Evaluation of Different Techniques for PMT Optimization Using the Attune Nxt Flow Cytometer

Optimize detectors for high-quality flow cytometry data

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In a flow cytometry experiment, high-quality data can only be obtained when the instrument and its individual components are optimized. The detector is one of the critical components of a flow cytometer. A detector captures the photons that are emitted by the excited fluorophores and scattered laser light and converts them into photocurrent, which is then passed to the electronics system.

The most commonly used detectors for flow cytometry are the photomultiplier tubes (PMT) that reside in each channel of the instrument (Figure 1). In addition to converting the photons to photocurrent, the PMT amplifies the signal, a process that requires the application of a steady-state voltage to the detector. PMT sensitivity is controlled by the material used to construct it and the wavelength of light entering it, as well as the amount of voltage applied to it. Therefore, optimization of that applied voltage (or gain) is required for each PMT detector in order to obtain the best-quality data in each channel of the flow cytometer.

Optimizing the voltage

As the voltage applied to the PMT is increased, the fluorescent signal is increasingly separated from background noise, providing greater resolution of the positive signal. At a certain voltage, however, the increasing separation of fluorescent signal from background will plateau, and the separation of fluorescent signal from background will remain constant. This voltage level is called the minimum voltage requirement (MVR); an ideal MVR setting will amplify dim signals above background but will not be so high that the fluorescent signals exceed the upper range of PMT detection linearity. Additionally, the MVR should allow the fluorescence signal of unstained and brightly stained cells or beads to be seen in plots on the same numeric scale. If the voltage is set too far above the MVR, the dim signal will still be separated from background but the positive signal may go off scale. Conversely, setting the voltage too far below the MVR will most likely result in compromised detection of dim signals.

Voltration

A common method for setting the MVR is the “Peak 2” method [1]. With this method, dimly fluorescent beads are run using a series of different voltage settings (also called voltration or voltage walk), and the spread of the signal (or the coefficient of variation, CV) is plotted against the voltage series. An example of a voltration experiment is shown in Figure 2. While this method does result in setting the MVR to get clear resolution of the dim fluorescent signal from the background noise of the detector, it does not address the need to ensure that the brighter fluorescent signals do not exceed the upper limit of the PMT detection range. This drawback has led to the development of a variety of methods in which unstained and brightly stained cells or beads are both used to determine the MVR [2-4].

Comparison of methods to determine MVR

Recently our scientists presented a study in which they compared different sample types as well as various calculated parameters to determine the MVR on the Invitrogen Attune NxT Flow Cytometer and any significant differences between the methods [5]. The samples tested included: different types of internally dyed microspheres (hard-dyed beads) that are detected in all detectors but do not include fluorophores used for typical experimentation; antibody-capture beads labeled on the bead surface with fluorescent antibody conjugates that are detected in a specific detector; lymphocytes that were either unstained or stained with fluorescent antibody conjugates; and a combination of the different samples.

The parameters derived from the data included calculation of the standard deviation of the electronic noise (SDEN), as well as of the staining index (SI), alternative staining index (Alt SI), and voltration index (VI) (Figure 3). In this article we will focus on a subset of these data, mainly the comparison of SI, Alt SI, and VI using antibody-capture beads (Invitrogen AbC Total Antibody Compensation Beads), lymphocytes (unstained and stained Beckman Coulter CYTO-TROL Control Cells), and a combination of the two (Table 1).

Table 1. Minimum voltage requirement (MVR) for the BL1 channel, determined with different parameters and different sample types.

The resulting MVR values

The results of the study are shown in Figure 4, and the MVR obtained for the BL1 detector (the fluorescein channel) using each method on three different sample types is summarized in Table 1. On the Attune NxT Flow Cytometer, the MVR for the AbC Total Antibody Compensation Beads alone (Figure 4A and 4B) using SI, Alt SI, or VI values was found to be ~400 mV. The MVR for the CYTO-TROL lymphocytes alone (Figure 4C and 4D) and for labeled AbC Total Antibody Compensation Beads with unstained CYTO-TROL cells (Figure 4E and 4F) was determined to be ~450 mV. All three methods (SI, Alt SI, VI) provide a working range of voltages that can be used to achieve high-quality flow cytometry data.

MVR methods and more

Instrument settings play a critical role when designing a flow cytometry experiment and must be refined for each application depending on the target specificity and abundance, specific antibodies, cell function probes, and fluorophores in the study. Using MVR settings that have been optimized for each PMT detector in each flow cytometer channel helps to minimize day-to-day variation in instrument performance and ensure that the resolution sensitivity for each parameter will be consistent from experiment to experiment.

References

1. Maecker HT, Trotter J (2006) Cytometry A 69:1037–1042. 
2. Chase ES, Hoffman RA (1998) Cytometry 33:267–279. 
3. Maciorowski Z, Chattopadhyay PK, Jain P (2017) Curr Protoc Immunol 117:5.4.1–5.4.38. 
4. Perfetto SP, Ambrozak D, Nguyen R et al. (2012) Nat Protoc 7:2067–2079. 
5. Bradford JA, Shallice M, Diz R et al. (2018)J Immunol 200 (1 Supplement):120.37.