Many intracellular organelles have an intraluminal acidic environment, but a full understanding of its functional role is lacking. A new report details a method involving a light-activated proton pump fused with a pH-sensitive pHluorin (pHoexin) to study the relationship between vesicle pH and neurotransmitter content. By changing neurotransmitter content in vesicles by optogenetically altering acidification, the authors found that glutamatergic synaptic vesicles that were full of neurotransmitter had a higher release probability than those that were incompletely full, and suggest that this technique could be applied to the study of other organelles.