The article provides an advanced guide to using μManager, an open-source, cross-platform desktop application for controlling various microscope components. μManager supports a wide range of hardware and offers a user-friendly graphical interface and programming interfaces for plugins and scripts. The guide covers several advanced features, including: 1. **High-Content Screening Plugin**: This plugin enables the use of multi-well plates for automated acquisition of large datasets, facilitating high-throughput imaging. 2. **High-Speed Imaging**: μManager is optimized for continuous acquisition from high-speed scientific CMOS cameras, allowing for data rates of up to 100 frames per second. 3. **Slide Explorer**: A plugin for acquiring large areas of the microscope slide beyond the camera's field of view, using a mosaic of individual images. 4. **Localization Microscopy Plugin**: Automates the analysis of images containing single molecule fluorophores, quantum dots, or other localizable particles, providing advanced tools for filtering, inspecting, and manipulating localization data. 5. **Hardware Synchronization**: Enhances the timing of Multi-Dimensional Acquisition (MDA) sequences by leveraging hardware triggers, reducing latency and improving accuracy. 6. **Multiple Cameras**: Supports simultaneous imaging of multiple fluorescence channels using either dichroic mirrors or synchronized cameras, enhancing the field of view and reducing delays. 7. **Phototargeting**: Utilizes light projection devices for photobleaching, photoactivation, and photoswitching, enabling advanced techniques like Fluorescence Recovery After Photobleaching (FRAP). The article also includes detailed setup instructions and troubleshooting tips, emphasizing the importance of proper hardware configuration and eye safety.The article provides an advanced guide to using μManager, an open-source, cross-platform desktop application for controlling various microscope components. μManager supports a wide range of hardware and offers a user-friendly graphical interface and programming interfaces for plugins and scripts. The guide covers several advanced features, including: 1. **High-Content Screening Plugin**: This plugin enables the use of multi-well plates for automated acquisition of large datasets, facilitating high-throughput imaging. 2. **High-Speed Imaging**: μManager is optimized for continuous acquisition from high-speed scientific CMOS cameras, allowing for data rates of up to 100 frames per second. 3. **Slide Explorer**: A plugin for acquiring large areas of the microscope slide beyond the camera's field of view, using a mosaic of individual images. 4. **Localization Microscopy Plugin**: Automates the analysis of images containing single molecule fluorophores, quantum dots, or other localizable particles, providing advanced tools for filtering, inspecting, and manipulating localization data. 5. **Hardware Synchronization**: Enhances the timing of Multi-Dimensional Acquisition (MDA) sequences by leveraging hardware triggers, reducing latency and improving accuracy. 6. **Multiple Cameras**: Supports simultaneous imaging of multiple fluorescence channels using either dichroic mirrors or synchronized cameras, enhancing the field of view and reducing delays. 7. **Phototargeting**: Utilizes light projection devices for photobleaching, photoactivation, and photoswitching, enabling advanced techniques like Fluorescence Recovery After Photobleaching (FRAP). The article also includes detailed setup instructions and troubleshooting tips, emphasizing the importance of proper hardware configuration and eye safety.