The reproducibility of three-dimensional hydrogel surfaces based on polyurethane, poly(vinyl alcohol), and polyacrylamide (HydroGel) with respect to scanning mode (confocal vs nonconfocal), alignment (circular vs irregular), and influence of fluorescent background was investigated. It is demonstrated that, if even probe spots are provided, fluorescence intensities measured at the same photomultiplier tube gain are similar for confocal and nonconfocal scanning modes. Uneven probe spots, however, cause reduced fluorescence with confocal scanners, greater spot-to-spot variation, and higher degrees of intra- and interexperimental variability (%CV among three experiments). By using irregular instead of circular spot alignment, reproducibility (%CV) is improved for good- and bad-quality spots, in the latter case by up to three times as much. In addition, circularity can be used together with the mean-median correlation of pixel intensities as a quality measure.
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