If there is no fluorescein associated with the liposomes after dialysis, then either it never got in during the formation process, or the liposomes are leaky and it all came out again. The composition of the lipids and the surrounding medium and the temperature affect how well the liposomes retain their contents.

Did you prevent fluorescein photobleaching by doing all the preparatory steps in the dark? Did you buffer the fluorescein solution at an alkaline pH to assure deprotonation of the fluorescein?

If the liposomes contain fluorescein but you can't see them by microscopy, are they large enough to resolve and are you being careful not to photobleach the fluorescein too quickly to detect it? To test how well you can visualize the liposomes themselves, try incorporating a fluorescently labeled lipid or fluorescent membrane-binding dye into the liposomes.

I have dissolved the fluorescein in water, as i used the electroformation method for making GUV. What should be pH of the solution for fluorescein? I have used SOY PC and used room temperature for making liposome and using all the prevention to protect photobleaching. When i placed it under confofocal it is showing all green and no liposomes are there?? what could be the reason. 

The pH should be 7 or higher inside the liposomes.

If the external fluorescein was not completely removed by dialysis or later leaked out of the liposomes, the whole field will be fluorescent.

If the liposomes are too small to be resolved by confocal microscopy, then you will see only an unresolved green field. Use a phase contrast microscope to see if the GUVs are there.

You can try adding a quencher to the outside to eliminate the fluorescence of external fluorescein without quenching the fluorescence of internal fluorescein.

Do you mean that the pH of the CF solution which is used for the hydration media should be 7 or greater than that? Should i dissolve the CF in buffer instead of water and maintain the pH at 7 or higher? Can i used tris HCl buffer to dissolve the CF?

Since the solution that is used to rehydrate the lipids ends up being the solution inside the liposomes, then to assure a suitable pH of 7 or higher for ionization of CF you should rehydrate the lipids in a buffer of pH 7 or above. You can use this buffer to prepare the CF solution. You can use Tris-HCl as the buffer. In that case, use buffer with a pH adjusted to somewhere between 7.5 and 8.5. This is because the pKa of Tris is about 8.1 (varies depending on temperature), and to get the highest buffering capacity you should stay close to the pKa of the buffer.

Thanks Adam.. I just have one more question. you told about the addition of quencher to eliminate the external flourescence. I am not getting the concept. How to add this quencher and the how it works? can you please let me know? 

Certain substances will quench the fluorescence of CF on contact. If you are working in small volumes, probably the best one is anti-fluorescein antibody, which is commercially available. The antibody can't get through the liposome membrane, so it can only quench the fluorescence of external CF, leaving only the internal CF fluorescence.

thanks a lot Adam.

Hi Mohana, Do you have idea Phospholipids of  X, Y, and Z molar ratio (X:Y:Z) you have taken to to prepare liposome? Thanks

I have used only one lipid to prepare liposome and it is 5 mg/ml concentration of lipid.

Thanks Mohana. If you clarify me additional one more question, please let me know how much CF you have taken to encapsulate liposome so that i can figure out to help you.

I have used 1mM concentration of CF. I ml of this aqs solution of CF i used for doing the electroformation. Should i need to decrease the conc. of CF? 

1mM is very high. Can you try ranging from 5uM to 25uM. Most of the literature used uM conc. like to 0.5 to 1ml. Thanks

Dear Adam,

please introduce me the flourescent dyes in the  liposome and microemulsion preaparation?

Hi Abbas,

This is a big topic, with much literature on it. Can you be specific about what you want to know about?

Dear Adam,

thanks. my meaning is the flourescent dye that be  soluble in oil?

Hi Abbas,

A good dye to try is TMA-DPH.

http://www.sigmaaldrich.com/catalog/papers/6197175

Dear Adam,

Thanks.

Can I know the concentration of Tris-HCL that was used during 

dialysis to be left overnight for the liposome preperation. Thanks