(a) Experimental strategy for co-encapsulating an enzyme (carbonic anhydrase, CA, black spots) inside lipid vesicles, followed by adding a membrane-permeable substrate (6-carboxyfluorescein diacetate, CFDA). Note that the external enzyme molecules have not been removed and can react outside vesicles. (b) Details of the system under study, showing that CFDA can either react outside vesicles with free CA either permeate through the lipid membrane, reach the vesicle lumen, and then react with the encapsulated CA. The green-fluorescent product, 6-carboxyfluorescein, has been marked by a green box.