To stain the vesicle membrane, add the fluorescent lipid dye FM4-64 to the purified vesicles at a final concentration of two micromolar and incubate for 10 minutes before imaging. After isolating the protein filled vesicles from Escherichia coli culture, pipette the purified vesicles onto a less than one millimeter thin circular 2%LB agarose pad that has been allowed to form and set on a clean glass slide. Once the liquid has dried, place a 50 millimeter by 25 millimeter cover slip over the vesicles on the pad.
Hold the cover slip in place with spacers and adhesive tape. Then mount the slide onto an inverted microscope using an oil immersion objective, and leave the sample for two to three minutes to settle and the temperature to equilibrate. For single frame images, use three image averaging to reduce hardware dependent random background noise.
For time-lapse imaging, allow three to five minutes between individual frames. Widefield fluorescence microscopy imaging showed the purified amnion green containing vesicles. The lipophilic fluorescent dye, FM4-64, confirmed the presence of vesicle membranes.
Structured illumination microscopy of the live bacterial cells expressing the inner membrane protein, CydB, fused to amnion green, and VNp6-mCherry2 showed vesicle production and cargo insertion.
