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For both microbubble types, 30 randomly chosen microbubbles were analyzed. In a first step, the center point of the microbubble was manually annotated in one of the xz-slices. Next, the program computed the radial intensity profiles for 100 angles (i.e., at multiples of 2��/100 radians), using the manually selected center as starting point. After this, a circle was fitted through the intensity maxima using a MATLAB routine based on the method as described by Taubin [35], from which the radius (Rfit) and origin (Ofit) of the fluorescent contour within the xz-slice were obtained. All the pixels located within 90?nm from Rfit, defined as the region of interest (ROI), were included in the heterogeneity analysis. The ROI was subdivided https://www.selleckchem.com/products/midostaurin-pkc412.html in 32 angular parts (i.e., each ��/16 radians); for each angular part, the mean fluorescence pixel intensity (Ipart) was calculated. This procedure was repeated for a range of xz-slices spatially distributed around the equatorial plane of the bubble, whereby the origin of the best circle fit (Ofit) within a slice was used as starting center point for the radial intensity https://en.wikipedia.org/wiki/Ketanserin profiles in the adjacent xz-slice. Only xz-slices with a value for Rfit, which was larger than 75% of the value of Rfit in the equatorial plane of that particular bubble were included in the analysis. This was because toward the caps of the microbubbles, the xz-slices had a fluorescence pattern of filled circles, instead of a fluorescent rim surrounding a dark core, which could not be processed with the same contour tracking algorithm. On average, 35 xz-slices were included per bubble, resulting in on average 35?��?32?=?1120 angular parts per bubble, from which the median part intensity per bubble (?median) was calculated. An individual angular part was classified as an inhomogeneity when the absolute difference between the mean fluorescence https://www.selleckchem.com/products/liproxstatin-1.html intensity of this part (Ipart) and the median part intensity of the bubble (?median) was more than two-third of the value of ?median (i.e., |Ipart????median|>2/3?��??median). From this, the percentage of parts classified as an inhomogeneity per microbubble, being a measure for inhomogeneous ligand distribution, was calculated for both microbubble types. Statistical analysis was performed using IBM SPSS Statistics 20. First, Shapiro�CWilk normality tests were performed to determine if the SD and mean were significantly different from a normal distribution. Both distributions were not normally distributed (DSPC: p?=?0.001, DPPC: p