Table 2.

| Troubleshooting table

Step	Problem	Solution
9	wafer sticks to photomask	Increase the soft bake time, cooling time, and/or the mask-substrate distance
11	the SU-8 structures lift-off easily from the wafer	Increase exposure time (step 9). This could also be caused by improper wafer cleaning (if an oxide layer is present, it may need to be removed)
50	the flow cell leaks	Check tubing (step 20). Use a new PDMS block and/or clean the bottom of the block (channel side) using Magic Tape. Apply Dow Corning RTV 3140 coating to where the tygon tubing meets the PDSM block, as this helps distribute stress caused by moving the tubing. Try not to move the tubing much. Don't use high flow rates.
49–61	air bubbles are sucked into the flow cell	Degas the PDMS and the solutions. Apply Dow Corning RTV 3140 coating to where the tygon tubing meets the PDSM block. Use a slower flow rate and/or larger flow channel dimensions.
53	no fluorescence recovery after photobleaching	The SBL may simply be too dim. Increase the NBD fraction, laser intensity, EM gain, and/or the camera exposure time. After ascertaining lack of recovery is not due to a technical imaging issue, check for leaks, or bubbles. If vesicles are adsorbed but not burst, introducing 2–5 mM free Mg2+ and/or 2–10 times diluted buffer may help bursting them. If none of these seem to help, clean a new set of coverslips and restart.
58–61	too much fluorescence background	Lower the v-SUV density and/or the LR-PE density in the v-SUVs. Bleach the background faster using higher laser intensity (but check the effect on the fusion rate and the docking-to-fusion delays). Increase the temperature. Use better filters.