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Abstract BackgroundTo address the limitations of large-scale high quality microscopy image acquisition, PSSR (Point-Scanning Super-Resolution) was introduced to enhance easily acquired low quality microscopy data to a higher quality using deep learning-based methods. However, while PSSR was released as open-source, it was difficult for users to implement into their workflows due to an outdated codebase, limiting its usage by prospective users. Additionally, while the data enhancements provided by PSSR were significant, there was still potential for further improvement. MethodsTo overcome this, we introduce PSSR2, a redesigned implementation of PSSR workflows and methods built to put state-of-the-art technology into the hands of the general microscopy and biology research community. PSSR2 enables user-friendly implementation of super-resolution workflows for simultaneous super-resolution and denoising of undersampled microscopy data, especially through its integrated Command Line Interface and Napari plugin. PSSR2 improves and expands upon previously established PSSR algorithms, mainly through improvements in the semi-synthetic data generation (“crappification”) and training processes. ResultsIn benchmarking PSSR2 on a test dataset of paired high and low resolution electron microscopy images, PSSR2 super-resolves high-resolution images from low-resolution images to a significantly higher accuracy than PSSR. The super-resolved images are also more visually representative of real-world high-resolution images. DiscussionThe improvements in PSSR2, in providing higher quality images, should improve the performance of downstream analyses. We note that for accurate super-resolution, PSSR2 models should only be applied to super-resolve data sufficiently similar to training data and should be validated against real-world ground truth data.