| | How does ConfoCor 3 work? | |  |
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| Non-invasive Study of Molecular Interactions |  FCS with ConfoCor 3
 What is FCS?
How does FCS work?
How does ConfoCor 3 work?
What can you do with ConfoCor 3? |
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| ConfoCor 3 in combination with the LSM 510 incorporates a series of improvements for FCS in single living cells. Positioning is done pixel precise with the scanning mirrors, sensitivity is improved by optimized beam path design and cross-correlation is enhanced with the new highly corrected C-Apochromat objectives. | |
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| Confocal optics reduce the probe's volume size | | |
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| FCS relies on the detection of fluctuations in the fluorescencence emission of a particle which provide information on the particle's diffusion properties. An absolute prerequisite for the detection of such fluctuations is a sufficiently small and well defined measuring volume to avoid noise and signal neutralization by too many simultaneously emmiting particles.
In ConfoCor 3, focusing of the laser beam to a diffraction limited point through advanced confocal optics reduces the sample volume element to 1/4 femto liter (smaller than the E.coli cell body). The average occupation of a thus sized element with fluorescent molecules goes down to single particles even at nanomolar concentrations of the sample molecule. | | |
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| Cross Correlation improves Sensitivity | | |
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| FCS monitors the diffusion time of a fluorescent particle and changes therof upon interaction with a non-fluorescent binding partner. As outlined above, ConfoCor 3 exhibits optimized confocal optics that reduce signal contamination by excess free fluorescent ligands. This improvement not withstanding, one limitation has remained to date which is inherent to the technique: FCS monitors changes in the fluorescent particle's mass upon complex formation and the extent of this change is strongly dependent on the relative sizes of the two ligands. This dependency renders FCS analysis of interactions between one small flourescent molecule and a large non-labelled ligand an ardous task. | | |
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| Cross Correlation: Two separate Detection Channels for Improved Analysis of Molecular Interactions | | |
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| ConfoCor 3 overcomes this limitation by incorporating two avalange photo detectors (APDs) allowing for the simultaneous detection of two fluorescence signals. This permits analysis of two interaction partners labelled with different fluorescent dyes. In this setting, the APD-pair now receives a threefold signal from both free ligands and the ligand complex. Thus, in contrast to the classical FCS approach with one fluorescent binding partner, the doubly-labelled complex submits an autonomous fluorescence signal that reaches both APDs.
Clearly, the software used for the processing of the raw experimental data must now cope with an additional task: the discrimination of signals emanating from free ligands versus the complex. The new ConfoCor 3 software performs this so-called cross-correlation leading to signal deconvolution. With other words, ConfoCor 3 no longer necessarily relies on the evaluation of changes in a ligand's diffusion properties: by use of the two-channel principle and the new software, ConfoCor 3 can ignore the single-labelled free binding partners and directly track the appearence of the bimolecular complex providing a signal which is directly proportional to the extent of complex formation.
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