How To Use A Threshold To Reduce Background Noise In Flow Cytometry

On most flow cytometers, the photomultiplier tube (PMT) is the interface between the fluidics system and the electronics system. It is the PMT that converts the photons emitted from the fluorochromes into the electronic current that is digitized and ultimately converted to the value stored in the listmode file.

Any stray photon of light or random electron emission from a dynode will cause a cascade, and ultimately a photocurrent. This is often known as dark current. The figure below shows the idealized idea behind this concept.

Dark current signal noise in a flow cytometer

Figure 1: Stylized signal coming off a PMT showing the dark current and actual signals of cells passing the laser intercept.

As this figure shows, if each of these peaks is counted, there would be over 50 ‘events’ seen by the flow cytometer. Most of these events would be considered junk or debris.

Imagine if each of these events was recorded in the listmode file — how large would the file be?

To reduce this background noise in the system, we can use something called a threshold.

The threshold is a value that the signal must be above before the system will call a pulse an ‘event’.

If one enforces a threshold, the resulting pulse would look like this:

Stylized flow cytometer signal with threshold added

Figure 2: Stylized signal with a threshold added.

This threshold now reduces the signal from over 50 events down to just two. A much more manageable file size and analysis will be able to be performed by removing this noise.

1. Thresholding reduces background noise in the flow cytometer.

Thresholding is a powerful tool for reducing the signal caused by debris and dark current present in the flow cytometer.

Thresholding is a useful tool in reducing the debris that can overwhelm your datafile.

When computer storage was more expensive, and computers less powerful, it was much more heavily used.

Judicious use of threshold is warranted. However, it is also important to remember that if the flow cytometer doesn’t see an event, it doesn’t mean that the event is not present in the sample. This is especially true when sorting cells.

Here is an example of the effects of increasing the threshold on populations. These are CS&T beads run on a FACSAria, with an increasing threshold from 5,000 to 50,000 on the forward scatter parameter. A total of 20,000 events was collected for each file. Two gates (small and sort) are indicated.

Effect of increasing a threshold on flow cytometer signals

Figure 3: Effects of increasing threshold on CS&T beads.

As can be seen by this data, increasing the threshold, decreasing the amount of debris seen in these beads, and the percentage of events in each gate, changes. These values are shown below.

Table 1: Percentage of cells in the two gates from Figure 3.

Cells in 2 gates at the cytometer

2. Thresholding removes smaller events.

Thresholding increases the percentage of target events in the datafile by removing the smaller events.

If one was performing immunophenotyping analysis, for example, the increased threshold resulting in a loss of the events in the ‘small gate’ would probably not be of concern. However, if one was to sort based on these different thresholds, a very different picture emerges.

The sort logic for this experiment is shown below, as generated in DIVA.

Threshold sorting strategy

Figure 4: Sorting Strategy for Threshold Sorting Data.

After sorting the beads at different threshold levels, a post-sort analysis was performed on the instrument. Before the beads were placed back on the system, the threshold was reset to 5,000 and a wash was performed for two minutes, and the data from that wash shows the amount of background noise in the system.

A total of 89 events were observed in two minutes, with two of these events in the ‘small gate’ and none in the ‘sort gate’.

Water wash background noise in a flow cytometry experiment

Figure 5: Results of a two-minute water wash showing the background noise in the system after sorting beads.

The results of the pre- and post-sorts are shown in figure 6. This is data from the ‘bright cells’ sort is shown, and the beads were sorted on purity mode.

Flow cytometry experiment sort analysis chart

Figure 6: Results of pre- and post-sort analysis at either 10,000 or 50,000 threshold on forward scatter. Post sort analysis was performed with a threshold of 5,000.

Table 2: Data from sort gates in Figure 6

Data from sort gates

The numbers tell the tale.

At a low threshold, the purity of the post-sort population of interest is extremely high, with only a minor contamination of the events in the ‘small gate’. However, when the instrument is blinded to the small events (via a high threshold), the post-sort analysis shows that there is significant contamination and much lower purity.

Since the system could not see the small events, it was not possible for these events to be excluded, thus the small events were sorted AT RANDOM into the collection tube, because the system did not abort those droplets where a small event was in the leading or lagging droplet.

While increasing the threshold will speed up the rate of acquisition of the events of interest, the effect of increasing the threshold must be weighed against the sensitivity of the downstream application.

If the cells are to be cultured, this debris may be tolerated. If the downstream analysis is a very sensitive technique, such as RNAseq, this debris might not be tolerated. It pays to be careful with the threshold to avoid surprises — like having your highly purified cell population contaminated with a host of unexpected genes (say 𝛃-globin).

Adding a threshold when acquiring flow cytometry data is like putting on sunglasses on a sunny day. It reduces the number of events by setting a bar that a signal pulse must clear before it is counted as an event. Depending on the importance of the data, the downstream applications for the data (or sorted cells) will dictate how critical the threshold is. Threshold wisely and practice proper sample preparation to reduce the debris in the tube to ensure the best outcome.

To learn more about how to use a threshold to reduce background noise in Flow Cytometry, 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.

Join Expert Cytometry's Mastery Class
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.

Similar Articles

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

By: Heather Brown-Harding, PhD

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

Top Technical Training eBooks

Get the Advanced Microscopy eBook

Get the Advanced Microscopy eBook

Heather Brown-Harding, PhD

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.

Get The Free Modern Flow Cytometry eBook

Get The Free Modern Flow Cytometry eBook

Tim Bushnell, PhD

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more.

Get The Free 4-10 Compensation eBook

Get The Free 4-10 Compensation eBook

Tim Bushnell, PhD

Advanced 4-10 Color Compensation, Learn strategies for designing advanced antibody compensation panels and how to use your compensation matrix to analyze your experimental data.