Measurement accuracy in confocal microscopy

Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three-dimensi onal visualization and three-dimensional quantitative analysis. On human ly mphocytes, we measured geometrical features, cell contents in DNA and in cy clin A and CDK1 proteins, localization and colocalization of these two prot eins. Cells were acquired at a vertical sampling step of 0.5 mu m, which gi ves sufficient information about cell labelling. For the purpose of obtaini ng fast and reliable data at a reduced time cost, we examined various possi bilities to simplify acquisition. For example, it might be possible to incr ease the vertical sampling step to 2.0 mu m while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central s ections appeared to give only rough estimations about the whole cells. Fina lly we compared confocal microscopy to conventional two-dimensional epifluo rescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three-dimensional structures and, more especially, localization of proteins .