M.Sc. Biochemistry - Module Biophysical Chemistry - Spectroscopic/Biophysical Methods

Background / Contents of the course

In conventional fluorescence microscopy only the information conferred by the fluorescence intensity is exploited. But also other intrinsic properties of a fluorophore such as the fluorescence lifetime can be used to retrieve additional information about the sample. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are too difficult (or impossible) to observe by fluorescence intensity measurements.

Since the lifetime of a fluorophore depends on its local environment it can be used as a sensor for properties of the solvent such as the viscosity and dissolved compounds such as ions or oxygen. By exploiting physical phenomena such as Förster energy transfer (FRET), FLIM can be used to test if molecules are in close vicinity to each other. In this way molecular interactions can be “imaged”.

This course gives a short introduction into the measurement of fluorescence lifetimes by using techniques established in the Biomolecular Photonics group (see the page Fluorescence lifetime measurements for more details). Examples treated in the course are (depending on your interest and our current research projects) fluorescence lifetime-based ion concentration measurements, phosphorescence-based oxygen concentration measurements, time-resolved anisotropy measurements and fluorescence lifetime based FRET measurements. As well mathematical algorithms for analyzing the data will be discussed.

Course overview

The course consists of introductory lectures given at the beginning of the winter term and a practical part taking place in the lab of the Biomolecular Photonics group. The lectures will give a short introduction into modern microscopic methods and into the basics of measuring fluorescence lifetimes. Lectures will be given jointly to participants of this course and students of the M.Sc. Medical photonics programme (see the M.Sc. Medical Photonics website for more details and additional materials). The practical part will be organized upon mutual agreement during one week within the lecture free period. A detailed schedule can be found below.

Prerequisites

To be able to participate in the practical course knowledge about the microscopic techniques employed in the course is essential. Therefore attendance to all lectures marked below as "mandatory" (and, of course, understanding of the topics discussed in the lectures) is a requirement for participating in the practical part of this course.

For analyzing the data basic knowledge in mathematics (especially exponential decay functions) and statistics (Gaussian error distribution) is necessary. Algorithms used to evaluate the data (i.e. curve fitting) will be discussed in one of the lectures. Participants should be able to work with Microsoft Excel (or a comparable spreadsheet program) for loading, analyzing and plotting the data obtained in the course.

All this should not intimidate you. We are happy to teach you all the knowledge necessary to work with a laser scanning fluorescence microscope, to do fluorescence lifetime measurements and analyze the data. However, we expect you to actively participate in the course and to have enough stamina to learn the theory and analyze the data. If you are in search for some course, which is easy to complete, this is definitely not the course you should choose. But, if you are interestested in learning more about modern fluorescence microscopy techniques we are happy to share our knowledge and enthusiasm with you.

Additional material

Copies of the slides used in the lectures can be downloaded from the website of the M.Sc. Medical Photonics programme. Please, follow this link. Manuals for the devices used in the practical course and data obtained in the course can be found on the download page of this module. Students interested in this practical course will receive login and passwords upon request.