Part 1 Principles
Confocal Microscopy tutorial
Part 2 application of confocal microscopy
6. fluorescence detecting in confocal microscopy
Cross-talk correction 2: cross
emission (emission bleeding through)
As the curve shows: considerable portion of the FITC emission is from DAPI, and so does TRITC from FITC, and vice versa. Cross emission causes false positive co-localization and false high intensity of the emission.
If you want to eliminate the overlapped emission, it
is extremely harmful for the second emission because only a small portion is
The following images taken from specimen of FITC or TRITC single stained fluorescence beads. It shows how the data can be erroneous collected and how results can be mis-interpreted, and the final effect after cross-talking correction by adjusting laser intensity, gain and offset on PMT.
|This is FITC fluorescence beads excited by 488 nm laser
line, showing considerable amount of bleeding-through into red channel and
present a false co-localization in overlay image
Image after applying correction, red emission bleeding from FITC is eliminated, no false co-localization exist anymore.
| This is Rhodamine
fluorescence beads excited by 568 nm laser line, showing considerable amount
of bleeding-through into green channel and a false co-localization in
Image after applying correction, green emission bleeding
from Rhodamine is eliminated, there is no false co-localization in overlay
The correction on above image can be done easily and effectively because there is only one fluorescence staining existing. By using single-stained specimen, you know for sure any signal in other channel is false and should be removed. It serves you as reference to adjust laser intensity, gain and offset level of PMT to get rid of emission bleed-through.
| When two fluorophores co-existing in specimen and
two excitation lasers are used simultaneously, the situation become more complicated.
There is no reference for you to make a clear judgment that how much is the
bleeding-through from another channel. Indiscriminately reducing laser
intensity or change detector gain and offset will also reduce the intensity
of specific signal.
Whenever possible, in multiple labeling experiment, single stained specimen has to be prepared and used as reference for cross talking correction.
Besides adjusting laser intensity, gain and offset of PMT, there is an additional way in Sp2 confocal microscope for cross-emission correction: moving or narrowing the detecting range of the spectrometer to avoid collecting those overlapped spectra area according to the knowledge of how much they overlapped. Here the advantage of spectrometer based system become more apparent.
If the cross-talking can not be removed successfully by those above-mentioned methods, or the signal become very weak after an aggressive suppression because your specimen do not have enough intensity for the removal, sequential scan has to be used.
In sequential scan mode, you can use single excitation at a time and collecting wider range since there is no emission from another channel. The consecutive collected data will be added together for overlay as they were collected in a single scan, although there is a short time delay.
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