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A Guide To Establishing Baseline PMT Voltages
In any lab, reproducibility is crucial for long term success. Since many flow cytometry instruments are highly mutable, reproducibility can become a challenge. Unknowingly having a filter set swapped out or an insufficiently powered laser can be detrimental. Therefore, it is vital to ensure your highly tuned instrument is performing at its best, so you have no doubts about the data after you run your sample.

The Lord of The Rings, New Line Cinema.

To assure that instruments are performing optimally over a long period of time, beads with embedded fluorescence are often used to find voltages that both minimize electronic noise and maintain the fluorescence signal in a linear range for each detector. Typically baseline assessments of the specific channels are performed when any major change occurs to the instrument itself. A common misconception is that the voltages automatically assigned by these beads are suitable for your particular experiment. This is not the case, as these beads are used to follow an instrument's health and should not be utilized to establish experiment-specific PMT voltages. If you were to use the baseline voltages for your multiple color assays, you would have optimized your assay for beads rather than your cells.

Comic by Sidney Harris.

There are multiple ways to assess these baseline voltages. Some instruments use specific beads called Cytometer Setup and Tracking (CS&T) beads to determine baseline voltages known as "application settings". These beads are composed of three different fluorescent intensities ranging from dim to bright. After running CS&T baseline, the bead-specific software calculates the standard deviation of electronic noise. For these beads, the PMT voltage that is selected is 10 times greater than the electronic noise for each channel.
If your current setup does not use CS&T beads or you would like to perform this type of baseline assessment manually, you're in luck. There are several other methods for calculating these values using fluorescent beads. In Stephen Perfetto's 2012 Nature Protocol paper, the authors validated a method where you run both a bead that has multiple fluorescent intensities and an unstained non-fluorescent compensation bead in 50 V increments from 350 to 800 V. The unstained compensation beads are meant to represent the background noise. Once these runs have been completed, you then plot the negative population and the positive populations for each voltage increment. The baseline voltage range for each channel is then selected based off the voltage increment that has the greatest separation between the negative and positive populations (based on MFI or SI), while the linearity (the MFI between the different fluorescent intensities) remains consistent. While this protocol is highly practical, it does require a large amount of time and only delivers a baseline range rather than an absolute baseline that the CS&T beads and software provide.

Alternatively, if you are interested in a method that is a bit less cumbersome, you could instead establish the baseline voltages off the inflection point of Rainbow Calibration Particles, peak 2. Similar to the Perfetto method, this baseline assessment would still require you to acquire the beads at varying voltages. But, instead of determining the maximum separation between negative and positive peaks, you would only need to collect the CV of the dim population. For each voltage increment, both the CV and PMT voltage are recorded. These data points would then be plotted for each specific detector. An inflection point at which the CV remains consistent would serve as the baseline voltage for each specific channel. It's worth noting that this method relies entirely on the dim population of beads.

Dexter's Laboratory, Cartoon Network.

While there are tradeoffs to all three of the methods mentioned above, each provides a unique way to standardize your flow cytometer. These baseline settings will help monitor for any changes or issues that might occur due to problems associated with the laser, electronic noise, filters, and PMTs. Any swings in these voltages should be investigated further. Lastly, it's important to remember that these baseline settings are not meant to be experiment-specific. However, following these protocols will set you on the right track to producing reliable, consistent data with your flow cytometer. Stay tuned for a follow-up blog that delves into voltage and PMT settings for experiments.

 

References:
  1. Quality assurance for polychromatic flow cytometry using a suite of calibration beads 
  2. Flow cytometry controls, instrument setup, and the determination of positivity 
Contributed by Sean Cosgriff.
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