In the earliest years of microscopy, it was well understood that visualization of the finest details was often limited by the ‘intrusion’ of light from out-of-focus planes. For many decades the only solution to this dilemma was to limit microscopic studies to very thin samples or to physically squash or slice thicker samples. More recently, biomedical research has driven the need for microscopes that can resolve very fine detail in 3 dimensions within intact, and often living, specimens. The use of fluorescence labeling further exacerbates the problem of out-of-focus light because signal is generated throughout the volume of the sample. The following article describes the most prevalent techniques for 3D imaging and important optical considerations for achieving highest image quality.