Spectral Unmixing in FCS Express provides a way to easily discriminate the individual emission spectral signature of a specific fluorochrome or dye using single stained controls.

In this chapter we want to add more details on two scenarios that you might face while running the Unmixing Wizard into FCS Express 7.

•Duplicate Brightest Detector warning message

•Low Abundance Populations

 

•Duplicate Brightest Detector warning message

The peak channel in the emission spectral signature is automatically identified for each dye when using the Spectral Unmixing Wizard.

Under some circumstances, auto-detection of peak channels in the wizard can identify multiple dyes with the same peak channel and identify these with a warning message "Duplicate brightest detector..." (Figure 9.47).

 

 

 

This mainly happens in the following two scenarios:

1.The spectral signatures of those dyes, actually share the same brightest detector. This is unlikely, since in most experiments, dyes are carefully selected upfront to not share the same brightest detector. Having said that, even when that happen, the unmixing might still be possible if the remaining part of the spectra of those dyes is different enough.

2.The Spectral Unmixing Wizard considers a channel being the brightest detector even if it is not. This can mainly happen in two cases:

a)when the negative control is set to "Use Self As Negative" and the autofluorescence of the particle is particularly high in some channels. Since the brightest detector is chosen before identifying the stained and the unstained population, its selection is biased by the autofluorescence.

b)When the automatically-created Scatter gate does not properly fit the particles of interest and maybe includes multiple populations of particles, each with different autofluorescence. Since the brightest detector is defined by the events in the Scatter gate, the brightest detector could be selected based on a non-target population.

 

Scenario #1 can be avoided by an accurate selections of the dyes to include in the experiment.

 

Scenario #2a can be either avoided, managed or ignored:

oIt can be avoided, by using an external negative control (i.e. "Step 1" of the Unmixing Wizard). This way, the spectral signature of the negative population (i.e. the autofluorescence) will be taken into account before the identification of the brightest channel and thus will not be a bias.

oIt can be managed, by manually selecting the brightest detector (i.e. "Step 5" of the Unmixing Wizard). I.e. if you know upfront which is the best channel to identify the positive and the negative population (i.e. the expected brightest detector), said channel can be manually chosen and displayed on the histograms automatically generated by the Unmixing Wizard.

oIt can be ignored, if the brightest channel detected by the Automatic Wizard is not the expected one, chances are that it still allow a proper discrimination of negative and positive particles.

 

Scenario #2b should managed by adjusting the Scatter gate so that it encompass the appropriate particles. This can also be made by leveraging on Data Specific Gates (please to the "Step 3" paragraph in the Creating an Unmixing Definition Using Single Stained Controls chapter). Note that upon adjusting the Scatter gate, the Brightest detector won't be recalculated. However, all plots and histogram gated on the Scatter gate, will display the appropriate particles. At that point, the user can still put in practice the actions presented for Scenario #2a.

 

 

•Low Abundance Populations

Low abundance populations can represent a challenge for the automatic peak detection on histograms. When this happens, is likely that that the automatic peak detection places the marker around the wrong population simply because the latter is recognized as a clear peak on the histogram.

 

When this happen, it is a good practice to first check whether the population of interest (i.e. the population we would like to place the marker around), is expected to be rare or, in contrast, if it results in being rare just because

othe Scatter gate has not been placed properly,

oand/or the channel displayed on the histogram is not ideal to properly distinguish the population of interest.

 

 

Scatter gate has not been placed properly

In the example below (Figure 9.48), the single stained control for fluorochrome V450, has been made using an antibody specific for a Monocyte antigen. However, the Scatter gate has been placed around Lymphocytes. This resulted in a positive peak being almost absent, and the automatic peak detection failing to detect the positive peak. The marker was automatically placed around the very clear negative one. However, the root issue of this failure is not the rarity of the population of interest, it is simply the wrong position of the Scatter gate.

A soon as the Scatter gate is properly placed around Monocytes (i.e. the population expected to express high level of the target antigen), the positive peak is revealed and the automatic peak detection properly placed the marker around it (figure below, lower pane). Note that, if the position/shape of Scatter gate should only be modified for this single stained control, Data Specific Gates can be used.

 

 

 

 

 

 

 

 

The channel displayed on the histogram is not ideal to properly distinguish the population of interest

If the channel displayed on the histogram is not ideal to properly distinguish the population of interest, another channel can be selected instead of the automatically selected one. To select a different channel, please refer to the Step 5 - Adjust parameters section of the Creating an Unmixing Definition Using Single Stained Controls chapter.

 

 

None of the above: the population of interest is expected to be rare

If the rarity of the population of interest is instead expected, the inappropriate position of the marker can be easily rescued by manual adjustment.

In the example below (Figure 9.49), the single stained control for fluorochrome cFluor BYG667, has been made using an antibody specific for an antigen expressed by a rare Lymphocyte population. The Scatter gate has been properly placed around Lymphocytes, so no adjustments of the Scatter gate position are needed.

To properly handle the scenario above, simply manually adjust the position and the width of the marker on the histogram to properly include the rare positive population of interest.