Written by Tim Bushnell, Ph.D
When a researcher first sits down in front of the flow cytometer, they are faced with several choices.
One of the most confusing is, “What voltage do I set my detector at?”
Unless the researcher is running a fixed voltage system (Accuri, and others), this choice can dramatically impact the sensitivity of the instrument, making or breaking the experiment.
In the days of the analog flow cytometers, voltages were set by placing a quadrant gate on a bivariant plot, with the lower left quadrant encompassing the first log decade. Unstained cells would be run on the instrument and the PMT voltage set until these cells were contained within that lower left quadrant.
Figure 1: Schematic of setting voltages ‘Old School’.
Because of how the data was processed in older generation instruments, a portion of the population was ‘off-scale’ and accumulated in the first channel.
Using this method, the highly autofluorescent cells would drive the voltage, often causing a compression of the less autofluorescent cells on the axis. However, this was not always obvious because of how the data was plotted.
It should be remembered that with these systems, the data was log transformed (and compensated) in the hardware, and stored that way. This prevented much post-acquisition manipulation.
Enter the digital cytometer age, in which the data is processed post-acquisition.
This paradigm change in data acquisition and storage also meant that traditional methods for setting voltages needed to be reviewed and new methods developed.
1. Peak 2 optimization.
All PMTs have a sensitive sweet spot: a voltage at which the detector is most sensitive for the fluorochrome emission being measured.
The first paper to discuss a practical application of determining the voltage sweet spot was published by Maecker and Trotter in 2006. In this paper, the authors discuss what is termed the ‘Peak 2’ method for determining sensitivity.
In this method, a very dim particle (peak 2 of the Spherotech 8 peak bead set) is run over a series of voltages, and the Coefficient of Variance (CV) of the bead is plotted versus the voltage.
A typical graph is shown in Figure 2.
Figure 2: Results of a Peak 2 optimization of a PMT.
As PMT voltage increases, CV of the beads decreases until it hits an inflection point, and there is no improvement in the CVs from that point. The red arrow indicates the ‘optimal’ sensitivity.
This graph is interpreted by identifying the inflection point on the graph (shown in the red arrow).
Increasing the voltage from this point does not improve PMT sensitivity (with a caveat, discussed later), so the best voltage to start at is just below that inflection point.
2. Cytometry setup and tracking.
BD ran with this idea, and implemented a different method in their Cytometry Setup and Tracking.
When a CS&T baseline is run, the optimal voltage is determined by first determining the Standard Deviation of Electronic Noise (SDEN).
The software calculates the voltage necessary to set the baseline at 10X SDEN. The results of this method are stored in the CS&T report, and a typical curve is shown in Figure 3.
Figure 3: Results of a CS&T optimization baseline report.
If you have a digital machine, made by anyone other than BD, what can you do?
Option one is to follow the peak 2 method, which works very well.
A second option is to measure the electronic noise in your system and generate a voltage via CS&T.
One method to measure electronic noise is to run a negative particle, (better yet, unstained cells), over a voltage range. Then, generate a plot of (1/mean2 vs CV2), and calculate a regression line.
The slope of the line is the Variance of Electronic Noise, and the standard deviation is the square root of the variance. This gives you a channel value for electronic noise. Multiply by ten to get the target value for each channel.
Now put your unstained cells on the instrument, adjust to the target value, and away you go.
Figure 4: Calculating SDEN. Results of calculating the SDEN using PBMCs run over a voltage series.
You can improve these target values by performing a voltage optimization using the target cells stained with the properly titered reagents for the specific panel.
This can help improve sensitivity because of the fact that the spectrum of fluorochrome of interest in your panel may not be accurately modeled using the beads.
As shown in the figure below, two different fluorochromes give very different results.
Figure 5: PMT optimization.
Cells were labeled with optimal concentrations of antibodies of either CY7-APC (Left) or BV650 (Right), and a voltage optimization was performed, starting below the recommended peak 2 value.
The separation index was plotted against the voltage and the curves above generated.
Particularly with the newer dyes, an improvement in the separation index identifies a better voltage for this cell/fluorochrome combination.
3. Setting consistent target voltages.
The last step in voltage setting on a digital instrument is to have some way to consistently set those target voltages over time, without having to go through a long, tedious process.
Beads again come to the rescue. In this case, a very bright bead (some favor the 6th peak of the 8 peak bead-set, but you can choose your favorite), that is fluorescent in the channels to be used is run when the optimal voltages are identified. This results in a value for each detector — the ‘target value’.
Then set up a template with the appropriate plots and target values and remember to save it for later use!
When you come back to the instrument, open up the template and run this bright bead. Adjust the voltages (as necessary) to achieve the target values in each channel and you’re good to start acquiring your samples.
The other advantage of this method is that you can become cross-platform compatible.
The best way to take out the fear and agony of setting voltages is to use some optimization methods. The peak 2 method, as described above, is a useful and robust method of identifying optimal PMT voltage ranges. Refining that to the voltage walk with the actual cells and fluorochromes of interest will further improve sensitivity, which is especially critical for rare cell populations or emergent antigens (like activation markers). Of course, don’t forget to set-up a way to monitor and maintain those voltage settings.
To learn more about how to set and monitor optimal voltages for a flow cytometry experiment, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.
My other passions include grilling, wine tasting, and real food. To be honest, my biggest passion is flow cytometry, which is something that Carol and I share. My personal mission is to make flow cytometry education accessible, relevant, and fun. I’ve had a long history in the field starting all the way back in graduate school.
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