AI Article Synopsis
- Dual-color Fluorescence Correlation Spectroscopy (FCS) is a technique used to observe protein interactions in live cells by analyzing the fluorescence signals from two different fluorescent proteins.
- The method grapples with the issue of spectral bleed-through, where overlapping emissions interfere with accurate readings, but Fluorescence Lifetime Correlation Spectroscopy (FLCS) provides a solution.
- FLCS allows for better analysis of protein interactions without needing extensive bleed-through calibration, and can be executed using various confocal microscopy setups, simplifying the experimental process.
Article Abstract
Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples.
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| http://dx.doi.org/10.1007/978-1-62703-649-8_31 | DOI Listing |
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