| Popis: |
To improve our understanding of proteins interactions with cellular membranes, we have developed a method based on confocal microscopy that enable us to correlate the number of proteins bound to a lipid vesicle with the vesicle properties (membrane integrity, membrane potential, vesicle size¼). This method consists in the simultaneous recording of confocal images in two different channels, and in the coincidental detection of single mobile particles in these channels. To demonstrate the usefulness of this method, we have used a model system consisting of small unilamellar liposomes (∼ 100 nm in diameter) rendered fluorescent by the addition of a small percentage of fluorophore and interacting with fluorescently labeled proteins.To optimize image acquisition parameters for the detection of single diffusing liposomes, we systematically varied the fluorophore to vesicle ratio, the fluorescence excitation intensity, and the confocal pixel size and dwell time. We analyzed the images using different pixel binning values and different detection threshold. We then determined the range of parameters for which we were able to detect particles with independent positions. We also verified that the particle concentration and average particle fluorescence emission extracted from the images using this single particle detection method were consistent with results obtained by fluorescence correlation spectroscopy (FCS), raster-scanning image correlation spectroscopy (RICS) and fluorescence intensity distribution analysis (FIDA). Using vesicles labeled with two different fluorophores, we measured the probability that a vesicle detected in one channel would also be detected in the second channel, and compared our results with those obtained by fluorescence cross-correlation spectroscopy (XCS).Our method should be useful for studies of protein or peptide insertion and pore formation into lipid membranes. |
