openFrame: A modular, sustainable, open microscopy platform with single-shot, dual-axis optical autofocus module providing high precision and long range of operation.

"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 2-step autofocus mode which we have shown can operate with defocus up to ±68 μm. We have used this to implement automated single molecule localization microscopy (SMLM) in a relatively low cost openFrame-based instrument using multimode diode lasers for excitation and cooled CMOS cameras. This article is protected by copyright. All rights reserved.