Tracking neuronal content using capillary electrophoresis with multiphoton excitation of fluorescence
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Capillary electrophoresis with multiphoton-excited fluorescence detection (CEMPE) allows low-background analysis of many spectrally distinct biological fluorophores using a single long-wavelength laser. This work demonstrates the methodical transformation of CE-MPE from a proof-of-concept instrument to a reliable and powerful workhorse for complex cellular samples. Preparation of cell extracts and their long-term storage prior to CE-MPE analysis have also been exhaustively characterized (Chapter 4). The process is suitable for extractions at 2 to 3 hour intervals over a day or more, or as frequently as every hour for shorter durations. With these methods, answers were obtained for hypothesis-driven research—answers not readily available from other techniques. For example, evidence suggested intracellular levels of vitamin B3 (nicotinamide) derivatives might exhibit a circadian rhythm in suprachiasmatic nuclei neurons. Therefore, Chapter 4 presents the tracking of these cofactors over 24 – 48 h periods in extracts prepared from an immortalized biological clock cell line. Chapter 5 extends this single-fluorophore work to investigate hypothesized intracellular changes in both indole and nicotinamide derivatives during depolarization-induced upregulation of serotonergic phenotype, using cells immortalized from the raphe nuclei of the brain. Chapter 5 also demonstrates detection of riboflavin (vitamin B2) derivatives in cell extracts, and proposes several relevant continuation experiments. Finally, Chapter 6 broadens the capabilities of CE-MPE to neutral analytes, such as melatonin, for the circadian investigation of multiple analytes in cells immortalized from the pineal gland, another clock-like area of the brain.