Recently, single-particle cryo-electron microscopy emerged as a technique capable of determining protein structures at near-atomic resolution and resolving protein dynamics with a temporal resolution ranging from second to milliseconds. This thesis describes the development of a novel method, microsecond time-resolved cryo-electron microscopy, that extends the time resolution to the microsecond regime. Following thorough characterization, the method is leveraged to produce biological insights by studying the microsecond dynamics of a viral system. Chapter 3 describes the development and demonstrates the experimental feasibility of microsecond time-resolved cryo-electron microscopy. Its extraordinary temporal resolution is achieved by revitrification, the local melting of a cryo-sample with a heating laser and subsequent cooling and vitrification due to rapid heat transfer, within a few microseconds. Notably, this is achieved within the vacuum of an electron microscope. After characterizing the physical processes and their timescales, transient protein configurations induced by electron beam damage are trapped and imaged in a proof-of-principle experiment. Chapter 4 provides a detailed description of the typical phenomena and practicalities encountered during microsecond revitrification experiments using microsecond time-resolved cryo-electron microscopy. Insights into microsecond nucleation and crystallization of water are described and interpreted to form a guiding principle to reproduce the experiments with different experimental equipment. The novel approach is easily scalable to the scope necessary for single-particle cryo-electron microscopy and integrates seamlessly into the conventional cryo-EM workflows. Chapter 5 describes the application of methodology from structural biology to confirm that protein structures and virus structures are conserved during rapid melting and vitrification. Even though samples crystallize before melting, the investigated biomolecules can be resolved to resolutions comparable to conventional cryo-electron microscopy. During these experiments, further qualitative observations regarding revitrified cryo-samples are made. Foremost, revitrified cryo-samples exhibit different beam induced motion, suggesting structural changes in the vitreous ice due to rapid revitrification. Moreover, particles can clump and reshuffle suggesting that the vitreous ice film is trapped in a disequilibrium which opens the possibility to study technical phenomena like preferred orientation. Chapter 6 details the application of microsecond time-resolved cryo-electron microscopy to resolve the dynamics of a biological system with state-of-the-art instruments. Cowpea chlorotic mottle virus particles are exposed to a sudden change in pH triggering conformational changes on the microsecond scale. Transient conformations are trapped and imaged with microsecond time-resolved cryo-electron microscopy elucidating the mechanics of the virus capsid protein detrimental to its life cycle and biology.