Part 1 Principles
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
6. fluorescence detecting in confocal microscopy
Cross-talk correction 1: cross-excitation
Cross-talk is a common problem for multiple fluorophore labeling experiment. In a specimen labeled by more than one fluorophores, the interaction between their excitation and emission is complicated. They can be divided into three categories:
1. Cross-excitation: a fluorophore is not just excited by wavelength at its peak value, but also by wavelength at certain range around the peak, which can extends into the area used by other fluorophores. For example
FITC has an excitation peak (100% of relative intensity) at 490 nm, it has range of excitation between 470-540 and is still excited 20% at 546 nm which is the peak value used by TRITC.
General speaking, cross excitation is not harmful in terms of specificity of the emission. Anyway, It does not matter very much by which excitation wavelength the fluorophore is excited, it is still emitted specifically from the fluorophore itself. Since excitation has additive effect, i.e. excitation from different wavelength will be added together, for emission coming from excitation wavelength other than its peak wave, it is just an extra butter on the cake. Although harmless, cross excitation make situation more complicated when dealing with cross emission correction. That means, when setting up PMT's gain and offset level to remove cross emission for a single channel, all lasers used in simultaneous excitation should be taken into consideration.
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