Today label-free bioimaging has been leading to widespread and fast-growing applications, which demands for a more efficient way to keep up such momentum. To this end, the research in this thesis will study the techniques of efficiency improvement for advanced label-free bioimaging, including the time efficiency, cost efficiency and information efficiency. Optical coherence tomography (OCT) is one of the most valuable label-free bioimaging modalities to provide noninvasive cross-sectional assessment of biological tissue. In many occasions, these applications demand for three dimensional (3D) imaging at video-rate in order to perform real-time diagnoses, which can be overcome by MHz-OCT. Here we demonstrate inertia-free all-optical ultrahigh-speed swept-source optical coherence tomography (OCT) based on amplified optical time-stretch (AOT). More importantly, the key significance of AOT-OCT is its broadband amplification stage, which greatly enhances the detection sensitivity compared with the prior attempts to employ optical time-stretch to OCT. We report an AOT-OCT system which is operated at an A-scan rate of multi-megahertz with high sensitivity (>80 dB) and perform time-stretch-based OCT of biological tissue in vivo. Moreover, using a more stable and coherent mode-locked fiber laser, we can achieve better performance without the compromise of averaging for supercontinuum-generation-based AOT-OCT system. It represents a major step forward in utilizing AOT as an alternative for achieving practical time-efficient OCT imaging at multi-MHz speed. For the further development of this ultrahigh-speed OCT, we present a theoretical analysis of the AOT-OCT system. The spectral resolution, coherence length and sensitivity of AOT-OCT system have been discussed in detail. By theoretical model of the noise sources based on Raman amplifier, we also quantify how the input signal, amplifier gain, A-scan rate affect the sensitivity of AOT-OCT imaging. These simulation results are expected to be valuable for optimizing the design of AOT-OCT. We also investigate in cost-effective implementation to realize efficient optical time-stretch process based on dispersive fiber. We explore and demonstrate the feasibility of using the standard telecommunication single-mode fibers as few-mode fibers (FMFs) for optical time-stretch confocal microscopy in the 1m range. It can provide sufficiently high dispersion-to-loss ratios for practical time-stretch imaging at 1 m, without the needs for high-cost specialty 1 m single mode fiber. In addition, Coherent anti-Stokes Raman scattering (CARS) microscopy is another attractive efficient tool for label-free biochemical-specific imaging, which can bypass laborious steps of preparing and staining in routine standard histopathology. Here we further explore ultrabroadband hyperspectral multiplex (HM-CARS) to perform chemoselective histological imaging with efficient information in fingerprint region. In order to unravel the congested CARS spectra, we employ phase-retrieval algorithm based on Kramers–Kronig (KK) transform and principal component analysis (PCA) to display the key cellular structures with components distribution. All these research efforts are aiming at improving the efficiency, from theory to implementation, for label-free bioimaging technology such as OCT and CARS. These schemes demonstrate great potential to realize powerful label-free bioimaging with high efficiency, including ultrafast 3D OCT imaging at video-rate, cost-effective optical time-stretch imaging and HM-CARS imaging with richness of biological fingerprint information. / published_or_final_version / Electrical and Electronic Engineering / Doctoral / Doctor of Philosophy

Thesis (Ph. D.) -- California Institute of Technology, 2010. / Title from home page (viewed 05/13/10). Advisor and committee chair names found in the thesis' metadata record in the digital repository. Includes bibliographical references.

With the wide-spread adoption of exogenous fluorescent indicators – and more recently genetically encoded fluorescent proteins – over the past two decades, there exists a diverse chemical toolkit with which to probe biological systems. Individual cell types and sub-cellular compartments can be targeted in an increasingly wide range of model organisms. However, imaging these samples is often an exercise in balancing the needs of any given experiment against the constraints of the chosen imaging technology. For example, a volume of brain tissue is host to neurons, glia, vascular compartments and red blood cells that all occupy discrete locations in 3D space, but must work together to support healthy organ function. Single-cell activity on the order of milliseconds can trigger downstream processes that unfold over the course of multiple seconds or even minutes. The development of a technique capable of providing depth-resolved, volumetric imaging with scalable spatiotemporal resolution is crucial to developing a proper understanding of such biological systems. Bottlenecks in the throughput of existing technologies stem from a combination of inefficient illumination and volume acquisition strategies, and insufficient sensor read-out speeds. Light sheet microscopy is a promising solution, but individual designs tend to be highly specialized to specific types of samples and do not easily adapt to a wide range of experimental settings. In this thesis, I detail my work in developing swept, confocally-aligned planar excitation (SCAPE) microscopy from a first-generation prototype into a versatile, easy-to-reproduce, easy-to-use system for high-speed, 3D imaging. The first chapter introduces the challenges of designing optical systems capable of high-speed, volumetric imaging. An introduction to design choices faced in the construction of fluorescence microscopes, and current approaches to 3D imaging are discussed. The second chapter describes the progression from the 1st to 2nd generation SCAPE system. Improvements made through ray-tracing models and an enhanced optomechanical design are described, and results from this system in a number of model organisms are presented. The third chapter presents results from a range of biological applications to which SCAPE microscopy has been applied. Work in imaging the zebrafish heart to demonstrate the system’s improved imaging speed, the C. elegans to show the system’s resolution, and finally a number of examples of large field-of-view and high-resolution structural imaging are all described. Finally, the fourth chapter concludes with an overview of the work that lies ahead to both further develop of SCAPE microscopy, as well as to bring the existing system’s strengths to bear in a wider range of environments.

Existing optic fibre-bundle based imaging probes have been successfully used to image biological signals from tissue in direct contact with the probe tip (Hirano et al. 1996). These fibre-bundle probe systems employed conventional fluorescence microscopy and thus lacked spatial filtering or a scanned light source, two features used by laser scanning confocal microscopes (LSCMs) to improve signal quality. Improving the methods of imaging tissue in its natural state, deep in-vivo and at cellular resolution is an ever-present goal in biological research. Within this study, a novel (580 μm diameter) optic fibre-bundle direct-contact imaging probe, employing a LSCM, was developed to allow for improved imaging of deep biological tissue in-vivo. The new LSCM/probe system possessed a spatial resolution of 10 μm, and a temporal resolution of 1 msec. The LSCM/probe system was compared to a previously used direct-contact probe system that employed a conventional fluorescence microscope. Quantitative and qualitative data indicated that the LSCM/probe system possessed superior image contrast and quality. Furthermore, the LSCM/probe system was approximately 16 times more effective at filtering unwanted contaminating light from regions below the imaging plane (z-axis). The unique LSCM/probe system was applied to an exploratory investigation of calcium activity of both glial and neuronal cells within the whisker portion of the rat primary somatosensory cortex in-vivo. Fluorescence signals of 106 cells were recorded from 12 female Sprague Dawley rats aged between 7-8 weeks. Fluo-3(AM) fluorophore based calcium fluctuations that coincided with 10 - 14 Hz sinusoidal stimulation of rat whiskers for 0.5-1 second were observed in 8.5% of cells (9 of 106). Both increases and decreases in calcium levels that coincided with whisker stimulation were observed. Of the 8.5 % of cells, 2.8% (3 cells) were categorized as glial and 5.7% (6 cells) as neuronal, based on temporal characteristics of the observed activity. The remaining cells (97 of 106) displayed sufficient calcium-based intensity but no fluctuations that coincided with an applied stimulus. This was partially attributed to electronic noise inherent in the prototype system obscuring potential very weak cell signals. The results indicate that the novel LSCM/probe system is an advancement over previously used systems that employed direct-contact imaging probes. The miniature nature of the probe allows for insertion into soft tissue, like a hypodermic needle, and provides access to a range of depths with minimal invasiveness. Furthermore, when combined with selected dyes, the system allows for imaging of numerous forms of activity at cellular resolution.

Wang, Jianpeng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Understanding the relationship between neural activity and cortical hemodynamics, or neurovascular coupling is the foundation to interpret neuroimaging signals such as functional magnetic resonance imaging (fMRI) which measure local changes in hemodynamics as a proxy for underlying neural activity. Even though the stereotypical stimulus-evoked hemodynamic response pattern with increased concentration of oxy- and total-hemoglobin and decrease in concentration of deoxy-hemoglobin has been well-recognized, the linearity of neurovascular coupling and its variances depending on brain state and tasks haven’t been thoroughly evaluated. To directly assess the cortical neurovascular coupling, simultaneous recordings of neural and hemodynamic activity were imaged by wide-field optical mapping (WFOM) over the bilateral dorsal surface of the mouse brain through a bilateral thinned-skull cranial window. Neural imaging is achieved through wide-field fluorescence imaging in animals expressing genetically encoded calcium sensor (Thy1-GCaMP). Hemodynamics are recorded via simultaneous imaging of multi-spectral reflectance. Significant hemodynamic crosstalk was found in the detected fluorescence signal and the physical model of the contamination, methods of correction as well as electrophysiological verification are presented. A linear model between neural and hemodynamic signals was used to fit spatiotemporal hemodynamics can be predicted by convolving local fluorescence changes with hemodynamic response functions derived through both deconvolution and gamma-variate fitting. Beyond confirming that the resting-state hemodynamics in the awake and anesthetized brain are coupled to underlying neural activity, the patterns of bilaterally symmetric spontaneous neural activity observed by WFOM emulate the functionally connected networks detected by fMRI. This result provides reassurance that resting-state functional connectivity has neural origins. With the access to cortical neural activity at mesoscopic level, we further explore the cortical neural representations preceding and during spontaneous locomotion.

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain.

In this work, a series of DTPA based Ln complexes containing one or two chromophores with different degrees of conjugation were synthesized. The proton relaxivities of Gd(III) analogues were investigated as potential MRI contrast agents while the photoluminescence of Eu(III) and Tb(III) analogues were studied for their applications in optical probes for cellular imaging. Later investigation indicates that only emissions from the chromophores could be measured upon long wavelength photon excitations in the microscope. With suitable ligand design, novel dual functional imaging probes were finally synthesized and these showed good luminescence intensity and image contrast in both in-vivo and in-vitro studies. Eight DTPA based Ln (III) complexes LnL1-L8 containing one or two chromophores which include benzene, 2-aminopyridine, 3-amino-pyridine and 4-aminopyridinewere synthesized. The syntheses, relaxometric properties, hydration numbers, quantum yields, sensitization efficiencies, brightnesses, cytotoxicities and cellular uptake properties were discussed. Those mono-substituted complexes show higher relaxivity, while the di-substituted complexes show lower relaxivity than Gd-DTPA (4.17 mM-1 s-1),a clinically used MRI contrast agent(CA).The di-substituted Tb(III)/Eu(III) analogues show lower sensitization efficiency than the mono-substituted ones in the energy transfer process. Therefore, the experimental results clearly illustrate that the complex with one chromophorein the DTPA system is a better option for being used as a MRI contrast agent and an optical probe. Another eight new mono-substituted DTPA based Ln(III) complexes LnL9-L16 containing extended conjugated chromophores were synthesized and investigated. The phenyl derivatives and naphthyl derivatives were added onto the para-position of 2-aminopyridine that was employed as the chromophore. All GdL9-L16possess one bound water molecule and show higher relaxivity than Gd-DTPA. The relaxivities at 300 MHz at 25oC are in the descending order of GdL15(5.37 mM-1s-1) > GdL16(5.23 mM-1s-1) > GdL13(5.12 mM-1s-1) > GdL14(5.06 mM-1s-1) > GdL11(4.96 mM-1s-1) > GdL12(4.83 mM-1s-1) > GdL10(4.80 mM-1s-1) > GdL9(4.50 mM-1s-1). Their quantum yields, sensitization efficiencies and brightnesses are greatly improved because of the highly conjugated chromophores. Moreover, they all showed low cytotoxicity to cells in a MTT assay and a high accumulation in cells in cellular uptake studies. However, no emission from the Eu(III) ion was detected from the Eu(III) analogues upon long wavelength photon excitation in the cell imaging studies, only the emissions from the chromophores were observed. Two mono-substituted DTPA based Ln(III) complexes containing anthracenyl derivatives as the chromophore LnL17-L18 and two DTPA-based binuclear Ln(III) complexes LnL19-L20were synthesized and investigated. Among the four complexes, GdL18 shows the highest relaxivity (4.65 mM-1s-1) and the highest fluorescent quantum yield (2.45%).It also has low cytotoxicity to cells in MTT assay and high accumulation in cells in cellular uptake study. In addition, GdL18shows very strong binding interaction towards serum albumin, i.e. 318,400mol-1dm3for HSA and 90,200 mol-1dm3for BSA. In preliminary studies, GdL18can both give good luminescence intensity and image contrast in both in vitro cell imaging and in vivo MRI studies. Therefore, GdL18 is considered as a potential candidate for use as a dual functional MRI/optical imaging probe. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010. / Committee Chair: Dickson, Robert; Committee Member: Fahrni, Christoph; Committee Member: Payne, Christine; Committee Member: Petty, Jeff; Committee Member: Srinivasarao, Mohan. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI Company.