AbstractBackgroundThe comet or single‐cell gel electrophoresis assay is a sensitive method for the detection of DNA damage. The main drawback of comet sampling is the low cell density necessary to prevent nucleus overlap after electrophoresis, which limits large‐scale high throughput screening. Another problem may be inconsistent comet focusing. We investigated whether an approach based on three‐dimensional (3D) confocal microscopy might be beneficial for these concerns.MethodsA vertical comet assay enabling three‐dimensional confocal comet imaging of nuclei seeded at very high density was developed together with dedicated software algorithms to retrieve quantitative data at the single cell level.ResultsThree‐dimensional confocal comet imaging greatly relieved the user interactions of our nonautomated two‐dimensional comet sampling procedure. Batches of comets were blindly sampled, and confocal sectioning improved the clarity of the images and the accuracy of comet sampling. A 1‐Gy dose response was readily established. The sampling speed was competitive with that of commercial packages.ConclusionsVertical comet imaging is a new concept for fast and user‐friendly comet sampling that allows miniaturization of the assay. It may become an essential step toward high throughput screening and exploit the benefits of confocal imaging. Cytometry Part A 51A:26–34, 2003. © 2002 Wiley‐Liss, Inc.