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A perfect scratch becomes unusable if the field drifts out of focus, the illumination shifts between time points, or the wound edges are too low-contrast to segment. The biggest scratch assay imaging mistakes aren't dramatic failures, they're small inconsistencies that quietly distort wound area. This guide covers the imaging principles behind reproducible wound healing assays on any microscope: imaging mode, objectives, Köhler illumination, and locking your optics.