Description

The Advanced Fluorescence Microscopy course provides a comprehensive understanding of both fundamental and advanced principles of fluorescence microscopy, with a strong emphasis on live imaging and cutting-edge technologies. Students will explore basic concepts, including fluorescence spectroscopy, spectral analysis and unmixing, microscope architecture and adjustments, sample preparation, and the selection of appropriate fluorophores, endogenous probes, and biosensors. The course will cover wide-field versus optical sectioning microscopy, with an in-depth examination of confocal microscopy. Advanced microscopy modalities will include super-resolution techniques (SIM, STED, SoRa, STORM) and the latest innovations in confocal super-resolution microscopy. The course will introduce Total Internal Reflection Fluorescence (TIRF) microscopy, with applications in single-molecule studies and live imaging, with a particular focus on protein dynamics in tissues, cells, and small organisms, such as actin cytoskeleton remodeling. Live imaging techniques will include spinning disk microscopy with SoRa super-resolution, light-sheet microscopy, dual-camera systems, multiphoton imaging and high-content imaging. Students will also gain hands-on experience with advanced technologies such as ratiometric imaging (e.g., calcium, pH, NADH), ablation techniques, fluorescence recovery after photobleaching (FRAP), Förster resonance energy transfer (FRET), and fluorescence lifetime imaging microscopy (FLIM)—all taught by experts in their respective fields. Beyond imaging, the course will emphasize proper image analysis and visualization in alignment with current microscopy guidelines to ensure data quality and reproducibility. By the end of the course, students will be equipped to integrate state-of-the-art microscopy techniques into their research, generate hypothesis leveraging these technologies, and produce high-quality microscopy data that adhere to rigor and reproducibility standards.