| A new method for the ultrasensitive analysis of single cells has been established, which involves the separation of the cellular content in a capillary, one cell at the time, and detection of any fluorescently tagged analytes by laser induced fluorescence. The present work focuses on instrumentation development and validation, which further demonstrates the applicability of our technology in problems with biological relevance. First, the linear dynamic range of the analysis by capillary electrophoresis with laser induced fluorescence (CE-LIF) was improved to an unprecedented nine orders of magnitude; this was done with the use of beam-splitters and detectors added into our existing technology. Using a lipid substrate GM1 labeled with tetramethylrhodamine (TMR), the upgraded instrument demonstrated that only the catabolic compartments of the cell were probed in neurons from the rat cerebellum. New strategies for probing anabolism were evaluated, including the use of Bodipy-labeled substrates, which did allow probing the anabolic compartments within the cell. This lead to our second instrumentation upgrade: the design of a CE-LIF system capable of two-color detection, which enabled the simultaneous analysis of concurrently occurring degradative and biosynthetic pathways within a single neuron, while maintaining the sensitivity that is the hallmark of this technology with zeptomole (10-21) to yoctomole (10-24) limits of detection. |
