What Is Photon Counting And How To Use 8-Peak Rainbow Beads

For decades, flow cytometry professionals have been using 8-peak quality control beads to quickly check PMT sensitivity, resolution, and linearity on their flow cytometry and cell sorting systems.

It’s only recently that these beads have become respected not only as a diagnostic tool but also as a flow cytometry educational tool.

As scientists have continued to learn more about the physics of photon collection and conversion to electrons, they have started to realize that 8-peak beads can teach a very compelling lesson.

The lesson involves photoelectron generation and the inherent error in this process that fundamentally governs all flow cytometric measurements.

Photoelectron is the term we used to describe an electron that was generated at the photocathode of a flow cytometer’s photomultiplier tubes (PMTs) as a result of the process that converts a photon to an electron.

What Are 8-Peak Rainbow Beads?

8-peak beads, sometimes called “rainbow” beads, are a set of beads in a single vial that contains 8 different populations that differ only in the amount of fluorophore contained within them.

One of the peaks, termed Peak 1, is unlabeled, and the additional seven, termed Peaks 2-8, contain increasing amount of fluorophore.

8-peak beads are designed to fluoresce in all channels on most flow cytometers and cell sorters.

Traditionally, they have been used to check fluorescence sensitivity and resolution by measuring the position of the unlabeled peak and the separation between all of the peaks, respectively.

Additionally, they have been used to check linearity in fluorescence detection channels by correlating the amount of fluorophore on each population of bead, which differs in a linear fashion, with the position on the scale onto which the flow cytometer places the beads.

Although there are more accurate methods that have been developed for sensitivity and resolution testing, an 8-peak bead test still remains the gold standard for estimating how a flow cytometer is performing in its ability to measure subtle differences in fluorescence levels.

The image below shows a plot of 8-peak beads in a channel with a 525/15 bandpass filter, usually used to measure FITC, GFP, etc.

This 8-peak beadset run on an optimized instrument. The 8 peaks are labeled in red.

The spread of the data for peak 1 and peak 6 is indicated by the dashed red lines, with the blue arrow indicating the spread of the data.

Figure_1

Upon a closer examination of the above plot, you may notice something very interesting: the peaks have very different widths, depending on their intensity.

But, how can this be the case if the only difference between bead populations is the amount of fluorophore?

Why do the peaks with lower intensities (less fluorophore) appear to be wider than peaks with higher intensities?

Wouldn’t this indicate that within the dimmer population of beads, some of the beads have more fluorophore and some have less, leading to higher variation?

This is in fact NOT the case, and examining the reason for this gives us tremendous insight into how light is measured in a cytometer.

Although the dimmer peaks certainly have higher CVs, every bead in this population has essentially the same amount of dye bound up inside of it.

As a reference, the percent CV is the measurement of variation that is most commonly utilized in flow cytometry, defined as (standard deviation/mean of the values) × 100.

How A Flow Cytometer Counts Photons

The variation in CV values that is observed when using 8-peak beads has to do with something called photoelectron statistical error.

This effect comes into play at the photocathode of the PMT, which is the most critical point of the PMT.

It is here that a photon, from a fluorescence event, is converted into an electron, facilitating all downstream signal processing. The rest of the PMT, in comparison, is primarily devoted to amplifying the photoelectron into many photoelectrons, which is the first step in signal processing.

The below image graphically describe the process of photoelectron generation at the PMT in flow cytometers and cell sorters.

Figure_2

The process of photoelectron generation is governed by counting statistics. Essentially, all that a PMT does (at the photocathode) is to count photons, and a PMT is subject to counting error as any other counting process is.

The most critical aspect of counting statistics is the determination of the standard deviation of the count:

Given an average count of N events (photons), the standard deviation of the count can be determined as √Ν .

Therefore, the more photons that are counted per event, the lower the standard deviation (and CV). See the below table as an example (tabular values are theoretical counts for the purposes of this lesson and may not correspond to photon counts of actual measurements).

Events Counted[1]SD%CV (SD/Mean x 100)
1001010%
1,00031.63.16%
10,0001001%
100,000316.2.316%
1,000,0001000.1%

This estimation of the standard deviation (and thus CV) of a count explains fittingly why dimmer beads have higher CVs that brighter peaks.

The Difference Between Dim Versus Bright Rainbow Beads

Dim beads are dim because they contain less fluorophore and introduce fewer photons to the photocathode during illumination.

This low number of photons causes there to be a high error in counting; one bead event may produce 1100 photoelectrons, another may produce 900 while another produces 1050, even though each of these beads emits 1000 photons when illuminated.

On the other hand, the bright beads bombard the photocathode with a tremendous number of photons, leading to very little error in the counting process.

You may also ask why the brightest bead populations form a distribution themselves, albeit a narrow one.

The answer is simple—there is some intrinsic variation in the beads (they are not entirely identical within each of the 8 populations).

The CV of the brightest population, which is essentially not governed by photoelectron statistical error, reflects this intrinsic variation.

Another way to think about it is as follows: If a single bead was put through the cytometer 1000 times, this bead itself would form a distribution on a fluorescence plot with a measureable CV.

The magnitude of this CV is highly influenced by photoelectron statistical error.

This error is by no means restricted to the theoretical world of beads, and the lessons of 8-peak beads can absolutely be applied to real-world situations.

Dimly stained cells will have higher CVs than brightly stained cells (give nearly identical marker expression). The wider population of dimly stained cells will make resolution and sensitivity highly critical for proper subset identification. This is all the more reason to choose fluorophores and antibody panels wisely for these kinds of populations, pick channels with little spillover reception, which compromises resolution, and titrate antibodies to maximize the Staining Index.

To learn more about using 8-peak beads and other quality control reagents, 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

ABOUT 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.

Tim Bushnell, PhD

Similar Articles

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

By: Tim Bushnell, PhD

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

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 Industry Career 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.