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We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. The device is a 150 μm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover that has a similar refractive index (1.34) to water (1.33), making it compatible with top-down imaging used in light sheet microscopy. We provide a detailed fabrication protocol and characterize the optical performance of the device. We demonstrate that the device supports long-term imaging of cell growth and differentiation as well as the rapid addition and removal of reagents while simultaneously maintaining sterile culture conditions by physically isolating the sample from the dipping lenses used for imaging. Finally, we demonstrate that the device can be used for super-resolution imaging using lattice light sheet structured illumination microscopy (LLS-SIM) and DNA PAINT. We anticipate that FEP-based microfluidics, as shown here, will be broadly useful to researchers using light sheet microscopy due to the ability to switch reagents, image weakly adherent cells, maintain sterility, and physically isolate the specimen from the optics of the instruments.