How To Optimize Instrument Voltage For Flow Cytometry Experiments (Part 3 Of 6)

As we continue to explore the steps involved in optimizing a flow cytometry experiment, we turn our attention to the detectors and optimizing sensitivity: instrument voltage optimization. 

This is important as we want to ensure that we can make as sensitive a measurement as possible. 

This requires us to know the optimal sensitivity of our instrument, and how our stained cells are resolved based on that voltage. 

Let’s start by asking the question what makes a good voltage

Joe Trotter, from the BD Biosciences Advanced Technology Group, once suggested the following: 

Electronic noise effects resolution sensitivity  

  • A good minimal PMT voltage would place the unstained cells where the electronic noise is no more than 10% to 20% of the variance. 

Dynamic Range Of The PMT  

  • Leave room for an ~2 fold increase in the expression levels of the makers
  • Ensure the cells are within the linear range of the detector
  • Are the negatives (in the stained cells) too high?
  • Ensure that the compensation controls are within the linear range of the detector? 

You can see all these conditions met in the best settings. 

Now that we have defined a good voltage, the question is how do we determine that voltage?  

There are several paths that will get to that point. The first step for voltage optimization is determining the sensitivity of our detectors. The first method will be the instrument vendor’s recommendation for instrument voltage optimization. In some cases, this is the only method. The vendor identifies the best voltage and locks that down so the user cannot adjust the voltage.  

The second method for voltage optimization is to use a particle to identify where the detector is most sensitive. In PMT based systems, this has been popularized by using the 2nd peak of the 8 peak headset from Spherotech, as published in this paper by Maecker and Trotter (2006). Briefly you run these beads over a voltage range. Then calculate the rCV at each voltage and plot voltage vs rCV to generate a curve as shown in Figure 1.  

instrument voltage
Figure 1: Example of peak 2 characterization

Looking at this curve, we can identify a few important points. First, as the voltage increases the CV decreases until it gets to an inflection point. The slope of the line changes here. Beyond this point, the curve becomes flat and there is no improvement in the rCV. Thus, this value at the inflection point is the lowest minimum value that you should use.  

When you are start an experiment, this is the value that you should use. However, beads are not cells. They may not adequately represent the fluorochrome that is on your cells of interest. Especially true with many of the recently released fluorochromes.

Thus it is good to perform a voltage walk (a ‘voltration’). As you can determine if the sensitivity of the fluorochrome can be improved. In this case, one takes stained cells (stained based on titrated Ab concentration) and performs a similar voltage walk.  

voltage optimization
Figure 2: Voltage optimization for Brilliant Blue 510. Top panel shows concatenated files from 500 volts to 750 volts. As can be seen, as voltage increases, the spread of the background increases dramatically. Bottom panel shows the Staining Index (SI) plotted vs voltage. Based on this curve, the best SI is at 600 volts. While the SI at 650 is slightly higher, the top panel shows the increased spread of the data at that voltage, which could impact dim markers. 

Altogether, once you determine the PMT voltage for your experiment, set up a method to measure that over time. More on that in a future blog.  

Concluding Remarks

As one begins the process of optimization of a polychromatic flow panel, the systematic approach of determining the best antibody concentration (or other fluorescent markers), followed by characterization of the optimal voltage range for the specific fluorochrome in your panel start to provide a you with the data necessary to explain your decisions.

If you will be running this panel over time, it is worth the extra effort to ensure that your panel will be successful and that you will be able to answer the question you are interested in. 

To learn more about important control measures for your flow cytometry lab, 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.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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