Using ratio imaging to obtain quantitative information from microscope images is a powerful tool that has been used successfully in numerous studies. Although ratio imaging reduces the effects of many parameters that can interfere with accurate measurements, it is not a panacea. In designing a ratio imaging experiment, all of the potential problems discussed in this chapter must be considered. Undoubtedly, other problems that were not discussed can also interfere with accurate and meaningful measurements. Many of the problems discussed here were observed in the authors' laboratories. In our experience there are no standard routines or methods that can foresee every problem before it has been encountered. Good experimental design can minimize problems, but the investigator must continue to be alert. Progress in instrumentation continues to overcome some of the difficulties encountered in ratio imaging. CCD cameras with 12- to 14-bit pixel depth are being used more frequently, and several confocal microscope manufacturers are now also using 12-bit digitization. The dramatic increase in the use of confocal microscopes over the past decade is now causing microscope manufacturers to more critically evaluate the effect of axial chromatic aberration in objectives, and recent designs to minimize this problem are being implemented. Other developments such as the use of AOTFs to attenuate laser lines extend the applicability of ratio imaging. Ratio imaging is clearly applicable to a wide range of cell biological problems beyond its widespread use for measuring ion concentrations. Imaginative but careful use of this technique should continue to provide novel insights into the properties of cells. Copyright 1998, Elsevier Inc. All rights reserved.