Applications of fluorescence microscopy in biology range from macromolecular to multicellular, with living specimens in particular placing severe constraints on the resolution, precision, speed, and optical efficiency of the image-forming instrumentation. In most cases, a high-resolution optical system will produce images in which the axial position of a feature in the specimen is encoded through sharpness of focus, leading to inherently 3D data. Even in the case where quantitative analysis of only one particular plane of focus within the specimen is to be made, fluorescence from that stratum must be isolated from out-of-focus contributions. At the present time, (1) confocal scanning and (2) direct imaging used with computational deconvolution are the main methods by which object structure is extracted from serial-focus fluorescence image sets. In addition, fluorescence microscopes utilizing interferometric illumination, multi-photon excitation, composite apertures and image interferometry have been introduced specifically to improve 3D resolution. In this report, we look specifically at the constraints imposed by biological specimens on instrumentation used for 3D fluorescence imaging.