Seminar Series
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Date:
Time: Location: Speaker: Title: |
11 April 2019, Thursday
2-3 pm F2, Firth Court Professor Thorsten Wohland, National University of Singapore Basics and Applications of Fluorescence Correlation Spectroscopy |
Fluctuations in any observable of a system are caused by molecular processes and therefore are an important source of information. If one can find a sensitive observable that can be measured with single molecule sensitivity one can determine the characteristic fluctuations and determine thus the characteristics of the underlying molecular processes. And although single molecule sensitivity is not easy to achieve, fluorescence is one observable in which the first single molecule detection – labelled with many fluorophores – was achieved already in the 1970ies and the first detection of a single fluorophore in a cell was achieved in the 1990ies. Since then many fluorescence fluctuation techniques have been developed. One of the first and most widely used techniques is fluorescence correlation spectroscopy (FCS).
In this seminar I will introduce FCS basics and explain how we nowadays can collect FCS measurements in all pixels of an image, a method called Imaging FCS. For this purpose we have to collect images with a high frame rate of about 1000 frames per second with highly sensitive cameras and with illumination modes that minimize photobleaching and provide a high signal to noise ratio. As illumination modes we therefore use either total internal reflection or light sheet illumination. I will show how Imaging FCS has been calibrated against a wide range of other biophysical techniques, including fluorescence recovery after photobleaching, single particle tracking, or atomic force microscopy, and how structural, organizational, and dynamic information can be extracted simultaneously from image stacks. Finally, applications of FCS and imaging FCS will be discussed on the example of Wnt3, a morphogen responsible for brain development. We show Wnt3 organization and dynamics on cell membranes and in live zebrafish to demonstrate that Wnt3, which carries two lipid modifications, partitions into cholesterol dependent domains in dependence of the lipid modifications, that it nevertheless can be found as a secreted signalling protein in the interstitial spaces in live zebrafish, and that it interacts with receptor proteins on target cells.
In this seminar I will introduce FCS basics and explain how we nowadays can collect FCS measurements in all pixels of an image, a method called Imaging FCS. For this purpose we have to collect images with a high frame rate of about 1000 frames per second with highly sensitive cameras and with illumination modes that minimize photobleaching and provide a high signal to noise ratio. As illumination modes we therefore use either total internal reflection or light sheet illumination. I will show how Imaging FCS has been calibrated against a wide range of other biophysical techniques, including fluorescence recovery after photobleaching, single particle tracking, or atomic force microscopy, and how structural, organizational, and dynamic information can be extracted simultaneously from image stacks. Finally, applications of FCS and imaging FCS will be discussed on the example of Wnt3, a morphogen responsible for brain development. We show Wnt3 organization and dynamics on cell membranes and in live zebrafish to demonstrate that Wnt3, which carries two lipid modifications, partitions into cholesterol dependent domains in dependence of the lipid modifications, that it nevertheless can be found as a secreted signalling protein in the interstitial spaces in live zebrafish, and that it interacts with receptor proteins on target cells.
