Digital-holographic metrology enables quantitative phase contrast microscopy of reflective and (partially) transparent samples. In this way, new application fields are opened up for nondestructive investigations of technical samples as well as for marker-free and time-resolved analysis of cell biological processes. Studies on long-term biological processes require permanent focus position readjustment to maintain an optimum image quality. Digital holographic microscopy permits subsequent numerical focusing by variation of the propagation distance. Here, the determination of the optimal propagation distance for a sharply focused image is of particular importance. At the Laboratory of Biophysics image definition quantification algorithms were adapted to the requirements of digital holographic microscopy. In order to obtain robust and reliable algorithms, the object-dependent optical absorption properties were taken into consideration. Automatic focus tracking is demonstrated on investigations with digital holographic microscopy on both technical amplitude objects and cytological pure phase objects.© (2007) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.