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