Abstract: Structural dynamics of biological macromolecular is crucial to its structure-function relation, hence obtaining high resolution structures of the biomolecules is essential. The structure determination of biomolecules was dominated by x-ray crystallography over last few decades. In contrast, single-particle cryo-electron microscopy was mainly employed for understanding morphology of the biological macromolecules. With the advent of direct electron detectors, cryo-EM has become a popular choice for biomolecular structure determination as it can, (i) obtain near atomic resolution of biomolecules and, (ii) determine multiple structures or conformations co-existing in one sample. The sample preparation for cryo-EM takes several seconds. However, some biological processes are much faster than that time scale, and the ensuing short-lived states of the molecules are difficult to capture. To address this issue, we developed time-resolved (TR) cryo-EM using microfluidic chip to trap short lived intermediates from tens of milliseconds to second time window. In this presentation, I will discuss TR cryo-EM technique along with single particle analysis to visualize transient intermediate structures of translation initiation process during protein synthesis. While translation initiation phase of protein synthesis, a small, or 30S, subunit and a large, or 50S, subunit, join together to form a 70S initiation complex (IC) ribosome with the help of initiation factors. We were able to resolve discrete structural intermediates on the 50S subunit joining and 70S IC formation pathways with a time resolution of tens of milliseconds.