The use of non-invasive methods to visualise and monitor processes inside living organisms is vital in the understanding and diagnosis of disease. The work in this thesis details the synthesis and applications of a new imaging modality; carbon dots, whose inherent fluorescence and non-toxic nature makes them attractive alternatives to more traditional ‘quantum dots’. In this thesis, different methods of carbon dot synthesis were attempted in order to produce carbon dots of the desired size and morphology. Nitrogen-containing carbon dots generated from 1,4-addition polymers provided the most successful route with optical and structural characteristics studied by TEM, UV-Vis and fluorescence spectroscopy and XPS. The biological behaviour of the carbon dots produced by this method were also evaluated. The ability of these carbon dots to up-convert when excited at long excitation wavelengths was studied. A number of biologically relevant applications of the carbon dots were studied. Using amine-functionalised carbon dots, cell targeting cargoes were conjugated and the effects of the carbon dot-cargo conjugates on cell lines were studied.

I investigated cellular mechanisms of ion and acid-base transport in rainbow trout (Oncorhyncus mykiss), crabs (Neohelice granulata), zebrafish (Danio rerio), Pacific hagfish (Eptatretus stoutii), and mosquito larvae (Aedes aegypti) with a primary focus on discerning the mechanisms governing ion transport and acid base regulation. In rainbow trout I provide the first functional evidence for two physiologically distinct mitochondrion-rich (MR) cells at the gill and demonstrate a new model for transepithelial Na+ uptake from freshwater involving apical Na+ channels and basolateral Na+/HCO3- co-transporters. These data are supported by extensive thermodynamic consideration of Na+ uptake from freshwater. I also demonstrate functional Cl-/HCO3- exchangers in both MR cell subtypes with roles for Cl- uptake and intracellular pH (pHi) regulation respectively and I present the first evidence for a Cl- dependent Na+/H+ exchanger in gill MR cells. Finally I demonstrate a unique Na+ dependent pHi recovery mechanism that requires protein kinase C for activation. A major limiting factor in clarifying the mechanisms of Na+ uptake in freshwater fish is the lack of a typical Na+ channel in any of the fish molecular databases. My work on zebrafish, although preliminary, indicates that a member of the acid-sensing ion channel family could be responsible for Na+ uptake from freshwater. I then expanded my research outside the trout model using an isolated crab gill preparation. I provide a cellular model for H+ secretion in crab gills that supports the transepithelial Na+ transport model that I described in rainbow trout. In Pacific hagfish, I demonstrate that recovery from blood acidosis is dependent on a Na+/H+ exchanger in gill MR cells. This mechanism of regulation involves translocation from the cytoplasm to the apical membrane during acidotic stress. This data combines with other studies demonstrating the mechanisms of acid and base secretion from a single MR cell subtype. Finally, I show that serotonin stimulation alkalinizes the pHi of the anterior midgut cells in the larval mosquito to levels never before observed in cell biology. These data challenge the dogma of pHi regulation in cell biology and demonstrate the power of using a comparative approach to systems physiology. / Physiology, Cell and Developmental Biology

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La compréhension des processus cellulaires au niveau membranaire est un domaine d’étude important en recherche biomédicale. Contourner la limite de diffraction en microscopie de fluorescence est maintenant devenu possible en exploitant les transitions moléculaires du fluorophore. Ce travail présente le développement instrumental de deux techniques complémentaires permettant d’atteindre une résolution nanométrique, grâce à l'émission stimulée (STimulated Emission Depletion - STED) d’une part, et la microscopie de fluorescence aux angles supercritiques (Supercritical Angle Fluorescence, SAF) d’autre part. La microscopie STED est une méthode permettant de surpasser la barrière de diffraction et d’atteindre des résolutions latérales de l'ordre de 40 nm dans des échantillons biologiques. Ce dispositif de microscopie exploite les transitions moléculaires des marqueurs fluorescents pour surmonter la limite de résolution due à la diffraction. L'amélioration de la résolution est obtenue par déplétion de l'état excité du fluorophores dans les régions périphériques de l'espace du volume focal. Cependant, malgré l'amélioration importante de la résolution latérale avec la technique STED, cette dernière présente une réelle complexité de mise en œuvre qui a par conséquence un impact important sur le cout des instruments STED commerciaux. Dans ce contexte, la réalisation instrumentale et la performance en imagerie d'un dispositif STED sont présentées dans ce manuscrit. Bien que les microscopes STED classiques offrent une meilleure résolution latérale, la résolution axiale est toujours limitée par la diffraction. L’amélioration de la résolution dans cette direction implique une certaine complexité instrumentale. Dans ce cadre, nous démontrons une nouvelle approche utilisant l’imagerie SAF permettant d'obtenir un sectionnement axial de l'ordre de 150 nm. L’approche se base sur la propriété d'une molécule à émettre dans les angles supercritiques uniquement lorsqu’elle se rapproche de l'interface verre-eau. Le sectionnement axial est obtenu dans une configuration simple en détectant uniquement les composantes de l’émission supercritique. La combinaison de ces techniques d'imagerie donne un outil puissant pour étudier les phénomènes moléculaires sur les membranes biologiques. / Understanding cellular processes on membranes has been a key area of biomedical research. Circumventing the diffraction limit in fluorescence microscopy has now become possible by exploiting the molecular transitions of the fluorophore. In this context, this work presents the instrumental development of two complementary techniques for realizing nanometric all-optical resolution and axial sectioning, namely STimulated Emission Depletion (STED) and Supercritical Angle Fluorescence (SAF) microscopy. STED microscopy is an elegant method that has allowed us to break the diffraction barrier with light microscopes and has achieved resolutions of the order of 40 nm (transverse) in biological samples. In this technique, we exploit the molecular transitions of the fluorescent marker to overcome the resolution limit due to diffraction. Resolution enhancement is achieved by efficient depletion of the excited state of the marker in the peripheral spatial regions of the focal volume by using depletion beams in addition to the excitation beam. Despite the major resolution improvement demonstrated, the technique is not well spread out, mainly due to its apparent complexity; and the cost and limited tunability of the commercial system. In this context, the instrumental realization and the imaging performance of a cost-effective home-built STED microscope is presented in this manuscript. While conventional STED microscopes offer improved lateral resolution, an isotropic gain in resolution usually comes at the cost of complex instrumentation. In this regard, we demonstrate SAF microscopy as a powerful tool that achieves an axial sectioning of the order of 150 nm. This is done by exploiting the property of a molecule to emit into the supercritical anglesonly when near the glass-water interface. Axial sectioning is obtained in a simple configuration by detecting solely the supercritical components of radiation. A combination of these imaging techniques offer a powerful tool to study molecular phenomena on the biological membranes.

cAMP is a second messenger in a variety of chemosensory receptors, including taste buds and glucose-sensitive pancreatic beta-cells. cAMP is modulated during taste transduction, yet the significance of cAMP changes and the taste cell types in which they occur (Type I glial-like; Type II Receptor; Type III Presynaptic) remain unclear. I developed techniques to image real-time changes in intracellular cAMP in taste cells using genetically-encoded cAMP reporters. This FRET-based reporter permits one to measure single-cell cAMP levels with excellent spatial and temporal resolution (Zaccolo & Pozzan 2002, Science 295:1711). Using a biolistic approach I have transfected rat fungiform taste buds with cAMP reporter plasmids. Focal application of bitter tastant to living fungiform tastebuds in situ produced a decrease in [cAMP]i within individual taste receptor cells. These results are qualitatively similar to previous biochemical measurements from homogenized taste tissue (Yan et al. 2001, Am J Physiol Cell Physiol 280:C742) but are now allowing us to examine the cAMP response in individual, identified cells. I next explored the effect of elevating cAMP on calcium levels, using Fura-2 imaging of isolated mouse vallate taste buds. Elevating [cAMP]i in taste buds evoked calcium responses in presynaptic/Type III taste cells, which do not express GAD1. cAMP induced responses were generated by calcium influx. Using pharmacological antagonists, I determined that the calcium influx triggered by cAMP is through L-type calcium channels, whereas influx following depolarization is primarily through P/Q-type calcium channels. Consistent with these data, single cell RT-PCR showed that the L-type subunit (alpha 1C) was expressed primarily in GAD-negative Presynaptic cells, while the P/Q-type (alpha 1A) was expressed in all Presynaptic cells. Thus, cAMP may modulate the function of synapses in some taste cells. Finally, we have developed a mouse strain expressing a cAMP reporter in a tissue-specific and tetracycline-inducible manner. We crossed this mouse with another strain expressing tet-activator in beta cells of the pancreas. Such islets responded to increasing concentrations of glucose (5.5 to 35mM) with an increase in cAMP levels. The half maximum of 9mM glucose for the cAMP response corresponds well with reported glucose concentrations that elicit insulin release from whole islets. Stimulating pancreatic islets with glucose is known to drive calcium influx into beta-cells. When we simultaneously imaged both second messengers, we found that cAMP changes precede and are independent of calcium changes. In conclusion, these studies have outlined novel potential functions for cAMP signaling in the transduction of both primary tastant and plasma glucose information. In addition, the flexibility of the tet-system will enable cAMP reporter expression in numerous cell types, including those which mediate gustatory transduction.

Single cell manipulation techniques have important applications in laboratory and clinical procedures such as intracytoplasmic sperm injection (ICSI) and polar body biopsy for preimplantation genetic diagnosis (PGD). Conventionally, manipulation of cells conducted in these procedures have been performed manually, which entails long training hours and stringent skills. Conventional single cell manipulation also has the limitation of low success rates and poor reproducibility due to human fatigue and skill variations across operators. This research focuses on the integration of computer vision microscopy and control algorithms into a system for the automation of the following single cell manipulation techniques: (1) sperm immobilization, (2) cell aspiration into a micropipette, and cell positioning inside a micropipette, and (3) rotational control of cells in three dimensions. These automated techniques eliminate the need for significant human involvement and long training. Through experimental trials on live cells, the automated techniques demonstrated high success rates.

Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope. Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP. It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins. Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

Imaging of biomolecules on the nano-scale is a crucial developing technology with major implications for our understanding of biological systems and for detection and therapy of disease. Plasmonic nanoparticles are a key optical contrast agent whose signal is generated by the collective oscillation of electrons in the metal particle. The resonance behavior of the electrons depends strongly on the arrangement of neighboring nanoparticles in a structure. This property may be exploited in imaging applications to report information on nanoscale morphology of targeted biomolecules. While the effect of plasmon resonance coupling has been studied in dimers and linear arrays of nanoparticles, this phenomenon remains largely unexplored in the case of 2D and 3D assemblies which are important in molecular cell imaging. This dissertation demonstrates how the optical signal from assemblies of gold nanoparticles can be related to nanoscale morphology in cellular imaging systems. First, the scattering spectra from live cells labeled with gold nanoparticles were collected and compared to the nanoscale arrangement of the particles in the same cells as determined by electron micrograph. Then, trends in scattering spectra with respect to nanoparticle arrangement were analyzed using a model system that allowed precise control over arrangement of nanoparticles. Several approaches to creating these model systems are discussed including biochemical linking, capillary assembly of colloidal particles, and direct deposition of gold onto substrates patterned by electron beam lithography. Spectral properties of the assemblies including peak position, width, and intensity are gathered and related to model variables including interparticle gap and overall particle number. It is shown that the redshift in the scattering spectra from nanoparticle assemblies is derived from both the particle number and the gap and is due to near-field coupling of particles as well as phase retardation of the scattered wave. The redshift behavior saturates as the number of particles in the aggregate increases but the saturation point depends strongly on interparticle gap. The drastic dependence of the red-shift saturation on the gap between nanoparticles has not been previously described; this phenomenon can have significant impact on the development of nanoparticle contrast agents and plasmonic sensor arrays. / text

Single cell manipulation techniques have important applications in laboratory and clinical procedures such as intracytoplasmic sperm injection (ICSI) and polar body biopsy for preimplantation genetic diagnosis (PGD). Conventionally, manipulation of cells conducted in these procedures have been performed manually, which entails long training hours and stringent skills. Conventional single cell manipulation also has the limitation of low success rates and poor reproducibility due to human fatigue and skill variations across operators. This research focuses on the integration of computer vision microscopy and control algorithms into a system for the automation of the following single cell manipulation techniques: (1) sperm immobilization, (2) cell aspiration into a micropipette, and cell positioning inside a micropipette, and (3) rotational control of cells in three dimensions. These automated techniques eliminate the need for significant human involvement and long training. Through experimental trials on live cells, the automated techniques demonstrated high success rates.

Magnetic Resonance Imaging can now detect cells that are labeled with contrast agents such as superparamagnetic iron oxide (SPIO). Quantitative monitoring, which is desirable for evaluating cellular therapies, remains challenging. In this work, an MRI technique called TurboSPI is implemented for quantitative cellular imaging. TurboSPI acquires maps of the relaxation rate R2', which is directly related to SPIO concentration. Quantification of R2' is demonstrated using micron-sized iron oxide particles and SPIO-labeled cells. To explain experimental results that deviated from predicted behavior, an extended analytical description of MRI signal relaxation near SPIO was developed. This model compares well to Monte Carlo simulations and experimental data, and may allow improved quantification. The slow imaging speed of TurboSPI is overcome using a signal processing technique called compressed sensing to reconstruct undersampled data, enabling in vivo animal imaging with TurboSPI. Such images demonstrate detection of iron with improved specificity and good potential for quantification.