Vulnerable plaque is a clinically silent condition of atherosclerotic plaque that leaves a large number of patients at risk of a coronary event. A method to detect vulnerable atherosclerotic plaque would greatly enhance the ability of clinicians to diagnose and treat patients at risk. Fluorescence lifetime imaging microscopy (FLIM) offers a way to extract both spatial and biochemical information from plaque by taking several wide-field images over time. The goal of this study was to determine the potential of a FLIM angioscopy system to detect and differentiate coronary atherosclerotic plaques ex-vivo into several groups including thin, fibrotic, lipid-laden, thick-cap fibroatheroma (FA), and fibrocalcified. Samples were extracted post-mortem weekly and sliced open to have their lumens imaged. For each sample, 51 time resolved wide-field images were taken over 10 nanoseconds at 390 (±40) nm, 450 (±40) nm, and 550 (±88) nm wavelengths. To analyze the samples, the intensity map and lifetime map were created at each wavelength. The intensity map was simply the wide-field images summed in time and normalized. In order to calculate lifetime at each point, a fast, model-free Laguerre deconvolution algorithm was recently developed for FLIM data analysis and was used. This allowed for fast, efficient estimations of the fluorescence decay curves at each pixel of the FLIM images and facilitated the computation of quantitative parameters describing the fluorescence emission of the tissue, specifically, the relative fluorescence intensity and lifetime at defined emission bands. Statistical analysis on these FLIM derived parameters indicated that the autofluorescence emission of the plaques allows for distinguishing relative plaque thickness: thin plaque, whose signal is dominated by elastin fluorophores, shows a marked difference between thicker plaques, such as fibrotic, fibrocalcified and thick-cap FA (who are dominated primarily by collagen). However, the ability of the current FLIM system to differentiate vulnerable plaque remains in question due to the absence of thin-cap FA samples. Further work has also been proposed; of primary concern is gathering thin-cap FA plaque samples needed to validate the system’s ability to differentiate vulnerable plaques from other common groupings.

Single molecule studies on CdSe quantum dots functionalized with oligo-phenylene vinylene ligands (CdSe-OPV) provide evidence of strong electronic communication that facilitate charge and energy transport between the OPV ligands and the CdSe quantum dot core. This electronic interaction greatly modify, the photoluminescence properties of both bulk and single CdSe-OPV nanostructure thin film samples. Size-correlated wide-field fluorescence imaging show that blinking suppression in single CdSe-OPV is linked to the degree of OPV coverage (inferred from AFM height scans) on the quantum dot surface. The effect of the complex electronic environment presented by photoexcited OPV ligands on the excited state property of CdSe-OPV is measured with single photon counting and photon-pair correlation spectroscopy techniques. Time-tagged-time-resolved (TTTR) single photon counting measurements from individual CdSe-OPV nanostructures, show excited state lifetimes an order of magnitude shorter relative to conventional ZnS/CdSe quantum dots. Second-order intensity correlation measurements g(2)(τ) from individual CdSe-OPV nanostructures point to a weak multi-excitonic character with a strong wavelength dependent modulation depth. By tuning in and out of the absorption of the OPV ligands we observe changes in modulation depth from g(2)(0) ≈ 0.2 to 0.05 under 405 and 514 nm excitation respectively. Defocused images and polarization anisotropy measurements also reveal a well-defined linear dipole emission pattern in single CdSe-OPV nanostructures. These results provide new insights into to the mechanism behind the electronic interactions in composite quantum dot/conjugated organic composite systems at the single molecule level. The observed intensity flickering, blinking suppression and associated lifetime/count rate and antibunching behaviour is well explained by a Stark interaction model. Charge transfer from photo-excitation of the OPV ligands to the surface of the CdSe quantum dot core, mixes electron/holes states and lifts the degeneracy in the band edge bright exciton state, which induces a well define linear dipole behaviour in single CdSe-OPV nanostructures. The shift in the electron energies also affects Auger assisted hole trapping rates, suppress access to dark states and reduce the excited state lifetime.

Calcium (Ca2+), a crucial effector for many biological systems, has been associated with diseases such as cardiovascular disease, Alzheimer’s, Parkinson’s, cancer, and osteoporosis. It is important to develop calcium sensors to measure intracellular Ca2+ dynamics at various biological and pathological states. Our lab has engineered such probes by designing a Ca2+ binding site into fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) and mCherry. In this thesis, we aim to improve optical properties and metal binding properties of green EGFP-based sensor CatchER and mCherry based red sensors by site-directed mutagenesis and protein engineering, various spectroscopic methods and cell imaging. The green EGFP-based sensor CatchER, with a Ca2+ binding pocket charge of -5, displays the greatest affinity for Ca2+ and has the greatest fluorescence intensity change with Ca2+ when compared to its variants with a less negative binding pocket charge. In addition, we have also designed several SR/ER targeting CatchER variants using Ryanodine receptor and Calnexin transmembrane domains. These constructs were shown to display a strong presence in the SR/ER lumen and further designed for a new luminal orientation. Further, we have shown that the optical properties of two red calcium sensors can be significantly improved by modifying the local environment of the chromophore.

The technique of Fluorescence Lifetime Imaging Microscopy (FLIM) has been employed to quantitatively and spatially map the fluid composition and temperature within microfluidic systems. A molecular probe with a solvent-sensitive fluorescence lifetime has been exploited to investigate and map the diffusional mixing of fluid streams under laminar flow conditions within a microfluidic device. Using FLIM, the fluid composition is mapped with high quantification and spatial resolution to assess the extent of mixing. This technique was extended to quantitatively evaluate the mixing efficiency of a range of commercial microfluidic mixers employing various mixing strategies, including the use of obstacles fabricated within the channels. A fluorescently labelled polymer has been investigated as a new probe for mapping temperature within microfluidic devices using FLIM. Time Correlated Single Photon Counting (TCSPC) measurements showed that the average fluorescence lifetime displayed by an aqueous solution of the polymer depended strongly on temperature, increasing from 3 ns to 13.5 ns between 23 and 38 oC. This effect was exploited using FLIM to provide high spatial resolution temperature mapping with sub-degree temperature resolution within microfluidic devices. A temperature-sensitive, water-soluble derivative of the rhodamine B fluorophore, effective over a wide dynamic temperature range (25 to 91 oC) has been used to map the temperature distribution during the mixing of fluid streams of different temperatures within a microchannel. In addition, this probe was employed to quantify the fluid temperature in a prototype microfluidic system for DNA amplification. FLIM has been demonstrated to provide a superior approach to the imaging within microfluidic systems over other commonly used techniques, such as fluorescence intensity and colourimetric imaging.

Fluorescent nano-diamond(FND) is an unique fluorescence bio-labeling materials, which exhibit good fluorescence yield, excellent photostability, and non-toxicity. The emission color of FND is determined by the defect centers in the diamond crystal. When the defect center composed of one vacancy and two nearest-neighborhood nitrogen substitutes, it forms a H3 center. H3 center has a zero-phonon line at 496nm , and a broadband green emission around 530 nm,. When the FND contains lots of H3 centers, the emission color is green, hence it¡¦s called green FND(gFND). Since H3 centers composed of two nitrogen substitutes, it is naturally to fabricate the gFNDs by diamonds with high nitrogen substitutes. However, H3 center is not the only products when the diamond contains many nitrogen substitutes, and high density of vacancies. Other type of defect centers (NV-, NV0, ¡K) exhibit lower energy gap, and quench the emission of H3 centers. In this thesis, it aims to study the spectroscopic homogeneity of the gFNDs. Comparing the intensity of the scattering images and the corresponding fluorescence images, it provides the information of the relation between particle size and the density of color centers. Furthermore, images with different color filters are compared to provide the information of the composition of defect structures. Fluorescence lifetime image is performed for the emission dynamics of the nano-particle. The results indicate that the decay lifetime has an relation to the emission intensity. When the nano-particle contains more color centers, it quenches the emission from H3 centers more.

In this dissertation, we construct a temperature controlled sample stage that is compatible with high numerical aperture objective optical microscope, and perform single molecule experiments under the system. Mixing dilute fluorophore (CdSe/ZnS quantum dot, DiI, Rhodamine B) into the liquid crystal matrix (5CB), we monitor the fluorescence dynamics of the individual fluorophore at various temperature. Different from the thermodynamic states of conventional materials, those specific class of materials which we called ¡§liquid crystals¡¨ are attracted for their existence of unique liquid crystal phase, which exhibits a solid-state like higher orientation ordering, and a liquid-state like liquidity. Probe individual fluorophore allows us to monitor the nanometer length scale local structural and dynamic heterogeneity in the solid, liquid crystal and liquid phases. The operating temperature of the platform covers more than 20 oC to 40 oC range with stability much better than 0.1 oC. Quantum dot in PMMA exhibits a clear on-off blinking behavior, and the single exponential fluorescence lifetime relaxation. While in the solid phase of the liquid crystal matrix, quantum dot exhibits similar behavior, which indicates the quantum dot is confined in the matrix. However, there exists slightly difference in decay lifetime. On the contrary, in the liquid crystalline phase as well as the liquid phase, quantum dot exhibits bi-exponential relaxation behavior. Besides a similar time scale relaxation dynamics, there exists additional fast decay behavior, which is from the feasible rotational rotation in the non-rigid matrix. In particular, the anisotropic decay dynamics in the liquid crystalline phase indicates the orientation preference of the liquid crystal molecules. Fluorescence Correlation Spectroscopy (FCS) provides the information of local dynamics of various time scales. FCS results exhibit an unclear transition that crossovers several decades in time scale, which indicates the highly heterogeneity of the liquid crystal. The results of DiI exhibits different rising time in the fluorescence lifetime measurement, which implies the forming of aggregation due to the limited solubility of the DiI molecules in the liquid crystal matrix. Results of Rhodamine B exhibit a clear rotational diffusion dynamics at ~ microsecond scale and the corresponding translational diffusion dynamics at ~ mini-second scale. Moreover, the transition time scale of translational diffusion exhibits a temperature dependence. At higher temperature, it shifts to a shorter time scale.

We have constructed a single molecule detection system with the capability to simultaneously measure many parameters, including transient fluorescence intensity, fluorescence lifetime, and photon anti-bunching behavior via the Hanbury Brown-Twiss photon-correlation technique. In addition, we apply the system to study the single DiI (1, 1 '- dioctadecyl- 3, 3 , 3 ', 3 ' - tetramethylindocarbocyanine perchlorate) molecule, to characterize the photo-physical behaviors. Cyanine dyes are the molecules that constitute of two nitrogen centers, one of which is positive charged, and is linked by a conjugated chain with odd number of carbon atoms to the other nitrogen center. Cyanine dyes are interested in the photo sensitization, optical recording media, nonlinear optics, laser dyes, and many interesting photophysical and photochemical behaviors. Among them, DiI plays an important role in single molecule fluorescence investigations. The high photo-stability, good QE, and low inter-system crossing rates, make it a pioneer for the widely investigations in single molecule studies. Our experimental goal is to understand the characteristic of the monitored single molecule by the measuring photo-physical parameters. Our results include the typical behaviors in DiI molecules: clear on-off blinking, fluorescence anti-bunching, one-step photo-bleaching, and consistent fluorescence polarization orientation. In addition, we also observed some change during measurement, which indicates the corresponding change of structure. Few molecules also exhibit non-zero probability around the zero delay time, which indicates the simultaneous existence of more than one quantum emitters in the detected region. These results demonstrate that the parameters are essential for understanding and characterizing the observed molecules in single molecule level.

Out of the growing body of evidence demonstrating the role of higher-order chromatin organization within the nucleus in regulating the functions of the linear sequence of DNA emerges the genome as a physical entity. DNA packs into hierarchical levels of chromatin condensation, which then tailor accessibility to the linear sequence for nuclear processes while also serving as a central feature of nuclear organization. Further, varying condensation state alters the physical properties of the chromatin fiber. These may then exert or facilitate forces aiding in the spatial organization within the nucleus. Yet, this complex concept of nuclear structure even neglects the dynamic aspects of the genome continuously fluctuating and undergoing structural remodeling within the nucleus. Thus, while chromatin position within the nucleus is critical for biological functions including transcription, we must reconcile a particular position of a gene locus with the dynamic and physical nature of chromatin. Here we characterize the physical aspects of the genome associated with its dynamic properties that aid in regulation. We focus on developing techniques that measure the evolution of physical properties associated with nuclear processes. We leverage these techniques, capable of quantifying and spatially resolving its structural state within the nucleus and elucidating the underlying physics of its dynamics, to illuminate physical features associated with cellular processes. Specifically, we investigate the nuclear structural changes associated with growth factor stimulation on primary human cells known to impact large scale gene expression pathways. We also demonstrate dysfunction associated with these physical mechanisms accompany disease pathologies. Thus, we unify the biological understanding of cellular processes within the context of physical features of genome structure, organization and dynamics that are critical to human health and disease.

No description available.

Fluorescence lifetime imaging (FLIM) has the potential to provide rapid screening and detection of diseases. However, time-resolved fluorescence measurements require high-performance detectors with single-photon sensitivity and sub-nanosecond time resolution. These systems should also be compact, reliable, inexpensive, and easily deployable for laboratory and clinical applications. It is with these applications in mind that the development of single photon avalanche diodes (SPAD) and time-to-digital converter (TDC) prototype integrated circuits (IC) in standard digital CMOS have been pursued in this thesis. SPAD and TDC ICs were designed and fabricated in 130 nm IBM CMOS technology and then intensively studied. Several different SPAD pixels were modeled and designed, and the electro-optical performance was characterized and comparatively studied. By repurposing existing design layers of a standard CMOS process, the fabricated SPAD pixel test structures achieved up to 20× improvement of dark count rate (DCR) compared to previous designs. Optical measurements also showed up to 10× improvement in the detection limits for low-level light. Detailed dark noise characterization was performed at various temperatures using free-running and time-gated modes of operation. Optimal operating conditions were found for minimal afterpulsing effects. The SPAD’s capability to accurately measure fast fluorescence decays was also demonstrated in a practical setting with the lifetime measurements of two fluorophores, Rhodamine 6G and Ruby crystal, which have fluorescence lifetimes of approximately 4 ns and 3 ms, respectively. A fast and accurate TDC prototype circuit for time-correlated single-photon counting (TCSPC) applications was designed, fabricated and characterized. With a coarse-fine delay line architecture, the TDC size was reduced without compromising its linearity and jitter performance. Extensive characterization of the fabricated SPAD and TDC ICs shows that the measured performance met the stated design goals. / Thesis / Doctor of Philosophy (PhD)