In digital pathology, clinical specimen slides are converted into digital images by microscopic image scanners. Since random vibration and mechanical drifting are unavoidable on even high-precision moving stages, the optical depth of field (DOF) of microscopic systems may affect image quality, in particular when using an objective lens with high magnification power. The DOF of a microscopic system was theoretically analyzed and experimentally validated using standard resolution targets under 60× dry and 100× oil objective lenses, respectively. Then cytogenetic samples were imaged at in-focused and off-focused states to analyze the impact of DOF on the acquired image qualities. For the investigated system equipped with the 60× dry and 100× oil objective lenses, the theoretical estimation of the DOF are 0.855 μm and 0.703 μm, and the measured DOF are 3.0 μm and 1.8 μm, respectively. The observation reveals that the chromosomal bands of metaphase cells are distinguishable when images are acquired up to approximately 1.5 μm or 1 μm out of focus using the 60× dry and 100× oil objective lenses, respectively. The results of this investigation provide important designing trade-off parameters to optimize the digital microscopic image scanning systems in the future.