After panel design, and titration of the reagents, the next most important step in flow cytometry is setting the proper voltages on the photomultipler tubes (PMTs). These detectors take the photons of light emitted by the fluorochromes on the cells and convert them to electrons, which ultimately become the voltage current that is digitized and ultimately stored in the FCS file as the data output.
Voltage Setting Methods
The old days
In the old days, before the advent of digital electronics, setting voltages was a bit of the wild west. Researchers were often trained to set the voltages so that the unstained cells were located in the first decade of a plot (either uni- or bi-variate). Of course, this method often introduces error, especially in the far red region of the spectrum where there is very little autofluorescence.
Determining Optimal Voltage Setting Using The Peak Two Method
With the release of digital electronics, it became easier to measure and determine the optimal voltage settings. This has been discussed in a paper by Maecker and Trotter. In this paper, they use a dim particle (peak 2 of the Spherotech 8 peak bead set) and measure the spread of the data (CV) over an increasing voltage series. This generates data that looks like this:
Figure 1: Data from the URMC Flow Cytometry Facility. As this graph shows, increasing the voltage decreases the spread (CV) of the data up to an inflection point. At this point, increasing the voltage does not improve the signal any further.
This process can be easily replicated for any digital cytometer, and serve as a good starting point for any experiment. Rather than try to bring the unstained cells into the first decade (like was taught for analog cytometers) with digital instruments, if the PMT is optimized, the voltages should be set and not try to bring the negatives up or down into the first decade.
Determining Optimal Voltage Setting Using Cytometry Setup and Tracking
In the mid-2000’s BD Bioscience released a program call Cytometry Setup and Tracking (or CS&T). This program requires the use of FACSDiva 6.0 or later and using this program and the associated beads on a BD digital instrument it is possible to establish the optimal voltage to run samples on. With the CS&T, the optimal voltage is set at 10 times the standard deviation of electronic noise. These voltages become the baseline settings for the instrument. A typical result is shown in figure 2.
Figure 2: Data from the URMC Flow Cytometry Facility. As this graph shows, increasing the voltage decreases the spread (CV) of the data up to an inflection point. At this point, increasing the voltage does not improve the signal any further.
Why Choose One Method Over the Other?
If you’re not running a BD instrument, then CS&T will not work, which requires the peak 2 method. A second reason that peak 2 is preferred to CS&T is that in the BD method, there is a correction factor (the ‘bead file’) that is developed at BD and based on the characteristics of a specific filter set and laser power. If your instrument has different laser configuration, laser power or filter configurations, the peak 2 method is better for PMT characterization.
Of course, one of the limitations of these methods is that they both rely on dye beads. These beads contain specific fluorochromes that fluoresce in different channels. However, with the advent of new fluorochromes, these beads may not have the same fluorochrome that is present in the sample.
To resolve this issue, and to ensure the optimal voltage is obtained for the cells of interest and the fluorochromes of choice, one should perform a voltage optimization step. This process is similar to a titration experiment. However, rather than vary the concentration of antibody, the researcher varies the voltage and calculates the staining index for the experiment.
Figure 3. Fluorochrome voltage titrations.
Two different fluorochrome voltage titrations are shown in figure three. On the left, increasing voltages does not change over the voltage range, thus the optimal voltage suggested by the peak two method is the best voltage. On the right, increasing the voltage does improve the staining index, up till where it plateau’s. Thus, the optimal voltage is about 100 volts more than suggested by the peak two method. This makes sense, as the fluorochrome is a newer reagent, and has a different spectral profile from the fluorochrome on the beads.
Optimal voltage selection is essential for the best panel performance. The old school method of setting voltages should be thrown out the window. The starting point for any experiment should be based on the best voltage as determined using an optimization technique like peak two beads. To further improve panel performance, it is critical to optimize the voltage based on the actual cells and fluorochrome being used for the experiment.