Course catalogue doctoral education - HT17

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Title Functional Fluorescence Microscopy Imaging (fFMI) in biomedical research
Course number 2348
Program Allergi, immunologi och inflammation (Aii)
Language English
Credits 3.0
Date 2017-11-27 -- 2017-12-08
Responsible KI department Institutionen för klinisk neurovetenskap
Specific entry requirements
Purpose of the course This course is on advanced fluorescence microscopy imaging and spectroscopy techniques for quantitative characterization of molecular transport and interactions in cells. The purpose of the course is to give an introduction of the underlying physicochemical principles, hands-on experience and an overview of applications of these specialized techniques in biomedical research. The course is suitable for doctoral students lacking training in mathematics, physics, or optical engineering but want to apply these techniques in their research.
Learning outcomes At the end of the course the student will have hands-on experience with live cell imaging and specialized fluorescence microscopy and spectroscopy techniques and is expected to be able to:
1. Use fundamental aspects of molecular structure to describe light-matter interactions and the emission of fluorescence; use this knowledge to discuss fluorescent properties of a fluorophore.
2. Understand the buildup of fluorescence imaging instrumentation, identify different optical elements and describe their function.
3. Describe the theoretical background behind specialized fluorescence based methodologies for studying molecular interactions in live cells. Discuss pros and cons in relation to the biological problem studied.
4. Specify instrumental requirements and design a fluorescence imaging assay for a biological problem of interest.
5. Apply a specific labeling strategy and perform a fluorescence imaging assay.
6. Communicate the results in written and oral form.
7. Discuss the adequateness of the methodology used in the scientific literature concerned.
Contents of the course Fluorescence microscopy and associated techniques are indispensable research tools for investigating molecular mechanisms of biological processes. Versatility of fluorescence microscopy based techniques comes from the possibility to characterize fluorescence emission by spatial position, intensity, wavelength, lifetime and polarization. In addition, fluorescence microscopy and spectroscopy based techniques allow us to quantitatively study the cellular dynamics of molecules and the kinetics of their interaction with high spatio-temporal resolution and ultimate, single-molecule sensitivity. These techniques bring new biological insight at an unprecedented rate and are of crucial importance for the development of life sciences. The course covers the following topics: Luminescence and the nature of light (Fluorescence, Phosphorescence, Light scattering); Fluorescent markers and their photo-physical properties (Organic fluorescent dyes for covalent conjugation (Rhodamine 6G, Alexa dyes, Cyanine dyes); Quantum dots; Intrinsically Fluorescent Proteins (Aequorea victoria (GFP, YFP), Discosoma coral (DsRFP) and Montipora (Keima) families); Selectively binding dyes (DiI, DraQ 5). Instrumentation for Confocal Laser Scanning Microscopy (CLSM): Light sources, Optical Elements, Objectives, Detectors, Read-out devices); Quantization and Sensitivity in fluorescence imaging (Instrumental sensitivity, Method sensitivity, Absolute sensitivity); Factors affecting quantitative accuracy. Point Spread Function; Spatially resolved fluorescence imaging: Multi-photon excitation, Total Internal Reflection Fluorescence (TIRF) Microscopy, Super-resolution techniques (STORM, PALM and STED). Fluorescence based methods for studying molecular diffusion and interactions in live cells (FRAP, FRET, FLIM, FCS, FCCS, RICS). Image analysis techniques for quantitative characterization of cell phenotypes (CellProfiler).
Teaching and learning activities The course includes lectures, laboratory training, demonstrations, discussion sessions and short written assignments.
Compulsory elements All sessions are compulsory. Please report any absence to the course leader in advance by e-mail. Absence from any part of the course (lectures, laboratory sessions, discussion sessions and exam) is generally not accepted but could in special cases be compensated by an individually tailored additional module and a special written examination organized by the course committee.
Examination The final assignment consists of a written project report (5 pages) and an oral presentation of the project report (15 min).
Literature and other teaching material Recommended literature: Selected chapters from: Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, 2006. Pawley, James (Ed.) Handbook of Biological Confocal Microscopy, Springer, 3rd edition, 2006. On-line virtual microscopy interactive tutorials: http://www.olympusconfocal.com/java/index.html https://www.microscopyu.com/tutorials http://zeiss-campus.magnet.fsu.edu/tutorials/index.html http://bitesizebio.com/category/technical-channels/microscopy-imaging/ Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross-Correlation Spectroscopy (FCCS) http://fcsxpert.com/classroom/ Handouts: Scientific papers with related methodology.
Number of students 8 - 12
Selection of students Priority will be given to applicants who can concisely (max 250 words) show in their application an urgent need to begin their independent work with these techniques.
More information This is a two weeks course with 10 sessions that include: lectures, laboratory practice, written assignments, discussions, and time for self-study. The first week focuses on underlying physicochemical concepts, instrumentation and hands on experience. Specialized techniques are introduced and the details are discussed in the context of the broader body of available techniques. The second week is dedicated to expert lectures and advanced applications. The last session is reserved for assessment. Experimental exercises are carried out in the laboratory for Functional Fluorescence Microscopy Imaging (fFMI) at the Center for Molecular Medicine (CMM), Solna, L5:02, 020a. Lectures are conducted in the lecturing hall at the Center for Molecular Medicine (CMM), Solna, L8:01, 021.
Course directors
Earlier evaluation of the course Evaluation report
Course responsible Vladana Vukojevic
Institutionen för klinisk neurovetenskap
51771797
Vladana.Vukojevic@ki.se

CMM L8:01

17176
Stockholm
Contact person -