Minimal reproduction and calibration of a fluorescence microscope for single-cell imaging

Microscopy is a critical experimental tool in life sciences. However, when attempting to reproduce or scale-up a published method, researchers often meet difficulties in implementing specific protocols, often because they do not have full control over the hardware and software. We illustrate these challenges by demonstrating how we reproduced a single-cell fluorescence experiment from the literature. We describe how we leveraged Open-Source hardware and software tools to adapt the design of the original fluorescence microscope and the choices we made. Our modular approach gives researcher affordable tools to reproduce experimental setups and implement new protocols. It also gives a simple way to develop experiments with a persistent record of their prototype’s evolution, even at the developmental stage. We provide comprehensive guidance on building and calibrating the microscope hardware, implementing automation, and data analysis and storage methods. The highly modular Python software we introduce facilitate customization and reusability beyond microscopy, empowering researchers and engineers to adapt components for other scientific applications.