Table 1.

Troubleshooting table.

Step	Problem	Reason	Solution
42	Too few vesicles	Insufficient biotins on the surface	Surface functionalization with the biotin-PEG may not have been done properly. To test for this, use other fluorescent markers such as dye-labeled streptavidin or oligonucleotides that have been dual-labeled with biotin and fluorescent dye. If the surface biotin-PEG is fine, there may be a problem with NeutrAvidin or the biotin-lipid
	Large and bright vesicles	Aggregation of vesicles during reconstitution	Immobilize vesicles before reconstitution to identify the step at which the problem is caused. If the problem is during the reconstitution, the detergent concentration or the lipid composition may need to be optimized
	Too many vesicles	Incorrect vesicle concentration calculation	Use a fresh channel with a lower concentration of vesicles
47	Too much donor bleed-through	Intensity is too high	Reduce the laser intensity with neutral density filter or a half-wave plate/beam splitter
	Too many or too few docked vesicles	Unoptimized vesicle concentration or incubation time	Change the concentration or incubation time
	Too many docked vesicles	Nonspecific binding	Redo the PEGylation process. You may also run a control with a sample without Sec9c or SNAP-25 to check whether this is nonspecific binding