Direct methods for dynamic monitoring of secretions from single cells by capillary electrophoresis and microscopy with laser-induced native fluorescence detection
Is Version Of
Microscale separation and detection methods for real-time monitoring of dynamic cellular processes (e.g., secretion) by capillary electrophoresis (CE) and microscopic imaging were developed. Ultraviolet laser-induced native fluorescence (LINF) provides simple, sensitive and direct detection of neurotransmitters and proteins without any derivatization;An on-column CE-LINF protocol for quantification of the release from single cell was demonstrated. Quantitative measurements of both the amount of insulin released from and the amount remaining in the cell ([beta]PTC3) were achieved simultaneously;Secretion of catecholamines (norepinephrine (NE) and epinephrine (E)) from individual bovine adrenal chromaffin cells was determined using the on-column CE-LINF. No apparent relationship was found between the ratio of NE/E released and the original ratio of NE/E in the cell. Temporal information about the release was traced out with a dynamic stimulating method by pumping secretagogue continuously passing the cell with the aid of electroosmotic flow and measuring the peak broadening of the released catecholamines in the electropherogram;Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved by LINF imaging microscopy with high temporal and spatial resolution. Large variations were found among cells in the amount of catecholamines secreted and the rate of secretion. Local heterogeneity in the rate of secretion was also found;The secretion of serotonin from individual leech Retzius neurons was directly characterized by LINF microscopy with high spatial resolution. The results from images of co-cultured pressure sensory (P) (non-serotonergic) and Retzius (serotonergic) cells, as well as capillary electrophoresis, suggested that the major native fluorescence excited by the laser was derived from intracellular serotonin. A much more substantial release of serotonin was found at the axon stump left during cell isolation, a preferred region for synapse formation. The release of serotonin was also characterized to be calcium dependent.