Correlation of Cellular Measurements of Partial Exocytosis and Nano Vesicular Subcompartments between Amperometry and NanoSIMS

Electrochemical evidence since about 2010 has supported the view that most exocytosis from cellular vesicles is partial in nature. Intracellular vesicle impact cytometry combined with amperometry has been a key part in this progression. To support these observations, we have used correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. To do this, we have developed standards to carry out quantitative NanoSIMS and subsequently used it to quantify the chemical contents across the nanometer structure of vesicles. We then used NanoSIMS imaging of dopamine inside and drug into vesicles to quantify the fraction of messenger and demonstrate drug entry during exocytosis providing definitive evidence of partial release and a means to introduce drug into cells. We have developed an electrochemical sensing approach to discern the content of subvesicular compartments. We then combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line to localize 13C dopamine enrichment across nano vesicle domains. The absolute concentrations of 13C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to the electrochemical data. We found the relative amounts of catecholamine in each vesicular compartment were the same as found via the electrochemical sensor. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles correlating with electrochemical measurements.