Basics in fluorescence microscopy

Title: Basics in fluorescence microscopylogo_LCAM
Date: 31 Jan-2 Feb 2018 (annual)
Location: LCAM-FNWI, Sciencepark
Contact: Mark Hink
Previous editions: 1996-2013, 2017-



A basic course for graduate students, postdocs and lab technicians in biology, biophysics and (bio)medicine. It  provides detailed knowledge of the working principles of confocal imaging, with special emphasis on experiment related issues, such as optical aberrations, bleaching, specimen preparation and fluorescent probes. The course integrates theoretical lectures with hands-on experiments and practical experience, focused at cell biological studies. Experts in the field of microscopy will give an overview of “state-of-the-art” imaging techniques in biological research.


Basics in fluorescence microscopy

This 4 day course is a basic course for PhD students, postdocs and technicians in biology and biomedical science who need widefield and/or confocal microscopy for their experiments. It is our aim to teach how these light microscopy techniques can be used in celbiological research. After this course, participants will understand the basic optical principles of the widefield and confocal microscope and its limitations. In this course we will focus on basic technologies that can be used in biology such as multi-color (confocal) microscopy, ratio imaging, co-localization, phototoxicity and how to keep cells alive under the microscope. Furthermore, participants will be introduced to the latest developments in fluorescent probe & biosensor technology, sample preparation and an overview is presented discussing advanced technologies like FRET, FLIM, FRAP, FCS & super-resolution microscopy. The course consists of theoretical lectures (~50%) and hands-on experiments (~50%) via practical work at the microscope and an image analysis session. The teaching staff consists of experts in the field of light microscopy from the van Leeuwenhoek Centre of Advanced Microscopy and other institutes. Participants are encouraged to discuss their specific problems during the course.



– Light, optics, microscopy and contrast
– Confocal principle and the effect of the pinhole
– Aberrations, magnification and resolution
– Fluorescence, spectra and filters
– Microscopy hardware: Lasers, lamps and detectors
– Nyquist sampling theorem
– Image deconvolution and restoration
– Colocalisation and image analysis
– Dyes, fluorescent proteins and biosensors
– Microscopy sample preparation and incubation
– Functional imaging techniques FRET, FLIM, FRAP, FCS & SRM
– Image processing using ImageJ

After the course the participants will have practical experience in the basic operation steps of the confocal scanning and widefield microscope and basic knowledge of advanced functional imaging techniques, the preparation of biological specimen and be able to perform simple image processing steps.


Program (will be updated Nov 2017)

Day 1:
Lecture Microscopy principles
Lecture Confocal microscopy

Lecture Microscopy hardware & laser safety
Practical Confocal microscopy basics


Day 2:
Lecture Live cell microscopy and sample preparation
Lecture Fluorescent dyes, probes and biosensors

Practical Confocal microscopy advanced
Practical Co-localization, Spinning disk & Wide-field ratio imaging, Virtual Microscope/ImageJ session1


Day 3:
Lecture with overview of functional imaging techniques
Practical Co-localization, Spinning disk & Wide-field ratio imaging, Virtual Microscope/ImageJ session2

Practical Co-localization, Spinning disk & Wide-field ratio imaging, Virtual Microscope/ImageJ session3
Practical Co-localization, Spinning disk & Wide-field ratio imaging, Virtual Microscope/ImageJ session4


Day 4:
Lecture Microscopy data analysis
Lecture Microscopy data representation

Practical Advanced image processing using ImageJ  -> laptop required
Final discussion


Participants: Minimum 12, Maximum 16 PhD students, technicians and postdocs.
Level: Starting microscopist  (next level: Functional Imaging course)
Registration: will open in November 2017
Organiser & info: LCAM, Mark Hink