in image acquisition speeds and new contrast agents, in both clinical and basic research settings, have enabled entirely new approaches to functional imaging in living systems. Analysis of dynamic and multidimensional data requires very different approaches to the classical segmentation and visualization tools developed for purely structural or anatomical imaging. This thesis details the development of two different spatiotemporal analysis approaches for high-speed in-vivo dynamic optical imaging. Optical imaging is a diverse, versatile, and generally inexpensive modality that can take advantage of a wide range of endogenous and exogenous sources of optical contrast within living tissue. While light scattering can limit resolution and sensitivity of imaging in deeper tissues, optical imaging is well suited for small animal studies where it can be used for studies of physiology and disease processes, for pharmaceutical development and as a test-bed for translation to clinical applications. In the first part of this work, we present and apply spatiotemporal analysis techniques which we define as `dynamic contrast enhancement' methods. We apply these methods to in-vivo whole body small animal molecular optical imaging to demonstrate that dynamic analysis can be used for longitudinal assessment of organ function. We then demonstrate the equivalence of our approach to dynamic contrast enhanced magnetic resonance imaging. This optical technique could allow for better informed drug development and longitudinal toxicity evaluation. This technique could also serve as a platform for the development of functional imaging methods using dynamic MRI. We then apply spatiotemporal analysis techniques to high speed optical hemodynamic imaging data acquired on the exposed rodent cortex. The purpose of this work is to develop a mechanistically-based spatiotemporal model of neurovascular coupling, in order to better understand the basis of functional magnetic resonance imaging data in the human brain. Our results also provide new insights into potential links between neurovascular disruption and disease pathophysiology in the brain.

No description available.

Not available / text

Thesis (M.S.)--University of Texas at Dallas, 2008. / Includes vita. Includes bibliographical references (leaves 63-65)

This thesis is dedicated to developing novel photofunctional molecular materials for the applications in chemical sensing, bioimaging and electrochromic. To begin with, a brief introduction of photofunctional molecular materials and an overview of their applications in chemical sensing, bioimaging and electrochromic were presented in Chapter 1. In chapter 2, we have synthesized a series of water-soluble phosphorescent cationic iridium(III) solvato complexes (1-7) as multicolor cellular probes for imaging in living cells. All of these complexes can be dissolved in PBS. The emission of complexes can be tuned from green to red by changing the chemical structure of cyclomedtalating ligands. All complexes exhibit low cytotoxicity to living cells and exhibit cell membrane permeability and specific staining of cytoplasm. They enter the cells by the mechanism of energy-independent passive diffusion mechanisms. More importantly, complex 7 can act as a two-photon phosphorescent cellular probe, and fluorescence lifetime imaging microscopy is successfully applied for bioimaging in the presence of short-lived background fluorescence. We developed two excellent optical probes for CO2 detection in Chapter 3. The first one for the CO2 detection is a phosphorescent probe based on an iridium(III) complex with 2-phenylimidazo-[4,5-f][1,10]phenanthroline. After bubbling CO2 into the detection solution, the quenched phosphorescence by the addition of CH3COO can be recovered. Photobleaching experiment demonstrates that this phosphorescent CO2 probe shows higher photostability than some of the reported organic probes. More importantly, the time-resolved PL experiment demonstrates that this probe can be used to detect CO2 in the presence of strong background fluorescence, which improves the sensitivity and signal-to-noise ratio of the sensor in complicated media. The second one is a water-soluble fluorescent probe based on tetraphenylethene derivative. After bubbling CO2 into the detection solution, remarkable color change and fluorescence enhancement could be observed. The response of this probe to CO2 in aqueous solution is fast and the detection limit is about 2.4 × 106 M. To emphasize the practical application of this probe, a porous film was successfully fabricated by mixing the dye with sodium carboxymethyl cellulose in water, which can serve as an efficient CO2 gas sensor. More importantly, this probe exhibits low cytotoxicity towards live cells and has the ability to monitor the external CO2 concentration changes of living cells. Chapter 4 focused on the development of novel soft salt based phosphorescent probe. This type of probe consists of two oppositely charged ionic complexes with two distinguishable emission colors, which makes it a perfect candidate as a ratiometric probe. The emission color of 10 changes from blue to red with increasing pH value. 10 is cell-permeable and exhibits low cytotoxicity, and it has been successfully applied for ratiometric pH imaging with the use of confocal microscopy, demonstrating its great potential for intracellular environment monitoring. Furthermore, phosphorescence lifetime imaging experiments can detect intracellular pH variations by photoluminescence lifetime measurements, which allowed for eliminating background fluorescence and selecting long-lived phosphorescence images. Quantitative measurement of intracellular pH fluctuations caused by oxidative stress has been successfully carried out for 10 based on the pH-dependent calibration curve. A series of cationic Zn(II) complexes has been designed and synthesized in chapter 5. The photophysical properties of these Zn(II) complexes are affected by the counterions. By altering the counterions, the emission peak can be changed from 549 nm to 622 nm. Interestingly, the CIE coordinate and the emission colors can be simply tuned by adjusting the concentration of 11d in the polyether. Under an electric field of about 15 V applied onto the electrodes, the emission color of the solution of 11b-11d near the cathode changed its original emission color to sky blue. Based on this interesting electrochromic fluorescence of 11d, a quasi-solid information recording device has been successfully designed. Furthermore, data encryption has been realized by combining 1d with BODIPY, and information decoding processed has been accomplished, for the first time, by employing TPA excitation techniques, in which the large TPA cross section of 11d is differentiated from small TPA cross section of common organic dyes. Finally, Chapters 6 and 7 present the concluding remarksand the experimental details of the work described in Chapters 25

Functional optical imaging encompasses the use of optical imaging techniques to study living biological systems in their native environments. Optical imaging techniques are well-suited for functional imaging because they are minimally-invasive, use non ionizing radiation, and derive contrast from a wide range of biological molecules. Modern transgenic labeling techniques, active and inactive exogenous agents, and intrinsic sources of contrast provide specific and dynamic markers of in-vivo processes at subcellular resolution. A central challenge in building functional optical imaging systems is to acquire data at high enough spatial and temporal resolutions to be able to resolve the in-vivo process(es) under study. This challenge is particularly highlighted within neuroscience where considerable effort in the field has focused on studying the structural and functional relationships within complete neurovascular units in the living brain. Many existing functional optical techniques are limited in meeting this challenge by their imaging geometries, light source(s), and/or hardware implementations. In this thesis we describe the design, construction, and application of novel 2D and 3D optical imaging systems to address this central challenge with a specific focus on functional neuroimaging applications. The 2D system is an ultra-fast, multispectral, wide-field imaging system capable of imaging 7.5 times faster than existing technologies. Its camera-first design allows for the fastest possible image acquisition rates because it is not limited by synchronization challenges that have hindered previous multispectral systems. We present the development of this system from a bench top instrument to a portable, low-cost, modular, open source, laptop based instrument. The constructed systems can acquire multispectral images at >75 frames per second with image resolutions up to 512 x 512 pixels. This increased speed means that spectral analysis more accurately reflects the instantaneous state of tissues and allows for significantly improved tracking of moving objects. We describe 3 quantitative applications of these systems to in-vivo research and clinical studies of cortical imaging and calcium signaling in stem cells. The design and source code of the portable system was released to the greater scientific community to help make high-speed, multispectral imaging more accessible to a larger number of dynamic imaging applications, and to foster further development of the software package. The second system we developed is an entirely new, high-speed, 3D fluorescence microscopy platform called Laser-Scanning Intersecting Plane Tomography (L-SIPT). L-SIPT uses a novel combination of light-sheet illumination and off-axis detection to provide en-face 3D imaging of samples. L-SIPT allows samples to move freely in their native environments, enabling a range of experiments not possible with previous 3D optical imaging techniques. The constructed system is capable of acquiring 3D images at rates >20 volumes per second (VPS) with volume resolutions of 1400 x 50 x 150 pixels, over a 200 fold increase over conventional laser scanning microscopes. Spatial resolution is set by choice of telescope design. We developed custom opto-mechanical components, computer raytracing models to guide system design and to characterize the technique's fundamental resolution limits, and phantoms and biological samples to refine the system's performance capabilities. We describe initial applications development of the system to image freely moving, transgenic Drosophila Melanogaster larvae, 3D calcium signaling and hemodynamics in transgenic and exogenously labeled rodent cortex in-vivo, and 3D calcium signaling in acute transgenic rodent cortical brain slices in-vitro.

In recent years, multi-resolution techniques have become increasingly popular in the image processing community. New techniques have been developed with applications ranging from edge detection, texture recognition, image registration, multi-resolution features for image classification and more. The central focus of this two-part thesis is the multi-resolution analysis of images. In the first part, we used multi-resolution approaches to help with the classification of a set of protein crystal images. In the second, similar approaches were used to help register a set of 3D image volumes that would otherwise be computationally prohibitive without leveraging multi-resolution techniques. Specifically, the first part of this work proposes a classification framework that is being developed in collaboration with NorthEast Structural Genomics Consoritum (NESG) to assist in the automated screening of protein crystal images. Several groups have previously proposed automated algorithms to expedite such analysis. However, none of the classifiers described in the literature are sufficiently accurate or fast enough to be practical in a structural genomics production pipeline. The second part of this work proposes a 3D image registration algorithm to register regions of emphysema as quantified by densitometry on lung CT with MR lung volumes. The ability to register quantitatively-determined regions of emphysema with perfusion MRI will allow for further exploration of the pathophysiology of Chronic Obstructive Pulmonary Disorder (COPD). The registration method involves the registration of CT volumes at different levels of inspiration (total lung capacity to functional residual capacity [FRC]) followed by another registration between FRC-CT and FRC-MR volume pairs.

Photophysical characterization of several species of fluorescent silver nanoclusters, encapsulated in oligonucleotide scaffolds, was achieved at the bulk and single molecule level. These studies reveal the presence of a short-lived microsecond blinking component which leads to higher emission rates than exhibited by common organic dyes. This dark state was found to be photo-accessible with a very efficient depopulation transition leading to repopulation of the emissive state. Secondary excitation on resonance with this transition significantly shortens the residence time in the dark state giving rise to as much as 5-fold fluorescence enhancement. Manipulation of the secondary laser can be used to impose a regularly modulated waveform onto the fluorescent signal. Signal processing techniques can be employed to extract the modulated signal from large backgrounds, leading to drastically improved sensitivity. This new imaging concept can be extended, beyond Ag nanoclusters, to common organic fluorophores that demonstrate large dark state quantum yields. These fluorophores simultaneously illustrate the utility of this technique and help to define a general set of parameters for engineering ideal dyes for modulated signal extraction. Ideally suited for fluorescence enhancement, FRET pairs can be used to engineer a wide range of modulatable systems, based on detecting donor emission in the presence of a laser directly exciting the acceptor. The utility of Ag nanoclusters, organic dyes, and FRET systems for improved sensitivity are investigated in this work.

Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio. Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence. The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio. By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 26, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Ping Yu. Vita. Includes bibliographical references.