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Advanced optical imaging techniques address important biological questions in neuroscience, where structures such as synapses are below the resolution limit of a conventional microscope. At the same time, microelectrode arrays (MEAs) are indispensable in understanding the language of neurons. Here, the authors show transparent MEAs capable of recording action potentials from neurons and compatible with advanced microscopy. The electrodes are made of the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) and are patterned by optical lithography, ensuring scalable fabrication with good control over device parameters. A thickness of 380 nm ensures low enough impedance and >75% transparency throughout the visible part of the spectrum making them suitable for artefact-free recording in the presence of laser illumination. Using primary neuronal cells, the arrays record single units from multiple nearby sources with a signal-to-noise ratio of 7.7 (17.7 dB). Additionally, it is possible to perform calcium (Ca 2+ ) imaging, a measure of neuronal activity, using the novel transparent electrodes. Different biomarkers are imaged through the electrodes using conventional and super-resolution microscopy (SRM), showing no qualitative differences compared to glass substrates. These transparent MEAs pave the way for harnessing the synergy between the superior temporal resolution of electrophysiology and the selectivity and high spatial resolution of optical imaging., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)