Resolution of Signal

Multicolor flow cytometry is the terminology used when analyzing multiple fluorescent parameters in one sample, may it be surface markers, intracellular markers, DNA or all combined. In addition to ensuring the right controls (discussed in Chapter 4), optimizing your experimental procedure (discussed in Chapter 5) and careful sample preparation, there are a few other considerations which should be taken into account to ensure meaningful results can be obtained. This is because each additional fluorophore you add to your flow cytometry panel has the potential to influence another fluorophore. The result of this is unwanted fluorescence in additional channels which has to be compensated for (discussed in Chapter 2) and a loss in resolution. High levels of noise caused by non-specific staining, high background staining, cell autofluorescence and spillover can all contribute to reduction in sensitivity and resolution.

Resolution can be described as the potential of an instrument together with a combination of fluorophores to separate a positive population from a negative population. To ensure optimal resolution there are a few simple rules that can be followed that will help form the basics of all panel design.  Whilst following these best practice rules may not result in a perfect panel first time every time, it will ensure you have solid starting point that should result in fewer errors due to incompatible reagents.

Fig. 29. Cell population resolution. Using an additional marker CD66b FITC (MCA216F), the granulocytes (circled in black) that express low levels of CD14 A647 (MCA1568A647) can be resolved from lymphocytes which are negative for CD14 and CD66b.

Instrument configuration

Instrument configuration is important to understand before you start to build your panel. You simply cannot use a fluorophore your instrument is not configured for, regardless of which is the theoretical best fit. Instrument configuration is simply the set-up of the lasers, optics and filters that are contained within the cytometer. This can vary significantly between cytometers. The S3e™ Cell Sorter from Bio-Rad has 2 lasers and has 4 fluorescence detectors, whereas the ZE5™ Cell Analyzer has 5 lasers and the potential to simultaneously detect 28 fluorophores.