We originally developed a tool to measure the chemical messenger contents of single nanometer vesicles starting with capillary electrophoretic separation, lysing and electrochemical detection. We called this flow vesicle impact cytometry (FVIEC). The method used a microfluidic device with sheath flow and electrode for detection. The next generation approaches led to static electrochemical measurements and mass spectrometry imaging of vesicular content. First, micro-/nano-electrochemical approaches have been developed to carry out quantitative measurements of intravesicular content and real-time monitoring of vesicle release dynamics. We have used stochastic vesicle impact electrochemical cytometry (VIEC) and intracellular VIEC (IVIEC) to provide a highly effective way to quantify the electroactive contents inside vesicles. This has allowed direct comparison of the quantity of molecules released by exocytosis to those in the vesicles. Partial release is observed across all cell types examined to date by the electrochemical methods and appears to be regulated.More recently, we have used mass spectrometry imaging with NanoSIMS as a complimentary approach to image the neurotransmitter dopamine across the internal volume of these incredibly small vesicular structures. 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.