Auto-tuning and system-independent focus hold system for fluorescence microscopes

Abstract

Single-molecule localization microscopy is a fluorescence microscopy technique that allows for the acquisition of images with resolutions beyond the diffraction limit of light. However, long acquisition times are necessary, during which the investigated specimen may defocus due to various reasons, resulting in corrupted data. To prevent this corruption, so-called focus hold systems are introduced to the microscopy set-ups. These automatically adjust the position of the objective to counteract defocusing motions. A focus hold system can be realized by tracking the position of the specimen along the optical axis with the help of a reflected laser signal. Depending on the deviation from the focal position, the objective is then adjusted accordingly. The goal of this thesis is to design a control software that determines the drift in focus and performs an appropriate objective actuation. Additionally, the software must be compatible with various operating systems and easily adaptable to different microscopy set-ups. To confirm these properties, multiple measurements on two distinct microscopy set-ups were conducted and analysed. The analysis demonstrated that the software was capable of maintaining the focus within the set-up requirements and was adaptable to both set-ups. Furthermore, potential improvements for future adaptations were identified.