Minimize—even eliminate—color compensation

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With the option to be configured with up to 4 lasers and 14 colors for multiparameter analysis, the Attune™ NxT Acoustic Focusing Cytometer was envisioned as a modular system to fit most experimental design needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of 4 spatially separated lasers onto the sample stream, enabling consistency in data over time along with superior performance and reliability. And for the researcher who prefers to avoid color compensation, the 14 colors spread across the 4 independent laser lines allow popular dye combinations to be used with minimal or no compensation.

Spillover and compensation defined

Spillover occurs when the emission spectrum of a given fluorophore is detected in a detector meant to identify signal from another fluorophore (Figure 1). In order to appropriately visualize flow cytometry data, spillover needs to be corrected for by a process called compensation. Compensation is a way to mathematically subtract the overlap of fluorescence emission colors that occurs with multilabeled samples in flow cytometry. For example, even though the emission peaks of fluorescein (FITC; emission maximum ~525 nm) and R-phycoerythrin (PE; emission maximum ~575 nm) are well separated, the emission spectra of these two dyes overlap significantly (Figure 1).

During data acquisition on a flow cytometer equipped with a single blue (488 nm) laser, optical filters are used to direct the FITC emissions to the FITC detector and PE emissions to the PE detector. But because of their spectral overlap, some FITC emission light passes through the PE filter, which can result in a FITC-labeled target being counted in the PE detection channel. When this happens, software algorithms utilize data collected from single-color stained controls to estimate the FITC signal collected by the PE detector and subtract it from the data—a process called color compensation. Appropriate compensation, however, not only requires extra controls and time but is also prone to artifacts and errors.

Spectra of fluorescein and R-phycoerythrin emission demonstrating spectral overlap 

Figure 1. Spectra of fluorescein and R-phycoerythrin emission, demonstrating spectral overlap. Emission spectra of fluorescein (FITC, green) and R-phycoerythrin (PE, yellow), both excited by light from a blue (488 nm) laser (blue vertical line). Note that a portion of the FITC emission spectrum overlaps most of the PE emission spectrum.

Uncouple the excitation and detection of multiple dyes

Compensation is not simple, requiring runs of positive and negative, color-matched controls in conjunction with careful monitoring of background fluorescence. The Attune™ NxT Acoustic Focusing Cytometer can be configured with up to 4 spatially separated lasers, giving you the flexibility to choose dyes and detection channels that are well separated spectrally and do not have significant overlap (e.g., Table 1).

For example, by exciting FITC along the blue (488 nm) laser pathway and PE along the yellow (561 nm) laser pathway, the two fluorophores can be independently detected without the need for compensation (Figure 2). We have taken advantage of the Attune™ NxT cytometer’s optical path layout to develop a no-lyse/ no-wash, 6-color immunophenotyping panel to identify human T cell subsets without the need for compensation at any step (Figure 3).

Table 1. Lasers, filters, and probes for the no-lyse/no-wash, 6-color immunophenotyping panel acquired on the Attune™ NxT Acoustic Focusing Cytometer and shown in Figure 3.

LaserDetection channelBandpass emission filterTargetAntibody labelAntibody Cat. No.
Blue (488 nm)BL1530/30 nmCD8FITC MHCD0801
BL3695/40 nmCD4PerCP–Cy®5.5A15858
Yellow (561 nm)YL1585/16 nmGly APE MHGLA04
Violet (405 nm)VL1440/50 nmCD62LPacific Blue™ MHCD0828
VL3603/48 nmCD45Pacific Orange™ MHCD4530
Red (637 nm)RL1    
RL3780/60 nmCD3APC–Alexa Fluor™ 750 MHCD0327
2-panel scatter plots showing elimination of FITC spillover into the PE channel using a yellow laser 

Figure 2. Elimination of FITC spillover into the PE channel using a yellow laser. When FITC and PE use the same optical detection pathways after excitation by the blue (488 nm) laser, there is significant spillover of FITC signal into the PE detector, requiring compensation. However, when excitation and detection paths of FITC and PE are uncoupled in the Attune™ NxT cytometer using the yellow (561 nm) laser to excite PE, there is little or no spillover of FITC signal into the PE detector; thus, no compensation is required. Data were acquired using (A) 488 nm excitation with a 530/30 nm bandpass (BP) filter to detect FITC and a 590/40 nm BP filter to detect PE, and (B) 488 nm excitation with a 530/30 nm BP filter to detect FITC, and 561 nm excitation with a 585/16 nm BP filter to detect PE.
5-panel histogram figure showing optimal design of a no-lyse/no-wash, 6-color immunophenotyping panel for human T cell subsets

Figure 3. Optimal design of a no-lyse/no-wash, 6-color immunophenotyping panel for human T cell subsets acquired on the Attune™ NxT Acoustic Focusing Cytometer without using compensation at any step. Human whole blood was stained and interrogated using the probes and configurations described in Table 1. (A) A fluorescence threshold was set on Pacific Orange™ fluorescence (CD45), and events coincident with red blood cells were excluded based on PE positivity (glycophorin A or Gly A). (B) Lymphocytes were gated based on scatter properties, from which (C) T cells were identified by CD3 expression. (D, E) T cells were then analyzed for their expression of the lineage markers CD4 and CD8 as well as the activation marker CD62L in order to identify naive/central memory T cells (CD62L-positive) and effector memory T cells (CD62L-negative). No compensation was required to analyze or display these data.

Basic rules to minimize need for compensation

  1. Match fluorophores to targets according to brightness needed (use the brightest dyes for least abundant or most disperse targets).
  2. Consider the emission spectrum of the donor fluorophore when using tandem dyes. If possible, refrain from using tandem dyes if compensation minimization is the primary goal.
  3. Know your instrument configuration—choose dyes that will work with the instrument’s laser and filter configurations and, if spatially separated lasers are installed, maximize the use of lasers to minimize spillover.

But if you need to use compensation anyway...

  1. Controls must be at least as bright as any samples to which compensation will be applied.
  2. Background fluorescence at each wavelength should be the same for both the positive and negative control populations.
  3. Use a tool such as the Molecular Probes™ Fluorescence SpectraViewer to determine the spillover of each dye in each detection channel.

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