Capillary electrophoresis with multiphoton-exited fluorescence : native fluorescence, enzymatic assays, and ultra-fast separations
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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.