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'openFrame' is a modular, low‐cost, open‐hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open‐source and proprietary hardware components or software enables low‐cost, yet research‐grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long‐term time‐lapse image data acquisition, slide‐scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single‐shot closed‐loop focus lock, which we have demonstrated to accommodate defocus up to ±37 μm with <200 nm accuracy, and a two‐step autofocus mode which we have shown can operate with defocus up to ±68 μm. We have used this to implement automated single molecule localisation microscopy (SMLM) in a relatively low‐cost openFrame‐based instrument using multimode diode lasers for excitation and cooled CMOS cameras. LAY DESCRIPTION: We present a modular, low‐cost, microscopy platform called 'openFrame' that can be configured or adapted to most light microscopy techniques and is easily upgradeable. openFrame‐based microscopes are designed to be used with open‐source software tools and low‐cost/self‐built hardware components, such that cost‐effective instruments can be assembled and maintained locally by skilled users instead of requiring visits from service engineers. The core openFrame hardware components can be fabricated using freely shared CAD files or they can be purchased by users to implement a wide range of instruments, which can utilise a mixture of open and proprietary hardware components as desired. We intend this platform to widen access to basic and advanced microscopy techniques, including in lower‐resource settings. We also expect it to be useful to researchers prototyping new microscopes, since it allows a core instrument to be rapidly assembled and then easily modified with minimal constraints, for example, from proprietary software or legacy design choices. The flexibility to reconfigure openFrame‐based microscopes and interchange/recycle components between different instruments reduces obsolescence. The openFrame‐based microscope presented here includes a novel open‐source optical autofocus unit (openAF) that may be useful for long‐term time‐lapse imaging, and in slide‐scanning or automated multiwell plate microscopes. We demonstrate the use of an openFrame‐based instrument for super‐resolved microscopy using single molecule localisation microscopy (SMLM) utilising low‐cost cooled CMOS cameras and multimode diode lasers for high power excitation. [ABSTRACT FROM AUTHOR]