Specialist Course on Light and Fluorescence Microscopy Edition February 2025

Target audience

PhD students with limited experience in light microscopy who (will) make extensive use of microscopic visualization techniques for lifescience research applications (in the near future). No prior experience or training is required.

PhD researchers conducting Life Sciences often get involved in the use of light microscopy: microscopic techniques offer ever increasing possibilities to study biological specimen in great detail and became a basic tool in medical and biological research. Yet, increasing technical possibilities also imply an increasing complexity in performing cutting-edge microscopy based research. A solid understanding of the underlying principles and practices is crucial to obtain the best results. Moreover, as the course is organized within GLiM, the participants become acquainted with the instrumentation available at Ghent University.

Organizing and scientific committee

Prof. Dr. Kevin Braeckmans (Faculty of Pharmaceutical Sciences)

Prof. Dr. Luc Leybaert (Faculty of Medicine and Health Sciences)

Prof. Dr. Alain Labro ( Faculty of Medicine and Health Sciences)

Prof. Dr. Ruslan Dmitriev (Faculty of Medicine and Health Sciences)

Prof. Dr. Andre Skirtach (Faculty of Bioscience Engineering)

Prof. Dr. Els Van Damme (Faculty of Bioscience Engineering)

Prof. Dr. Stefaan De Smedt (Faculty of Pharmaceutical Sciences)

Dr. Deep Punj (Faculty of Pharmaceutical Sciences)

Dr. Irina Okkelman (Faculty of Medicine and Health Sciences)

Dr. Herlinde De Keersmaecker Ghent Light Microscopy CORE)

Geert Meesen (Faculty of Bioscience Engineering)

Abstract

Light microscopy, and especially fluorescence microscopy, is an essential tool to investigate and characterize biological specimen in the life sciences. Modern microscopes offer an increasing range of technical capabilities, but thereby become gradually more complex and require appropriate training for correct scientific use. This specialist course provides doctoral students with limited experience in microscopic imaging with the understanding that is needed to perform light and confocal imaging at a research-grade level.

Objectives

Theoretical lectures offer the participants the background needed to perform light microscopy studies in a correct scientific way. Theory will be put into practice in demonstration sessions. The demonstration sessions, during which different imaging techniques are shown to small groups of 5-6 students, naturally complement the theoretical lectures and are intended to provide insight in the theoretical principles. Using real-world examples, the participants are given practical guidelines on correct data acquisition and obtaining optimal data quality. These sessions form a solid basis for further individual hands-on training offered by the Ghent Light Microscopy (GLiM) CORE. During groupwork sessions, the students discuss the newly obtained insights and apply them to their own research questions. The last half day comprises a guided computer session that introduces fundamentals of image processing. Focus will be on good practice in image processing and methods to extract quantitative data. This session will provide the participants with a basic knowledge allowing to implement image analysis through FIJI in their research.

Dates and venue

Programme

Day 1:

• Day 1: Theoretical session (February 3 2025, 9.30-12.30 a.m.)

Lecture 1: Fundamentals of light and imaging – prof. Kevin Braeckmans

The main physical principles involved in image formation are explained. After reviewing concepts as light refraction, diffraction, resolution, etc., the basics of image formation are addressed. The differences between the types of objective lenses are discussed and attention is drawn to correct sample illumination.

Lecture 2: Transmitted light microscopy and digital imaging – Geert Meesen

The visualization of cells often proves to be difficult. The use of dedicated illumination techniques can enhance the image quality significantly. This lecture furthermore focuses on contrast-enhancing techniques such as DIC and phase contrast microscopy that help to reveal the smallest details in a cell. Concepts as resolution and noise are explored further and  guidelines involving correct camera use and the impact of digital imaging are offered.

• Day 1: Demonstration sessions (September 7 2022, 1.30 – 4.30 p.m.)

Transmitted light microscopy – Dr. Herlinde De Keersmaecker, Geert Meesen, Dr. Deep Punj

In the first demonstration session cell visualization with transmitted light microscopy is covered. The participants will learn in groups of 5 to set the correct sample illumination and explore different transmitted light microscopy techniques. The advantages of different contrast-enhancing techniques will be demonstrated and the principles of digital imaging are put into practice.

Day 2:

• Day 2: Theoretical session (September 8 2022, 9.30 – 14.00 a.m )

Lecture 1: Epi-fluorescence and confocal imaging – prof. Kevin Braeckmans

Fluorescence microscopy allows to image specific parts of a specimen with unrivalled contrast. In this session the basic concepts underlying fluorescence imaging are reviewed. It is explained how, with the use of various confocal techniques, optical slicing and three-dimensional imaging is achieved. Furthermore it is pointed out that the combination of confocal imaging and spectral detection is an excellent way to differentiate between different subunits in a sample. Special attention is drawn to practical considerations that are inherent to confocal microscopy, such as photobleaching and bleed-through.

Lecture 2: Advanced fluorescence microscopy: Strategies to image deeper in the sample, with higher resolution or gather functional information, a focus on two-photon imaging,  STED nanoscopy and Fluorescence Lifetime Imaging: Dr. Deep Punj

In this lecture light is shed on three advanced light microscopy techniques that are based on confocal laser scanning microscopy and are available at Ghent University. The first technique is two photon imaging, where the excitation strategy is shifted to higher wavelengths to allow sectioning as well as deeper penetration into tissue. The second technique is STED nanoscopy, with an adapted excitation scheme which allows to achieve resolutions up to around 40 nm, way beyond the optical resolution limit described by the Rayleigh criterion (~300 nm). The third technique is FLIM, where the fluorophore lifetime is measured instead of the intensity of fluorophore emission allowing investigation of molecular function, interactions, and environment.

Lecture 3: Cell labelling and considerations for live cell imaging – Dr. Herlinde De Keersmaecker

This lecture explores the different properties of fluorescent labels and discusses the practical considerations when used for life science research. Different techniques used to introduce fluorescent dyes into a specific structure of both fixed and living cells are discussed. Attention is given to creating optimal conditions for live cell imaging.

Day 3:

• Day 3: Demonstrations and groupwork session (September 8 2022, 9.30 a.m. – 1.30 p.m.)

Epi- and confocal fluorescence imaging – Dr. Herlinde De Keersmaecker, Geert Meesen, Dr. Deep Punj, Dr. Irina Okkelman

The concepts of epi-fluorescence microscopy, confocal imaging and the three advanced techniques will be shown. Each session will demonstrate and focus on a specific capability and the related caveats of fluorescence imaging methods covered during the theoretical lectures with as goal to develop a correct scientific way of microscopic imaging. In between the demonstration sessions, two moments are foreseen to discuss and apply the new insights to a selection of the own research questions in small groups of 5 students. During the demonstrations time is foreseen to discuss questions from the groupwork session related to the demonstration.

Day 4:

• Day 4: Demonstrations and groupwork session (September 9 2022, 9.30 a.m. – 11.30 a.m.)

Epi- and confocal fluorescence imaging – Dr. Herlinde De Keersmaecker, Geert Meesen, Dr. Deep Punj, Dr. Irina Okkelman The concepts of epi-fluorescence microscopy, confocal imaging and the three advanced techniques will be shown. Each session will demonstrate and focus on a specific capability and the related caveats of fluorescence imaging methods covered during the theoretical lectures with as goal to develop a correct scientific way of microscopic imaging. In between the demonstration sessions, two moments are foreseen to discuss and apply the new insights to a selection of the own research questions in small groups of 5 students. During the demonstrations time is foreseen to discuss questions from the groupwork session related to the demonstration.

• Day 4: Groupwork session (September 9 2022, 11.30 a.m. – 2.30 p.m.)

Each group of 5 students gives a 5-10 min presentation on their problem statement and conclusion followed by a short discussion with all participants.

Day 5:

• Day 5: Guided computer session (September 9 2022, 9.30 a.m.– 12.30 a.m.)

Basics of image processing – Geert Meesen, Dr. Herlinde De Keersmaecker

Using a set of microscopy images, the fundamentals of image processing and quantitative image analysis are explained through a guided computer session. The session provides basic understanding and application of FIJI, an open source image processing platform widely used in biomedical research.

Registration

• Follow this link for the registration and waiting list. Your registration will be confirmed by separate e-mail (outlook invite).
• Cancellation of your registration can only be performed by sending an email to doctoralschools@ugent.be.

• The no show policy applies.

Registration fee

Free of charge for Doctoral School members.

Number of participants

Maximum 30

Language

English

Training method

Theoretical lectures (6 hours), demonstration sessions (7 hours, on campus), PC session (3 hours, on campus), Groupwork (4 hours, on campus)

Evaluation method

After successful participation, the Doctoral School Office will add this course to your curriculum of the Doctoral Training Programme in Oasis. Please note that this can take up to one to two months after completion of the course.