This course provides a comprehensive introduction to the principles and practical applications of light and fluorescence microscopy in biological research. Students will gain both theoretical knowledge and hands-on understanding of modern imaging techniques, with a strong focus on their use in contemporary experimental workflows.
A key feature of the course is the unique opportunity to learn and understand how advanced microscopy methods are applied in real research settings. Emphasis is placed on experimental design, image acquisition, and data interpretation, enabling students to understand the basic concepts, critically evaluate and effectively use fluorescence-based imaging approaches in their own scientific work.
• Introduction, discussing course requirements (not counted for mandatory attendance)
• Milestones in the development of microscopy techniques. Optical train in the microscope: advantages and drawbacks of the inverse and upright systems. Image formation in the microscope, numerical aperture and resolution.
• Image formation in the microscope, numerical aperture and resolution. The point spread function. The condensor and the correct Köhler illumination. Type and choice of objectives. Enhancing contrast in optical microscopy: phase contrast and DIC imaging. Practical usage and limitations in bright field microscopy.
• Basic concepts in fluorescence microscopy. Main characteristics of fluorochromes and fluorescent proteins. Optical highlighter fluorescent proteins: photoactivation, photoconversion and photoswitching.
• Optical elements of a fluorescent microscope: filters, fluorescent light sources. Choosing the right filters: crosstalk and bleed-through in fluorescent microscopy. Spectral imaging and linear unmixing.
• Optical sectioning microscopy I. Deconvolution. Structured illumination microscopy (SIM). Principles of confocal microscopy.
• Optical sectioning microscopy II. Point scanning and spinning disc confocal microscopy. Two- and multiphoton microscopy. Light sheet microscopy.
• Fluorescence live cell imaging microscopy. Technical and practical considerations and limitations. Optimizing signal-to-noise ratio in live-cell imaging. FRET microscopy: investigation of protein-protein interactions.
• Use and importance of fluorescent biosensors (Ca imaging, pH and ion-selective measurements). Photoactivation and photobleaching: FRAP, FLIM: use of optical highlighters in microscopy. Live cell applications and interpretation of the data.
• Superresolution in fluorescence microscopy – the basic concepts. SR-SIM, STED, STORM and PALM microscopy.
At the end of the course, the learner will be able to understand and apply the fundamental principles of light and fluorescence microscopy, and critically evaluate the capabilities and limitations of major imaging modalities used in modern biological research. The learner will be able to select appropriate microscopy techniques for specific experimental questions, interpret fluorescence-based imaging data, and recognize the practical considerations involved in live-cell imaging and advanced super-resolution approaches.
A background in biology, such as a BSc in Biology or a related field, is required to engage fully with the course material, but prior knowledge in microscopy or physics is not mandatory. This course is especially recommended for students interested in the application of light and fluorescence microscopy techniques via understanding their core principles.
The course is designed to provide a practical understanding of light microscopy methods necessary for designing and understanding their applications. Online tutorial videos, lecture materials and the interactive gGroup discussions support active learning and promote a deeper conceptual understanding of the material.
• online tutorial sites (see the links given in the presentations) like iBiology
• recommended textbook: Sanderson: Understanding light microscopy, 2019, Wiley
• lecture presentations, including homepage links
The course integrates lectures with interactive group activities to support a thorough understanding of the molecular and cellular aspects of how neurons function and adapt to the environment. Each session combines passive and active learning in a structured approach:
1. Pre-Lecture Preparation: Students are asked to review handouts and recommended readings beforehand, enabling more engaged and effective participation. Discussion topics are provided in advance to support individual preparation as well as group discussions.
2. Lectures: Core concepts are delivered through hybrid lectures (to be recorded in Teams). Key topics are introduced in group discussions (see the next point) and then synthesized in a lecture overview, guided by the instructor.
3. Group Discussions in Breakout Rooms: Collaborative discussions on Teams allow students to engage with peers, ask questions, and deepen understanding, structured to encourage active participation from all.
4. Supplemental Online Resources: Textbooks, lecture recordings, and additional resources support learning outside of class and prepare students for the oral exam.
This challenge-based, student-centered, and technology-enhanced approach ensures students gain both theoretical knowledge and practical insights, preparing them for advanced study and research. Group discussions foster transversal skills and promote intercultural learning. An inclusive environment is emphasized through equitable participation, support materials for diverse learning styles, and instructor openness to feedback, creating a welcoming space where all students can benefit from their active participation.
Transcript of records