# 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.

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.

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) |

100 | 10 | 10% |

1,000 | 31.6 | 3.16% |

10,000 | 100 | 1% |

100,000 | 316.2 | .316% |

1,000,000 | 1000 | .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.**

**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.

More Written by Tim Bushnell, PhD