Imaging Fluorescence Correlation Spectroscopy for the Investigation of Cell Membrane Organization
by
Prof.Thorsten Wohland(National University of Singapore)
→
Europe/Stockholm
FA32
FA32
Description
Fluorescence Correlation Spectroscopy (FCS) is a powerful tool to study molecular processes over a wide range of times (nanoseconds-seconds) with single molecule sensitivity. However, FCS is performed mostly in a confocal mode preventing large scale multiplexing. By using total internal reflection (TIR) or single plane illumination microscopy (SPIM) as illumination schemes and using fast and sensitive EMCCD or sCMOS cameras as detectors we can record thousands of FCS curves simultaneously with diffraction limited spatial resolution and a temporal resolution down to 0.5 ms. This is sufficient to study the movement of lipids and proteins within lipid bilayers and cell membranes and provides unique advantages of this technique compared to confocal schemes. First, a whole cell membrane can be measured simultaneously giving access to all parts of a cell at the same time; second, the large number of measurements provides excellent statistics; third imaging FCS provides spatial and temporal resolution and thus gives access to membrane organization and dynamics; and fourth, the illumination schemes inflict much less photodamage on the sample compared to confocal techniques, thus providing more measurements per sample.
In this seminar we will first cover basic aspects of imaging FCS and its different implementations before applying imaging TIR-FCS (ITIR-FCS) to the study of the dynamics and organization of live cell membranes. For that purpose we label SHSY5Y neuroblastoma cells with markers for the liquid-ordered phase (sphingolipid binding domain, SBD) or liquid-disordered phase (DiIC18) and observe membrane dynamics and organization via ITIR-FCS. By using a range of membrane composition influencing agents (methyl-b-cyclodextrin, latrunculin A, NB-DNJ, fumonisin B, sphingomyelinase) we can observe changes in membrane dynamics and organization during treatment. Our measurement show that there exist at least two different, ceramide and sphingolipid dependent, domain types on the plasma membrane of neuroblastoma cells, and that membrane fluidity and organization are not necessarily correlated and exist over different length scales.
