BIOMEDICAL IMAGE RESOLUTION IMPROVEMENTS BY COMBINED USE OF FOCAL MODULATION, PUPIL ENGINEERING, AND SPARSITY PRIORS.

Ph.DDOCTOR OF PHILOSOPH

MONITORING THE METABOLISM OF THIOLS/DISULFIDES IN THE EXTRACELLULAR SPACE OF ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES BY ONLINE MICROFLUIDIC ANALYSIS COUPLED WITH ELECTROOSMOTIC SAMPLING

Sulfur-containing compounds, such as glutathione (GSH), cysteamine (CSH), homocysteine (Hcy), cysteine (Cys) and their disulfides, are critical for maintaining various cellular functions. Concentration profiles of these compounds in biological samples are useful for evaluating cellular physiological status and diagnosing diseases. Traditional quantitative methods involve tedious sample pretreatment and are not suited to dealing with submicroliter samples. We have developed a microfluidic system for online analysis of thiol-containing compounds in the extracellular space of rat organotypic hippocampal slices cultures (OHSCs). This system is also capable of monitoring ectoenzymatic activity involved in the metabolism of thiols/disulfides. A microfluidic chip capable of derivatization, multiple injection, separation, and quantitation was designed and coupled to a homemade confocal laser induced fluorescence detector with a multifunctional control program. The microfluidic system was evaluated in an on-chip kinetic study of the reduction of oxidized glutathione (GSSG) catalyzed by glutathione reductase (GR, EC 1.8.1.7) followed by derivatization of GSH with ThioGlo-1. The separation of analytes was successfully achieved within 4.5 s and 9 mm separation length. The linear range of the system is up to 50 µM (GSH). The mass and concentration detection limits are 10-18 mol and 4.2 nM, respectively. Based on GSH growth rates, the apparent Michaelis constants of GR were determined to be 40 ± 11 μM (GSSG) and 4.4 ± 0.6 μM (β-NADPH). Electroosmotic sampling of the extracellular fluid of OHSCs was further coupled to a microfluidic device for in situ quantitation of endogenous aminothiols in OHSCs, by which CSH (10.6 ± 1.0 nM), Hcy (0.18 ± 0.01 μM) and Cys (11.1 ± 1.2 μM) were separated and evaluated simultaneously. Using this method, we also observed the complete biodegradation of coenzyme A (CoA) in the extracellular space of OHSCs and determined the kinetic parameters of this sequential multi-enzyme reaction in in situ. Metabolism of cystamine (CSSC) and pantethine (PSSP) in OHSCs have also been investigated and the percentage yields of CSH from CSSC and PSSP after ~55 s exposure time to OHSCs were 91% ± 4% and 0.01% - 0.03%, respectively, which explains the differences of the drugs in clinical effectiveness and toxicity

Nanoscopy in nonlinear scanning fluorescence imaging systems

In the last 30 years, superresolution in optical microscopy has been a major field of research. During this time, different techniques have been created to break the diffraction limit in order to make observations at a nanometric scale. Given that optical microscopy is non-invasive, those superresolution methods pave the way for a better understanding of biological mechanism at a molecular level. Most of those methods are based on a nonlinear interaction between the excitation light intensity and the sample response (often fluorescent signal). In the same time, nanodiamonds containing fluorescent defects have been proven to be a choice probe for superresolution nanos-copy since they exhibit a strong and stable fluorescent signal even under high light intensities exposure (often required to obtain nonlinear photoresponse). Nanodiamonds containing Nitrogen Vacancy (NV) defects that exhibit a red fluorescent signal had been previously shown to be a viable biomarker for STED superresolved image. First, we demonstrated that green fluorescent nanodia-monds containing Nitrogen-Vacancy-Nitrogen (NVN) defects can be used with a Stimulated Emission Depletion (STED) superreso-lution microscope. Then, we implemented a STED microscope in our lab and compared the properties of NVN and NV centers for STED imaging. We conclude that even if nanodiamonds with NVN defects are less intense, they can be used as a second color nonbleaching biomarker. To illustrate the potential use of green nanodiamonds as bio-compatible probe, we superresolved them internalized into a cell with STED microscopy. Second, we tried to work on one of the main limitation in STED nanoscopy: the lack of information in the axial direction within a single scan. We combined our home made STED microscope with a Double Helix phase mask that modifies the detection point spread function in order to obtain axial localization of the superresolved emitters. We achieved three dimensional localization of nanometric fluorescent emitters but we note that photobleaching was the main limitation of this approach with organic dyes. We discussed different solutions to limit the photobleaching and their feasibility. We also worked on a different superresolution technique that we named Computational Nonlinear Saturated (CNS) microscopy. We showed that with digital post treatment of the acquired data, a nonlinear photoresponse can be harnessed to any scanning microscope equipped with a camera detector to enhance the resolution. We demonstrated that increasing the excitation power and inducing fluorescence saturation, it is possible to break the diffraction limit in a conventional confocal microscope (after data post-treatment). However, with this method, we did not obtain a gain in resolution as high as with other superresolution tech-niques involving fluorescence saturation, such as saturated structured illumination microscopy. To understand the origin of this limitation, we carried out simulation to investigate the performance of CNS microscopy in noisy environments compared with wide field techniques. We propose alternative implementation and quantify the possible resolution gain with simulations. Finally, we demonstrated how a technique, initially created for optical microscopy, can be adapted to lensless endoscopic imaging..