The physical limitations of biological optical microscopy are well established. However, considerably less attention is paid to the fact that the biological nature of the objects studied imposes additional constraints on microscopic imaging of cells and tissues. Biological systems are, by definition, dynamic. Therefore, any experimental procedure should address the biological and chemical changes during measurement in the studied system. The imaging itself may induce some of such changes, whereas others variations occur independently of microscopic observations. The goal of this short course is to present the factors that limit the accuracy, resolution, and reproducibility of microscopic imaging of biological objects. The discussion will focus on two methods of 3D optical imaging: confocal microscopy and two-photon microscopy. The course will recapitulate the fundamental physical limitations of optical imaging, and reevaluate their meaning in the context of practical biological microscopy. The following subjects will be discussed: influence of photon statistics and instrumental noise on accuracy and resolution, photophysical and biochemical stability of fluorescence labels, photodamage and phototoxicity, autofluorescence, and intrinsic optical properties of biological specimens. The course will also address the important issues of calibration and standardization. The performance of microscopic imaging of biological samples is usually evaluated in qualitative and subjective manner. There is no versatile, widely adopted standard for evaluation of optical microscopes used for biological studies, or for the quality of biological images collected. One of the aims of this short-course is to identify a set of statistical procedures for evaluation of microscope performance in the context of cell studies.