| Phospholipid vesicles, or liposomes, are used in a wide variety of investigations and applications. As biomimetic models for living cells, small reaction vessels, and drug delivery vehicles, these self-assembled structures are increasingly studied and exploited.;Controlling and analyzing phospholipid vesicle structure is critical in many liposome applications, and extrusion of hydrated lipid suspensions is often employed to produce uniform populations of vesicles. A quantitative fluorescence microscopy method is developed for the analysis of size, lamellarity, and structure of vesicles. In contrast to methods for bulk analysis, microscopy provides information about individual vesicles that is not averaged, and heterogeneities in vesicle populations can be characterized.;Encapsulation of molecules within vesicles has been the objective of many studies and applications, and quantifying small quantities or even single target molecules enclosed with vesicles remains a challenge. Herein, a fluorescence imaging experiment is used to determine quantitatively the number of encapsulated fluors and the encapsulation results are interpreted using Poisson statistics.;Although many single molecule studies report quantification, binding or tracking target molecules, a method is needed to permit identification as well. A spectral imaging method is developed to obtain spectral information on immobile, fluorescent labels using a simple modification of a wide-field fluorescence microscope by including a diffraction grating. The Bragg equation is used to convert position and intensity in an image to pure component spectra. Principal components analysis is employed to unambiguously assign the identity of labeled polystyrene nanospheres and single fluorescent molecules.;The spectral imaging experiment is applied to monitoring the internal pH within gradient-loaded liposomes. Encapsulation of a small number (∼50,000) of pH-responsive fluorescent probe molecules and measurement of fluorescence emission spectral changes of the indicator allow the determination of the local pH. The spectra are analyzed as the pH of the interior solution of the vesicle is titrated. |
