Lab-on-chip systems have been developed for various applications like point of care diagnostics and compact imaging systems. Compact, on-chip imaging systems face a challenge in the integration of multicolor light sources on-chip. This is because of the unavailability of compact, individually addressable, multicolor light sources on a single planar substrate. Colloidal Quantum Dot based Light Emitting Diodes (QDLEDs), which have found wide appeal, due to their unique properties like their tunable and narrow emission bandwidth and easy fabrication, are ideal for lab-on-chip integration. Among different types of QDLED structures implemented, inorganic QDLEDs have shown great promise. We have demonstrated designs and fabrication strategies for creating QDLEDs with enhanced performance. In particular: (I) We introduce a sandwich structure with a spin coated inorganic hole transporting layer of nickel oxide underlying the QD layer and with a spin coated zinc oxide electron transporting layer, with patterning of anode and cathode on the substrate. Compared to the use of sputtered thin films, solution processed charge transporting layers (CTLs) improve robustness of the device, as crystalline ZnO shows low CB and VB edge energy levels, efficiently suppressing hole leakage current resulting in LEDs with longer lifetimes. We also use Atomic Layer Deposition to deposit an additional hole injecting layer to protect the QDs from direct contact with the anode. With this device design, we demonstrate a working lifetime of more than 12 hours and a shelf-life of more than 240 days for the devices. Our solution based process is applicable to micro-contact printed and also spin-coated QD films. QDLEDs with spin-coated CTLs show a lifetime increase of more than three orders of magnitude compared to devices made using sputtered CTLs. (II) We implement strategies of the enhancement of light extraction from the fabricated QDLEDs. We discuss the integration of a two dimensional grating structure based on a metal-dielectric-metal plasmonic waveguide with the metal electrode of a QDLED, with the aim of enhancing the light intensity by resonant suppression of transmitted light. The grating structure reflects the light coupled with the metal electrode in the QDLED and we found an increase of 34.72% in the electroluminescence intensity from the area of the pattern and an increase of 32.63% from photoluminescence of QDs deposited on a metal surface. (III) We demonstrate the capability of our fabricated devices as a light source by measuring intensity across stained cells with QDLEDs of two different wavelengths and show the correlation as expected with the absorption profile of the fluorescent dye. We measure the absorption from the biological samples using QDLEDs fabricated with various design modifications, as a quantification of the improvements in device performance, directly affecting to our target application. / text

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

No description available.

Diabetes epidemic is underway. Beta cell dysfunction (BCF) and loss of beta cell mass (BCM) are known to be key events in its progression. Currently, there are no reliable techniques to estimate or follow the loss of BCM, in vivo. Non-invasive imaging and quantification of the whole BCM in the pancreas, therefore, has a great potential for understanding the progression of diabetes and the scope for early diagnosis for Type 2 diabetes. Glucagon-like peptide-1 receptor (GLP-1R) is known to be selectively expressed on the pancreatic beta cells and overexpressed on the insulinoma, a pancreatic neuroendocrine tumor (PNET). Therefore, this receptor is considered to be a selective imaging biomarker for the beta cells and the insulinoma. Exendin-4 is a naturally occurring analog of GLP-1 peptide. It binds and activates GLP-1R with same the potency and engages in the insulin synthesis, with a longer biological half-life. In this thesis, Exendin-4 precursor, DO3A-VS-Cys40-Exendin-4 labeled with [68Ga], [68Ga]Ga-DO3A-VS-Cys40-Exendin-4 ([68Ga]Exendin-4), was evaluated in different species models, namely, immune deficient nude mice, rats, pigs, non-human primate (NHP), and clinically in one insulinoma patient by positron emission tomography (PET), for its potential in beta cell imaging and its quantification as well as for visualizing the insulinoma. From internal dosimetry, the possible number of repetitive [68Ga]Exendin-4-PET/CT scans was estimated. Pancreatic uptake and insulinoma tumor uptake of [68Ga]Exendin-4 were confirmed to be mediated by the specific binding to the GLP-1R. Pancreatic GLP-1R could be visualized and semi-quantified, for diabetic studies, except in rats. Nonetheless, we found conflicting results regarding the GLP-1R being a selective imaging biomarker for the beta cells. PET/CT scan of the patient with [68Ga]Exendin-4 has proven to be more sensitive than the clinical neuroendocrine tracer, [11C]5-HTP, as  it could reveal small metastatic tumors in liver. The kidney was the dose-limiting organ in the entire species model, from absorbed dose estimation. Before reaching a yearly kidney limiting dose of 150 mGy and a whole body effective dose of 10 mSv, 2–4 [68Ga]Exendin-4 PET/CT scans be performed in an adult human, which enables longitudinal clinical PET imaging studies of the GLP-1R in the pancreas, transplanted islets, or insulinoma, as well as in healthy volunteers enrolled in the early phase of anti-diabetic drug development studies.

The molecular cloning and subsequent engineering of the green fluorescent protein (GFP) of the jellyfish Aequoria victoria allowed a novel approach to the investigation of cell signalling. GFP and its mutants can now not only be used to target specific organelles in living cells but also function as a basis for a variety of sensors for biologically important ions and molecular interactions. GFP-based Ca2+- sensors have been successfully used for studies in mammalian and plant cells. In filamentous fungi, however, they have not yet been reported to work. Since only little is known about calcium signalling in filamentous fungi, this project aimed to improve existing GFP-based Ca2+- sensors by exchanging the original fluorophores for improved versions and expressing those in the filamentous fungus Aspergillus niger. During this project, the donor and acceptor fluorophores of 3 existing Ca2+-FRETprobes based on cameleons and troponin C-sensors, have been changed, 2 novel positive FRET controls have been designed and these , as well as donor and acceptor fluorophores alone, have been expressed in the filamentous fungus Aspergillus niger. The probes were assessed using different imaging techniques, such as conventional confocal laser scanning microscopy (CLSM), fluorescence lifetime imaging microscopy (FLIM) and spectral imaging using a Leica TSC SP5 confocal and IRIS, a novel spectral imaging device designed at Heriot Watt University. Problems were encountered that prevented FRET analysis using CLSM and IRIS. These were due mainly to the difference in expression level of the constructs and the distribution of the emission bandpasses of the IRIS system. Analysis of the spectral data obtained on the Leica confocal system and analysis of the FLIM results, however, revealed significant differences between the donor only and the positive FRET controls. Spectra of the positive FRET controls and the Ca2+-sensitive probes showed emission peaks of both the donor and the acceptor fluorophores upon excitation of the donor fluorophore alone while analysis of the FLIM results revealed an additional decay component in the positive FRET controls. Both results are very strong indicators that we can detect FRET in living hyphae of Aspergillus niger transformed with the probes designed during this project.

With the rapidly increasing interest in the field of glycomics, which is the comprehensive study of the roles carbohydrates play in a living system, urgent need for developing quick and highly selective carbohydrate sensors is growing. The boronic acid group, with its electron-deficient structure (6 valence electrons with an open shell), acts as a Lewis acid with high intrinsic affinity towards Lewis bases such as fluoride, cyanide and hydroxyl groups. Specifically, formation of a 5- or 6- membered ring between the boronic acid moiety and a1,2- or 1,3-diol in aqueous solution has been fully explored as a strategy of carbohydrate sensor design. Along this line, those binders were termed as ¡°boronolectins¡± because of their similar functions as lectins. One challenge in developing boronic acid-based carbohydrate sensors is to enhance the discriminating ability among various carbohydrate analytes with diverse building blocks and complex linkage patterns. One approach is using polypeptide or oligonucleotide as a backbone or scaffold with functionalized boronic acid moiety to create a molecular library, and then selecting binders with favorable properties. The work presented here includes three general research parts: synthesis of a naphthalimide-based boronic acid-conjugated thymidine triphosphate (NB-TTP), fluorescence property studies of NB-TTP incorporated DNA (NB-DNA), and cellular imaging studies using NB-TTP: 1) 4-Amino-1,4-naphthalimide (Nap) was chosen as the fluorophore because of its relatively long excitation and emission wavelengths, and stability. The synthesis of naphthalimide-based boronic acid (NB) followed similar route as previously published work. Tethering of boronic acid moiety and TTP was accomplished through Cu(I)-catalyzed azide-alkyne cyclization (CuAAC), known as click chemistry. The synthesized NB-TTP showed fluorescence enhancements at long wavelength (¦Ëem: 540 nm) upon sugar addition. 2) NB-TTP was incorporated into DNA through Klenow fragment-catalyzed primer extension reactions. Different DNA sequences were designed with varying number and spacing for NB-TTP incorporation. The preliminary study provided certain insight into several factors that affect the fluorescent properties of different NB -DNA. 3) NB-TTP was added into Hela cell culture medium to study its cell imaging properties. With the observation under fluorescent microscope, it was demonstrated that NB-TTP showed good cell membrane permeability and significant accumulation in cell nucleus.

This thesis describes the dynamics of mitochondrial RNA-binding protein 1 and 2 (MRP1/2) complex in different cell lines of Trypanosoma brucei under an optimized immobilized condition. This study reveals the influence of RNA on the complex's dynamics. Furthermore, the function of RNA-binding complex 1 (MRB1) core has been studied via reverse genetic, biochemical and molecular techniques, with its role in RNA editing being proposed.

Live cell imaging is a unique tool for cellular research with a wide variety of applications. By streaming digital microscopic images an investigator can observe the dynamic morphology of a cell, track cell movement on a surface, and measure quantities or localization patterns of fluorescently labeled proteins or molecules. Digital image sequences contain a vast amount of information in the form of visually detectable morphological changes in the cell. We designed computer programs that allow the manual identification of visible events in live cell digital image sequences [Davis et al. 2007]. Once identified, the data are analyzed using algorithms to calculate the yield of individual events per cell over the time course of image acquisition. The sequence of event data is also constructed into directed acyclic graphs and through the use of a subgraph isomorphism algorithm we are able to detect specified patterns of events originating from a single cell. Two projects in the field of cancer research are here discussed that describe and validate the application of the event analysis programs. In the first project, mitotic catastrophe (MC) research [Ianzini and Mackey, 1997; Ianzini and Mackey, 1998; reviewed by Ianzini and Mackey, 2007] is enhanced with the addition of live cell imaging to traditional laboratory experiments. The event analysis program is used to describe the yield of normal or abnormal divisions, fusions, and cell death, and to detect patterns of reductive division and depolyploidization in cells undergoing radiation-induced MC. Additionally, the biochemical and molecular data used in conjunction with live cell imaging data are presented to illustrate the usefulness of combining biology and engineering techniques to elucidate pathways involved in cell survival under different detrimental cell conditions. The results show that the timing of depolyploidization in MC cells correlates with increased multipolar divisions, up-regulation of meiosis-specific genes, and the production of mononucleated cell progeny. It was confirmed that mononucleated cells are produced from multipolar divisions and these cells are capable of resuming normal divisions [Ianzini et al., 2009]. The implications for the induction of meiosis as a mechanism of survival after radiation treatment are discussed. In the second project, the effects of long-term fluorescence excitation light exposure are examined through measurements of cell division and cell death. In the field of live cell imaging, probably the most modern and most widely utilized technique is fluorescence detection for intracellular organelles, proteins, and molecules. While the technologies required to label and detect fluorescent molecules in a cell are well developed, they are not idealized for long term measurements as both the probes and excitation light are toxic to the cells [Wang and Nixon, 1978; Bradley and Sharkey, 1977]. From the event analysis data it was determined that fluorescence excitation light is toxic to multiple cell lines observed as the reduction of normal cell division, induction of cell death, and apparent morphological aberrations.

The post-prognosis lifespan for those suffering with Glioblastoma (GBM) is approximately 13 months with current standard of care. Intratumoral heterogeneity is a common characteristic that hinders GBM treatment in the form of therapy resistant cell subsets and influence on cellular phenotypes. One cell subset in particular, glioma stem cells (GSCs), is frequently left behind in the brain parenchyma once the bulk of the tumor has been resected. Previous research has found that patient-derived GSCs displayed varying invasion responses with and without the presence of interstitial flow. Interestingly, GSCs from a single patient are heterogeneous, displaying differences among sub-colonies derived from the same parental line. To study the motility of cells under flow, PDMS microfluidics are commonly used. Unfortunately, this setup often involves active flow generation using pumps, limiting the number of cell lines that can be imaged at a time. To increase the throughput of GSC sub-colonies imaged simultaneously, we developed a bio-compatible, printable device fabricated to allow for passive, gravity-driven flow through a hydrogel that recapitulates the brain microenvironment, eliminating the need for pumps. Stereo lithography 3D printing was chosen as the manufacturing method for the device, and this facilitated design feature modification when prototyping, increased the potential complexity of future iterations, and avoided some of the hurdles associated with fabricating PDMS microfluidics. This printable imaging device allows for higher throughput live-imaging of cell lines to aid in the understanding of the relationships between intratumoral heterogeneity, invasion dynamics, and interstitial flow. / Master of Science / For those suffering with Glioblastoma, a high-grade brain cancer, the life span post treatment is approximately 13 months. The cells in this and many forms of cancer have physical and biological differences that make successfully eliminating the disease difficult. One of the cell types contributing to this are Glioma Stem Cells (GSCs) that are often left in brain tissue once most of the tumor has been surgically removed. Previous research has found that GSCs from different sources had different responses with and without the simulated or actual presence of flow in brain tissue. This was further complicated when different responses were observed in cells obtained when breaking apart one of the cell lines and propagating these into their own sub-colonies. The current standard for studying the movement of cells under flow is by using compact chips made of a clear silicone rubber. The setup with microfluidics typically requires connection to external tubing and pumps to create flow and this limits the amount of cell types that can be imaged at a time. In order to monitor more cells at a time we created a 3D printable device that uses gravity for flow to go through a gel that mimics brain tissue and these cells of interest. Resin 3D printing was used to make these small devices so that they could be easily re-designed for other experimental purposes in the future. Hopefully this device could be used to more rapidly gain an understanding of cell movement in GBM and other disease models.