This tutorial shows step-by-step, how to calculate a spectral cross-correlation curve and the corresponding autocorrelation curves of a dye-labeled DNA-oligonucleotide using the Grouped FCS script.
Fluorescence Cross-Correlation Spectroscopy (FCCS) is a method to determine molecular interactions between two molecules labeled with different dyes. Usually, the dyes attached to the two molecules of interest differ in their absorption and emission properties. They can also vary in their fluorescence lifetime, for more information please check the tutorials for Fluorescence Lifetime Correlation Spectroscopy (FLCS).
To quantify the cross-correlation signal and connect it to a binding fraction, a calibration of the effective confocal volumes for each dye and the overlapping confocal volume must be performed.
The calibration can be done using a molecule labeled with both fluorophores.
FCCS calibration dyes are available e.g. from IBA (http://www.iba-lifesciences.com/details/product/5-0000-604.html).
Note: The “Samples” workspace is delivered with the SymPhoTime 64 and on the DVD-ROM and contains example data to show the function of the SymPhoTime 64 data analysis. If you haven't installed it on your computer, copy it from the DVD onto a local drive before going through this tutorial.
Response: The files of the sample workspace are displayed in the workspace panel on the left side of the main window.
IBA488+547_crosslinked.ptuby a single mouse click.
Note: This file contains a FCS measurement of a FCCS positive control, a DNA oligonucleotide labeled with a green and a red fluorophore. The sample was excited with a pulsed 485 nm laser and a cw 560 nm laser.
Response: The Grouped FCS script is applied to the file
IBA488+547_crosslinked .ptu. Thereby, a new Window opens:
Note: The window contains three different regions:
Note: In green, the selected area of the TCSPC window for FCS calculation is marked. It is calculated on the assumption that the sample is excited with pulsed laser light. As for channel 1 (the red detection channel), this is not the case in our sample, we have to select the complete TCSPC window to avoid loosing fluorescence photons.
Note: It can clearly be seen that the cross-correlation is only ~half as high as the autocorrelations. While in autocorrelations, the amplitudes ρ[i] of the autocorrelations correspond to 1/N[i] (= 1 divided by the average number of the molecules in the focus for autocorrelation i=1=Channel A and i=2=Channel B) and are therefore directly linked to the concentrations (C[i]=N[i]/Veff[i]/NA), the relationship is slightly more complex for the cross-correlation: C[1×2]=ρ[1×2]/ρ/ρ/Veff. Ideally, the cross-correlation should match the autocorrelations. Deviations are due to the different effective volumes for both wavelengths and imperfections of the objective, but can also be caused by degraded test samples or too high laser power, which leads to photobleaching of the fluorescent labels.
Response: A result file is created.
Note: While the cross-correlation amplitude is decreased from its ideal value due to optical imperfections, spectral crosstalk (usually from the green into the red detector channel) can cause false positive cross-correlation. To measure this effect, a negative calibration probe, i.e. a mixture of two single labeled compounds needs to be measured. To demonstrate this effect, a measurement file of such a sample is provided in the demo workspace.
The correlation curves of this file are calculated. The cross-correlation amplitude of this file is ~20% of the autocorrelations.
Note: In a sample as measured here by a combination of pulsed and cw excitation, it is possible to remove the false positive cross-correlation, which significantly simplifies the binding analysis. This is explained in the tutorial Using the FLCS script for spectral crosstalk removal via FLCCS.