Confocal imaging of HAMLET binding to lipid membranes in GUVs.

<p>A. Differential interference contrast images and corresponding fluorescence confocal microscopy images. EYPC lipid vesicle membranes were visualized after application of Alexa-labeled HAMLET (I), HLA (II) or free Alexa 568 dye (III). HAMLET bound to multiple layers of lipid membranes in the multilamellar vesicles. Vesicles after application of Alexa-labeled HLA or free Alexa 568 dye indicated weak fluorescence association. B. Membrane detection of LUVs by fluorescence confocal microscopy, and corresponding intensity profile measurements, using ImageJ software and a median filter radius of 1.0 pixels to process the images before the profile measurements. I & II) Binding of HAMLET to EYPC vesicles; confocal micrograph and corresponding DIC microscopy images. White arrowheads illustrate unilamellar structures. III) Intensity profile measurement across the line shown in I, confirming the strong association of HAMLET to the membrane. IV) Binding of Alexa-labeled HAMLET to soybean phorphoryl choline vesicles. V) Corresponding DIC micrograph. Black arrowheads illustrate unilamellar structures and the black arrow indicates a multilamellar structure. VI) Intensity profile measurement of the line shown in IV. HAMLET bound strongly to soybean vesicle membranes.