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The use of confocal and multiphoton microscopy for in vivo studies in animals continues to be an area of exciting technical and commercial development. However, the application of these technologies at high resolution, such that molecular and subcellular information is collected, remains an elusive goal. This review discusses the practical and performance limitations and the potential uses of currently available systems. We also highlight the ongoing developments in both miniaturized and bench-mounted systems for single and multiphoton optical sectioning studies in animals and in human clinical trials.