Advanced Microscopy

Course manual 2020/2021

Course content

Aim:
The general aim of this course is to get hands-on experience and theoretical background in performing state-of-the-art advanced (light) 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 possible to image and 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) collaborate and organize their advanced microscopy infrastructure.

Covered microscope techniques:

• Confocal Laser Scanning Microscopy (CLSM)
• Confocal and widefield Fluorescence Resonance Energy Transfer (FRET) microscopy
• Spinning disk confocal microscopy
• Fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP) & photoacivation microscopy
• Fluorescence lifetime imaging microscopy (FLIM) & FRET-FLIM
• Stochastic and Optical Super-resolution microscopy (PALM, GS-DIM, RCM, SIM & STED)
• Fluorescence correlation spectroscopy (FCS)
• Live cell microscopy
• Total Internal Reflection Fluorescence (TIRF) Microscopy
• Correlated light and Electron Microscopy (CLEM)
• Electron Microscopy and tomography (EM)
• Flow cytometry and cell sorting (FACS)

Course setup:
For each day one microscopy technique is highlighted by giving the basic theoretical background including tutorials (morning) and hands on experience at the microscope (afternoon). The course starts with a theoretical introduction in optical spectroscopy (fluorescence, absorption, anisotropy), microscope optics, fluorescent probes, image processing & data statistics. Participants will learn the basic principles behind the technique, their applications and limitations. During the tutorial, linked to the lecture, the participants will answer theoretical questions to strenghthen their knowledge of the technique. An important part of the tutorial is to analyse, calculate and draw conclusions from experimental data. The participants will be instructed how to analyse microscopy data. The participants will handle the software program ImageJ, write a macro and perform data statistics. These theoretical courses are accompanied by hands-on practicals on advanced microscopy. The participants will together with the instructor prepare samples and handle the microscope. Measurements will be optimized for the technique discussed and the data will be analysed. Participants will answer questions from the practical reader to strengthen their knowledge and learn important aspects related to microscopy experiments in practice.

After three weeks there will be a written exam. In the final week the participants will work in small groups joining a mini-research project, focusing on a specific technique and topic of choice. Dependent of the chosen subject the participants will together with their advisor design and setup experiments (incl. cell culture, transfections, sample preparations). After measuring the samples at the microscope the participants will analyse their results and present the outcome and recommendations at the final day.

Study materials

Syllabus

• Handouts, (digital) papers

Practical training material

• Handouts

Software

• ImageJ variant Fiji (http://fiji.sc/)

• Matlab + statistics toolbox (UvA student licensed)

• Microsoft Excel (or comparable) (Surfspot)

• Adobe Reader

Other

• Participants should bring their laptop for tutorials & computer practicals and pre-install all software packages mentioned above

Objectives

• Participants will learn the basic principles behind the technique, their applications and limitations.
• The participants should be able to design new experiments to answer biological questions and take into account various aspects like cell type, hardware setup, technique of choice, type of dye, negative and positive controls, measurement conditions & methods to analyse the data.
• Participants will be able to interpret microscopy images, calculate related parameters and their statistical relevance and conclude/compare the overall outcome of the experiments

Teaching methods

• Lecture
• Computer lab session/practical training
• Presentation/symposium
• Self-study
• Working independently on e.g. a project or thesis

Lectures, Tutorials, Practicals and Mini-Research Project

Learning activities

Attendance

Requirements of the programme concerning attendance (OER-B):

1. Attendance during practical components exercises is mandatory.

Additional requirements for this course:

Absence needs to be communicated to the course coordinator.

Assessment

The written exam will test all theoretical and practical skills (from lectures, tutorials and practicals) obtained during the first three weeks of the course. The exam will contain 10-12 open questions including subquestions. An example exam is available at Blackboard.

The mini-research project will be graded by the daily supervisor on basis of active participation and skills shown. In addition all course lecturers, instructors and advisors will grade the project presentation at the the final day.

The final score will be 75% written exam grade + 25% mini-research project

Inspection of assessed work

The manner of inspection will be communicated via the digitial learning environment.

Assignments

Mini-research project

• In the last week students will work in groups of 3 to do a mini-research project. Dependent of the chosen subject the participants will together with their advisor design and setup experiments (incl. cell culture, transfections, sample preparations). After measuring the samples at the microscope the participants will analyse their results and present the outcome and recommendations at the final day.

Fraud and plagiarism

The 'Regulations governing fraud and plagiarism for UvA students' applies to this course. This will be monitored carefully. Upon suspicion of fraud or plagiarism the Examinations Board of the programme will be informed. For the 'Regulations governing fraud and plagiarism for UvA students' see: www.student.uva.nl

Course structure

Timetable

The schedule for this course is published on DataNose.

Additional information

Non-BMS-CBAM students should contact the coordinator for the selection procedure, since only a limited number of participants can join the course.

Location: Universiteit van Amsterdam, Science Park Amsterdam (FNWI), UMCA-AMC hospital & NKI.
Maximum: 30
Canvas: Course overview and materials (lecture slides, tutorials, results & test exam)
Language: English
Prior Knowledge: Cell biology (like BMS course Molecular Biology of the Cell)

Contact information

Coordinator

• dr. ir. M.A. Hink