Spectral Unmixing
Crosstalk, bleed-through and overlapping emission signals
An important consideration when imaging specimens labelled with two or more fluorophores is that of emission spectra overlap, which can lead to a situation called bleed through or crosstalk. An example of crosstalk can be demonstrated when visualising samples labelled with FITC and TRITC – the samples appear orange rather than separate red and green. Bleed through must be eliminated or controlled through when performing co localisation studies.
In widefield fluorescent microscopy, the problems of overlapping emission spectra may be reduced or avoided by judicious choice of filter combinations. In confocal microscopy this problem can be addressed in many cases through the control of the excitation power and specific gating of the emission prior to it reaching the photomultiplier. In addition to this, users can choose to collect multi channel images sequentially, again reducing the chance of crosstalk.
There may be situations where the above techniques are not possible so another method has to be employed.
Spectral unmixing
This is a powerful computational method that allows overlapping signals from fluorophores to be separated based upon their spectral profiles.
The method for unmixing overlapping spectral profiles requires the dyes concerned to be calibrated by acquisition of reference spectra of each individually under the same conditions as the multi-labelled specimen. Multi-labelled images are then acquired and after correction for the background, the unmixing algorithm redistributes the intensities of the dye channels according to the calibration data.
