This tutorial shows step-by-step, how to calculate a spectral fluorescence lifetime cross-correlation (FLCCS) curve from a measurement of a mixture of two labeled oligunucleotides moving independently from each other. As the green dye has been excited with pulsed 485 nm excitation, while the red dye has been excited with a 560 nm cw laser, the different excitation scheme is used to eliminate the unwanted spectral crosstalk.
Note: 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 differ in their absorption and emission properties but they can also vary in their fluorescence lifetime.
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 macromolecule labeled with both fluorophores.
FCCS calibration dyes are available e.g. from IBA (http://www.iba-lifesciences.com/details/product/5-0000-604.html).
Usually, a positive control is needed, in which both dyes are moving in a coordinated fashion.
Additionally, a negative control is need, if the spectral crosstalk is not negligible.
If one dye is excited with a pulsed and the other with a cw laser, fluorescence lifetime cross-correlation (FLCCS) is an ideal tool to remove the unwanted spectral crosstalk as demonstrated in this tutorial. As a consequence, only the positive calibration probe is necessary. For details please refer to: Padila-Parra et al., Microsc. Res. Tech. 2011, 74, 788-93.
The main idea of the method stems from the fact that if the fluorescence signal from the dye excited with a pulsed laser leaks into the second spectral channel, it will also show a decay behaviour corresponding to its fluorescence lifetime. If the second dye is excited with a continuous wave laser, the fluorescence response in the TCSPC window will be a homogenous background similar to roomlight.
Therefore, it is possible to generate two patterns: one for the fluorescence response after pulsed excitation (e.g. for the green fluorescent dye) and one for the fluorescence response after cw excitation.
After these TCSPC patterns have been generated, the green spectral channel using the pulsed response pattern is cross-correlation with the red spectral channel using the cw-response pattern.
Note: This way you can compare the data from the results originating from a standard spectral crosscorrelation and a correlation that involves the fluorescence lifetime information. As the fluorescence lifetime information is used to remove a false positive contribution due to spectral crosstalk, cross correlation calculated in a purely spectral fashion is always higher, if a significant amount of spectral bleedthrough is present.
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.
IBA488+IBA547_unlinked_mix.ptuby a single mouse click.
Note: This file contains a FCS measurement of a FCCS negative control (two DNA oligonucleotides labeled with a green and a red fluorophore, respectively). The sample was excited with a pulsed 485 nm laser and a cw 560 nm laser.
Note: The window contains three different regions:
Note: This window consists of different sections:
IBA488_IBA547_unlinked_mix.ptu(the same file again) and click “OK”.
IBA488+IBA547_unlinked_mix.ptu) is active.
Note: It has been demonstrated, that false positive cross-correlation due to spectral crosstalk can be completely removed using FLCCS. This effect is also visible in a positive control, but as here the main part of the correlation is due to coordinated movement, the amplitude is only slightly decreased compared to a standard spectral cross-correlation analysis.
Response: The correlation curves of this file are calculated. The cross-correlation amplitude is significantly higher than for the negative calibration dye as processed before, but lower than the corresponding FCCS file obtained with the “Grouped FCS” script.