It is a non-trivial task to solve crystal structures of short-lived transient intermediates in biological reactions. In this project, we propose to develop a new technology termed Time Resolved Serial X-ray Crystallography (TRSXC) by using a concentric Microfluidic Electrokinetic Sample Holder (coMESH) to mix a suspension of microcrystals of a target enzyme with a second solution containing its substrate to generate intermediates, which will be injected into a synchrotron X-ray beam for structural determination. By controlling the size of the aging chamber in the coMESH, a series of structural snapshots of reaction intermediates can be taken on the millisecond to the seconds time scale. They can be combined to generate molecular movies of biological reactions with atomic resolution. The TRSXC system will be integrated with a microscopic absorption spectrometer for in situ identification of the chemical nature of the intermediates. A spectroscopically well-characterize O2 reduction reaction of bovine Cytochrome c Oxidase (bCcO) will be used to test the utility of TRSXC in structural determination of enzyme intermediates. Three specific Aims will be carried out to develop this new technology. (1) We will determine the coMESH mixing performance by using three well-characterized solution reactions as test models. (2) We will optimize conditions for structural determination of bCcO with the coMESH. (3) We will demonstrate the utility of coMESH-based TRSXC using the bCcO reaction as a test model. Upon completion of the project this new technology will be made available to scientific community for structural determination of transient intermediates of biological reactions.