In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

Cell penetrating peptides have been the focus of drug delivery research for 15 years due to their apparent ability to deliver cargoes inside cells more readily than many other carriers. Using two of the most commonly studied peptides (tat47-57 and R9), the present study differs from previous work by deliberately choosing to observe uptake with lower peptide concentrations closer to potential therapeutic doses, and by implementing raster image correlation spectroscopy (RICS) on a commercial microscope to quantify uptake in parallel to other techniques such as fluorescence correlation spectroscopy (FCS), confocal microscopy, and mass spectroscopy.An initial study using mass spectrometry and ExPASy (Expert Protein Analysis System) revealed that the peptides are stable for at least one hour in PBS. Based on this initial information and other experimental conditions, the study took two main directions with regards to the peptide: the membrane interaction and accumulation in the cell.The peptides interaction with the cell membrane revealed that neither tat-TAMRA nor R9-TAMRA disrupts the membrane of cells: incorporation of FM2-10 in the membrane was not modified in K562 cells whilst it was in presence of the control lytic peptides GALA and mellitin. Based on this information confocal microscopy was utilised to assess the localisation on the cell membrane. Peptide binding to the membrane appeared to be heterogeneous in distribution at 1µM bulk concentration.Accumulation in cells of the peptides was observed incubated at 37°C, confocal microscopy showed punctuated distribution with intracellular aggregations of fluorescence measuring between 2.5-3.5µm in diameter. Co-staining with a nuclear dye revealed these aggregations to be focused around the nucleus of the cell. Initial FCS experiments indicated a concentration dependent accumulation of the peptide in the cells and a decrease of the intracellular diffusion coefficients at high concentration possibly corresponding with molecular crowding. Interestingly the anomalous diffusion model did not statistically improve the results.RICS was implemented to study the kinetics of entry of TAMRA labelled cell penetrating peptides in both Caco-2 and HeLa cells lines at concentrations between 500nM and 2µM. Concentrations above 1µM exhibited higher final intracellular concentrations, yet the measured diffusion coefficients were similarly independent of extracellular concentration. Both peptides appeared to enter the cell quickly with a fast initial uptake over the first 10 minutes, reaching a concentration maxima after 30 minutes.Overall, the study reveals that many published studies may be incorrect as they may only be reporting the presence of a fluorescent dye inside the cell not the peptide. The fast binding of the peptide to the membrane is likely to cause false positive results when traditionally studying internalisation kinetics such as using flow cytometry and confocal microscopy. Correlation spectroscopy techniques such as FCS, provide useful information on internalisation of the peptides, but the single spot measurement is limited when providing information on the entire cell. RICS is a progression of correlation spectroscopy and provides a more representative picture of the cell.

Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface proteins including transmembrane and membrane-anchored receptors, which play a vital role in their response to pathogenic stimuli. The plasma membrane is a highly fluid and dynamic environment, which facilitates the diffusion of lipids and proteins within the plane of the membrane. This study aims to measure the lateral diffusion of two types of plasma membrane receptors on macrophages, toll-like receptor II (TLR2) and cluster of differentiation 14 (CD14), to answer three main research questions: 1) Which type of fluorescence-based microscopy techniques is best suited for measuring the lateral diffusion of TLR2 and CD14 on macrophage plasma membrane? 2) Does culturing macrophages on different surface topographies impact the diffusion of TLR2 in the plasma membrane and its pro-inflammatory response, along with morphological changes? 3) Does aging alter the lateral diffusion of TLR2 in the plasma membrane of macrophages? To date, a variety of fluorescence-based methods have been developed to study the dynamics of cell membrane constituents. These techniques are based on either ensemble or single particle measurements. We have used single particle tracking methods to track the mobility of fluorescently labeled membrane receptors on murine bone marrow-derived macrophages. Total internal reflection fluorescence microscopy (TIRF) was used to visualize and capture the dynamics in live cells. Using a custom routine algorithm we detected, localized, and tracked the particles to calculate their diffusion coefficient, extracted from the mean-squared displacement as the most common measure of diffusion. We also measured the diffusion coefficient using an ensemble-based technique known as Raster Image Correlation Spectroscopy (RICS) with a confocal laser-scanning microscope. The use of confocal eliminates the out-of-focus signal and enables measurements that are confined to a narrow plane in the cell. Also, the ability of RICS to separate the slow and immobile fractions of particles makes it possible to detect heterogeneities in diffusion. To our knowledge, this is the first study that has utilized both SPT and RICS to directly compare receptors’ diffusion in different membrane sections. Moreover, this is the first study that has examined the diffusion of receptors on macrophages adhered to different surface topographies, and the first that has investigated the receptors’ diffusion in young and old macrophages. / Thesis / Doctor of Philosophy (PhD) / The immune system is highly dependent on a specialized subset of white blood cells known as macrophages that are capable of clearing damaged and dead cells as well as a wide range of invading micro-organisms. Specific receptor proteins present on the membrane of macrophages are involved in the recognition of particles and subsequent signaling to recruit other immune cells or to promote healing and wound repair. To date, a variety of fluorescence-based microscopy methods have been used to study the dynamics of cell membrane components. The mobility of several membrane receptors in macrophages has been studied using microscopy techniques, which have provided valuable insights into their function. However, there is still insufficient information about the behavior of two key receptors (TLR2 and CD14) that participate in signaling in response to bacterial products. This thesis aims to answer three major questions with regard to receptor mobility (i.e., diffusion) within macrophage membrane: 1) Which type of fluorescence-based microscopy technique is more suitable for measuring the mobility of TLR2 and CD14 receptors on macrophage membranes? 2) What is the impact of different surface topographies on TLR2 diffusion in adhered macrophages, as well as cell shape, and the ability of macrophages to internalize particles? 3) Does aging alter TLR2 mobility in the membrane of macrophages? The following chapters provide detailed answers to these questions. In brief, we have demonstrated that TLR2 and CD14 diffusion measurements in adhered macrophages highly depend on the membrane section chosen. In addition, our results show that micro- and nanostructured surface topographies alter the shape of adhered macrophages and yield higher bacteria internalization, while the diffusion of TLR2 is not changed. When comparing macrophages derived from young and old mice, we find similar diffusion rate of TLR2 in macrophages of the two age groups.