LCAM Functional Imaging (annual)

Title: Functional Imaginglogo_LCAM
Date: October (annual)
Location: FNWI, AMC & NKI
Contact: Mark Hink
Previous editions: –



The general aim of this course is to get hands-on experience and theoretical background in performing state-of-the-art advanced microscopy applied to cell biology. Due to the revolution in genetic encoded labeling (GFPs, switchable probes, FlAsH and AGT-tagging, etc) and the revolution in advanced (light) microscopy (confocal, super-resolution, FRET, dynamics etc) it is now not only possible to image the location of fluorescently labeled molecules but with the functional imaging techniques one can quantify the dynamics of molecular processes inside cells with unprecedented resolution, speed and accuracy. Because the application of advanced microscopy is essential for modern research in (clinical) cell biology, this course provides training in the diverse modes of advanced microscopy. The microscope setups on which the training is provided are part of the van Leeuwenhoek Centre for Advanced Microscopy (LCAM) in Amsterdam, in which the research groups of Molecular Cytology (Swammerdam Institute for Life Sciences, UvA), of Cell Biology and Histology of the Academic Medical Center (AMC) and the Cellular Biophysics Group, Dept of Cell Biology Netherlands Cancer Institute (NKI) .



Course content:

This 9 day course is an intermediate level course for PhD students, postdocs and technicians in biology and biomedical science who already have experience with widefield and/or confocal fluorescence microscopy but want to extend their knowledge with the functional imaging techniques. After this course, participants will understand the principles of these methods and their limitations. In this course we will focus on functional imaging technologies that can be used in biology such as FRET, FLIM, FRAP, FCS & super-resolution microscopy. Furthermore, participants will be introduced to the latest developments in fluorescent probe & biosensor technology, sample preparation, data-analysis and data statistics. The course consists of theoretical lectures & tutorials (~50%) and hands-on experiments (~50%) at the microscope, including an image analysis session. This course runs in parallel with the 4 weeks Advanced Microscopy course for UvA master students. The teaching staff consists of experts in the field from the van Leeuwenhoek Centre of Advanced Microscopy. Participants are encouraged to discuss their specific microscopy issues or future experiments during the course.


Day 1: Fluorescent dyes
Sample preparation
Fluorescence Resonance Energy Transfer (FRET) microscopy
Day 2: Biosensors & optogenetics
Sample preparation & Acceptor photobleaching
Day 3: Filter and spectral FRET
Day 4: Fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) & photoactivation microscopy
Day 5: Super-resolution microscopy (STED, SIM, PALM, GS-DIM)
Day 6: Fluorescence lifetime imaging microscopy (FLIM)
Day 7: Fluorescence correlation spectroscopy (FCS)
Data analysis using ImageJ
Day 8: Statistics for microscopy data
Day 9: Electron microscopy and Fluorescence Activated Cell Sorting (FACS)
Project discussion session with lunch


Requirements: Since this is not a basic but intermediate level course the participants should have experience with widefield
and/or confocal microscopy and be familiar with the following theory:

– microscopy: image formation by a lens/magnification/Numerical Aperture/phase contrast/ fluorescence/filter cube
/pinhole/scanning vs camera imaging
– detectors: PMT / CCD camera
– images: pixelation/8-bit, 12-bit/RGB/ LUT/ brightness, contrast
– image analysis: some basic experience with image analysis using Image J, Imaris or similar.


Date: Total 9 working days: Mon Oct 9th – Fri Oct 13th (Week 1) & Mon Oct 16th – Thu Oct 19th (Week 2)
Participants: Minimum 4, Maximum 8 PhD students, technicians and postdocs.
Level: Experienced microscopist  (previous level: Basics in Fluorescence Microscopy, next level: In the Footsteps of van Leeuwenhoek)
Costs: Besides full course attendance, the fee includes coffee breaks, the final lunch session, course materials and the use of the microscopes. Attendees will be responsible for their own travel, lodging and meals.
Application (deadline Aug 22nd):  closed
Selection criteria: Experience and motivation, to be included in application form
Organiser & info: LCAM, Mark Hink (