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Imaging flow cytometry is a powerful tool by virtue of its capability for high-throughput cell analysis. The advent of high-speed optical imaging methods on a microfluidic platform has significantly improved cell throughput and brought many degrees of freedom to instrumentation and applications over the last decade, but it also poses a predicament on microfluidic chips. Specifically, as the throughput increases, the flow speed also increases (currently reaching 10 m/s): consequently, the increased hydrodynamic pressure on the microfluidic chip deforms the wall of the microchannel and produces detrimental effects lead to defocused and blur image. Here, we present a comprehensive study of the effects of flow-induced microfluidic chip wall deformation on imaging flow cytometry. We fabricated three types of microfluidic chips with the same geometry and different degrees of stiffness made of polydimethylsiloxane (PDMS) and glass to investigate material influence on image quality. First, we found the maximum deformation of a PDMS microchannel was >60 μm at a pressure of 0.6 MPa, while no appreciable deformation was identified in a glass microchannel at the same pressure. Second, we found the deviation of lag time that indicating velocity difference of migrating microbeads due to the deformation of the microchannel was 29.3 ms in a PDMS microchannel and 14.9 ms in a glass microchannel. Third, the glass microchannel focused cells into a slightly narrower stream in the X-Y plane and a significantly narrower stream in the Z-axis direction (focusing percentages were increased 30%, 32%, and 5.7% in the glass channel at flow velocities of 0.5, 1.5, and 3 m/s, respectively), and the glass microchannel showed stabler equilibrium positions of focused cells regardless of flow velocity. Finally, we achieved the world's fastest imaging flow cytometry by combining a glass microfluidic device with an optofluidic time-stretch microscopy imaging technique at a flow velocity of 25 m/s. © 2019 International Society for Advancement of Cytometry.