Browsing by Subject "Capillary electrophoresis"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Applications of multiphoton-excited photochemistry to microsecond capillary electrophoresis, photolithography, and the development of smart materials(2010-08) Ritschdorff, Eric Thomas; Shear, Jason B.; Holcombe, James A.; Stevenson, Keith J.; Vanden Bout, David A.; Schmidt, ChristineLaser-based techniques have become essential tools for probing biological molecules in systems that demand high spatial and temporal control. This dissertation presents the development of micro-analytical techniques based on multiphoton excitation (MPE) to promote highly localized, three-dimensional (3D) photochemistry of biologically relevant molecules on submicron dimensions. Strategies based on capillary electrophoresis (CE) have been developed for the rapid separation and spectroscopic analysis of short-lived photochemical reaction products. High-speed separation and analysis are achieved through a combination of very high electric fields and a laser-based optical system that uses MPE for both the generation and detection of hydroxyindole photoproducts on the time scale of microseconds. MPE was also used for the development of photolithographic techniques for the creation of microstructured protein-based materials with highly defined three-dimensional (3D) topographies. Specifically, a multiphoton lithographic (MPL) technique was developed that used a low-cost microchip laser for the rapid prototyping of 3D microarchitectures when combined with dynamic optical masking. Furthermore, MPL was used to create novel “smart” biomaterials that reproducibly respond with tunable actuation to changes in the local chemical and thermal environment. The utility of these materials for creating biocompatible cellular microenvironments was demonstrated and presents a novel approach for studying small populations of microorganisms. Finally, through the development of a multifocal approach that used multiple laser beams to promote the photocrosslinking of biological molecules, the speed and versatility of MPL was extended to allow both the parallel fabrication of 3D microstructures and the rapid creation of large-scale biomaterials with highly defined spatial features.Item Capillary electrophoresis with multiphoton-exited fluorescence : native fluorescence, enzymatic assays, and ultra-fast separations(2001-08) Okerberg, Eric Steven; Shear, Jason B.Analysis of single neurons and/or single synaptic vesicles will uncover heterogeneity that is masked by the ensemble averaging required of many current techniques. In the Shear lab, we have developed instrumentation to perform capillary electrophoresis (CE) coupled to multiphoton excitation (MPE) as a means to improve detection limits for bioactive molecules in such volume limited samples. CE has provided significant advancements in this arena due to its ability to fractionate sample volumes of less than one picoliter and the relative ease with which highly sensitive detection strategies can be employed. Moreover, the use of extremely narrow separation channels (~600 nm) permits the collection of low volume samples without excessive dilution and provides the spatial and temporal resolution necessary to investigate heterogeneous microenvironments. Such low vii volume analyses necessitate the use of sensitive detection strategies due to the limited sample quantity within small inner diameter (i.d.) separation channels. Multiphoton-excited fluorescence is an excellent strategy for such low volume detection due to the inherently small excitation volumes, improved spectral isolation of the signal from the source, and the ability to probe diverse chromophores with a single, tunable source. Here we present the use of MPE with CE and demonstrate marked improvements in mass sensitivity for several neurotransmitters and neuropeptides (Chapter 2). Moreover, the versatility of this approach is demonstrated through the analysis of enzymatic reactions (Chapters 3 and 5) and photochemical derivatization (Chapter 4). We demonstrate a new platform for ultra-fast separations based on multiphoton-excited photochemistry in Chapter 4. Future efforts will demonstrate the capacity of CE-MPE to probe volume limited biological microenvironments such as single synaptic vesicles.Item Characterization of biological chromophores using fast electrophoretic analyses and multiphoton-exited fluorescence(2001-08) Gordon, Mary Jane Sia; Shear, Jason B.A fast and highly sensitive technique has been developed for characterizing mixtures of biological molecules that should be useful for various applications, including investigations of enzymatic reactions with fast chemical kinetics, analysis of transient photochemical reactions, and probing neurotransmitter secretion. In this dissertation work, multiphoton excitation (MPE), a high sensitivity detection approach, and capillary electrophoresis (CE), a fast separation strategy, have been coupled to rapidly characterize primary amine neurotransmitters, to perform fast electrophoretic analyses of photochemical reactions, and to probe changing biochemical microenvironments. Nonfluorescent neurotransmitter molecules have been separated and detected by virtue of fluorogenic-labeling strategies with mass detection limits (< 50,000 molecules) that should prove useful for various single-cell studies. Also, this technique has been extended to rapidly probe spectroscopically indistinct components in solution. Extremely short capillary lengths and large applied electric fields are used to extend the speed of CE to sub-second time regimes. Previously, others have demonstrated that fast CE can be accomplished using optical gating, a technique in which sample injection is controlled by modulation of photobleaching at the separation channel inlet. In these studies, this strategy has been adapted to an "inverse" optical photogating mode with CE, where fluorescent packets of photogenerated molecules are created and detected by MPE. Two instrumental configurations, defined by the alignment of the laser light with the capillary ("end on" and "side on" geometries) and the separation distance, are constructed to achieve fast separations. Using these approaches, this work demonstrates the capacity to analyze dynamic biochemical microenvironments and track transient reaction products on a several-hundredmicrosecond to several-hundred-millisecond time scale. These fast analysis strategies have provided additional information on transient species on millisecond and faster time scales, and may prove valuable in analyzing cellular processes in real-time.Item Tracking neuronal content using capillary electrophoresis with multiphoton excitation of fluorescence(2005) Wise, Dana Diane; Shear, Jason B.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.