Have you taken excitation and emission spectra of the fluorescence to conform that they have the expected wavelength maxima? My concern is that you are not detecting fluorescence at all, just the light scattering from the liposomes. When the liposomes are dissolved by the detergent, the light scattering is reduced, causing a decrease in the apparent fluorescence.

I'm guessing that the DPA and Tb3+ concentrations may be too low to see the fluorescence of the complex when the liposomes are broken open. The internal volume of liposomes is a very small fraction of the total volume of the suspension, so when the liposomes are dissolved, the Tb3+ and DPA concentrations are much lower than the internal concentrations.

This assumes that the liposomes actually had any internal volume. Some lipid compositions form unsealed lipid particles that do not retain anything. The solutions you are trying to incorporate can also have an influence on this. Try making liposomes with an inexpensive, water-soluble organic fluorophore like fluorescein or pyranine to see if they actually retain any of it.

Another approach to this experiment is to put a fluorophore inside the liposomes and a quencher outside. When the liposomes leak, the fluorophore is exposed to the quencher and the fluorescence decreases.

Have you taken excitation and emission spectra of the fluorescence to conform that they have the expected wavelength maxima? My concern is that you are not detecting fluorescence at all, just the light scattering from the liposomes. When the liposomes are dissolved by the detergent, the light scattering is reduced, causing a decrease in the apparent fluorescence.

I'm guessing that the DPA and Tb3+ concentrations may be too low to see the fluorescence of the complex when the liposomes are broken open. The internal volume of liposomes is a very small fraction of the total volume of the suspension, so when the liposomes are dissolved, the Tb3+ and DPA concentrations are much lower than the internal concentrations.

This assumes that the liposomes actually had any internal volume. Some lipid compositions form unsealed lipid particles that do not retain anything. The solutions you are trying to incorporate can also have an influence on this. Try making liposomes with an inexpensive, water-soluble organic fluorophore like fluorescein or pyranine to see if they actually retain any of it.

Another approach to this experiment is to put a fluorophore inside the liposomes and a quencher outside. When the liposomes leak, the fluorophore is exposed to the quencher and the fluorescence decreases.

Dear Adam,

Thank you so much for the suggestion. For the concern of excitation and emission spectra, yes they are correct as I am exciting at 290nm and emission maxima comes around 490nm, that is what I am using to measure the emission intensity. The role of scattering does come to play because when I checked different concentration of liposomes, concentrated liposomes showed more decrease in signal after triton addition than for the diluted liposomes. Also, even for liposomes without Tb inside, increase in concentration gives increase in signal proving that there is some scattering.

For idea about using higher concentration of flurophore, I tested different combination of Tb and DPA without liposomes and found that too low and too high concentration shows less signal. I got increase in signal after triton addition at very low concentration of liposome but that signal was too low to see increase in signal after toxic protein addition.

I liked the idea of using other fluorophore and also using quencher outside to find out whether the liposomes formed are good or not.

I hope I can figure out something from that. Thank you so much again for the suggestions.

Hi Ankit, just to expand on Adam's answer is that you could encapsulate quantum dots (QD's) into liposomes. QDs come in many distinct sizes and you could use them to evaluate the sizes of the membrane pores generated by the toxin.