LCAM Functional Imaging (annual)
Title: Functional Imaging
Date: October 2022 (annual)
Location: FNWI, AMC & NKI
Contact: Mark Hink
Previous editions: 2018, 2019, 2021
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 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 University Medical Center Amsterdam (UMCA) and the Cellular Biophysics Group, Dept of Cell Biology Netherlands Cancer Institute (NKI) .
This 8 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 probes, 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. Two special sessions will be dedicated to discuss each participant’s specific microscopy project & issues and data-analysis questions.
|Day 1 :||Fluorescence microscopy & probes|
|Fluorescence Resonance Energy Transfer (FRET) microscopy|
|Day 2||Super-resolution microscopy (STED, PALM & GS-DIM)|
|Day 3||Filter and spectral FRET|
|Day 4||Fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) & photoactivation microscopy|
|Day 5||Project discussion session with each participant|
|Project discussion session 2|
|Weekend||No course elements|
|Day 6||Fluorescence lifetime imaging microscopy (FLIM)|
|Day 7||Fluorescence correlation spectroscopy (FCS)|
|Day 8||Image processing and statistics for microscopy data|
|Customised data analysis session|
Requirements: Since this is not a basic but intermediate level course the participants should have some experience with widefield and/or confocal microscopy and be familiar with the following theory and terms:
|– microscopy:||image formation by a lens/magnification / Numerical Aperture / phase contras t/ fluorescence / filter cube
/ pinhole / scanning vs camera imaging / confocal vs widefield microscopy
|– detectors:||PMT / CCD camera|
|– images & data:||pixels / 8-bit, 12-bit/RGB / LUT / brightness / contrast|
|– image analysis:||basic experience with image analysis using Image J, Imaris or similar.|
Date: Total 8 working days in October 2022 (Dates to be announced, excluding the weekend)
Participants: Minimum 4, Maximum 8 PhD students, technicians and postdocs.
Level: Experienced microscopist
Costs: To be announced. Besides full course attendance, the fee includes course materials, use of the microscopes and the final lunch session. Attendees will be responsible for their own travel, lodging and meals.
Application: Application for the course will be opened late July 2022.
Selection criteria: Experience and motivation, to be included in the application form.
Organiser & info: LCAM, Mark Hink (email@example.com)