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Over a decade has passed since pioneering diffraction experiments on macromolecular microcrystals were performed at the European Synchrotron Radiation Facility. Since then the measurement of high-quality diffraction data for the purpose of structure determination has been transformed from a specialized experiment into routine practice at dedicated microfocus macromolecular crystallography beamlines at synchrotron facilities. In this article, we review the evolution of microfocus macromolecular crystallography over the past two decades, and explore the instrumentation and methodologies that have evolved to enable structure determination in the face of unfavourable odds. Several case studies are reviewed in depth illustrating the power of microcrystallography and microfocus instruments and their application in answering important biological questions. Challenges and limitations currently facing the community and areas of future development and problems that must be addressed in the future are also discussed.