How Flow Cytometry Converts Photons To Digital Data

How exactly does a photon of light become an electron, and eventually a number in the FCS listmode file?

In flow cytometry, cells are labeled with fluorescent tags, passed by an excitation light source, and the resulting emitted photons are collected to become the data that identifies characteristics about the cells in question.

These characteristics can include what proteins are expressed, the cell cycle state, the phosphorylation state of a protein, the calcium state of the cells, levels of RNA expression, and so much more.

If there is a fluorescent probe that can measure a specific characteristic, it can often be used in flow cytometry.

A great resource for reference is the Molecular Probes Handbook, as it contains a wealth of information about different fluorescent reagents that can measure everything from apoptosis to Zn++ ion concentration.

This process involves the detection system and the electronics, and basically following a bouncing photon to its ultimate digitization.

The role of the detector on a flow cytometer is to capture photons and convert them to electric current.

By and large, the two most common detectors on commercial flow cytometers are photodiodes and photomultiplier tubes.

In a PMT, photons of light enter through a window and hit the photocathode. If the photon is of sufficient energy, they eject an electron (due to the Photoelectric Effect).

The electron is focused to one of several ‘fins’ (called dynodes), where the electrons are multiplied via secondary emission. At the end of this chain is an anode, where the electrons are converted to an electronic pulse. This is shown schematically below.

Electrons striking photoanode and creating current flow

As is shown, there is a linear relationship with the photons hitting the cathode and the output photocurrent.

It should be noted that PMTs can turn any photon that strikes the photocathode into a photocurrent.

Thus, to control the photons that are measured by any given detector, optical filters are placed in front of the PMT.

Most commonly, these are band pass filters, and as a reminder and shown below, band pass filters allow light within a given range through. To describe these filters, manufacturers label these filters with the center of the range and the size of the window of light that can pass through the filter. So a 620/40 band pass filter will allow light between 600 to 640 nm through, as shown below.

Band pass filters allow light to pass within a given range

Current produced is proportional to intensity of the fluorescent signal:

At the end of the day, one has a current produced that is proportional to the intensity of the fluorescent signal, and each PMT measures a specific wavelength range because of the optical filters placed in front of the detector.

At this point, the photons of light have been turned into electric current.

The next question is, “How does that electric current get turned into a digital value that is sorted in the FCS file?” The image below shows what a typical electronic pulse looks like.

The current produced is proportional to the intensity of the fluorescent signal

This pulse has three characteristics that can be measured: the peak height, the pulse width and the pulse area, and the integral of the height and width. The boxes represent the fact that the electronic pulse is being sampled at some frequency, and with each sampling, the values are digitized by the Analog to Digital Converter (ADC).

The ADC has two characteristics: the sampling frequency and the resolution.

The sampling frequency is expressed in megaHertz (MHZ), and ranges from 10 to 100 on the current generation of instruments. This means that the electronic pulse is being sampled between 10 to 100 million times PER SECOND.

From this sampling, the value is digitized into a range equal to 2 to the power of the ADC. This can range from 10 to 24 bits or 1,024 to 16,777,216 discrete values.

The values for some common instruments are shown below:

InstrumentVendorSampling FrequencyADC
AccuriBD Biosciences80 MHz24
FACSCanto IIBD Biosciences20 MHz14
FACSDiva   (LSR-II, Aria, Fortessa)BD Biosciences10 MHz14
FACSVerseBD Biosciences25 MHz14
GalliosBeckman-Coulter40 MHz20
MACSQuantMiltenyi Biotec??32
MoFlo XDPBeckman-Coulter100 MHz16
SH800SONY100 MHz20

It is worth noting that BD instruments use a predetermined 18 bit log-lookup table so that the range of the data is 262,144 bins.

Frequency of sampling and resolution in a Flow Cytometer:

There are two important characteristics of the ADC: the frequency of sampling and the resolution.

Natural questions might include: What are all these bits, really? Or are they bins? Or channels?

In the case of a 10 bit ADC, this means there are 1,024 discrete values that the sampled pulse can be assigned. With a higher ADC (Accuri with a 24 bit ADC), it has over 16 million discrete values.

These are the ‘channels’ or ‘bins’ and represent the actual value that has been measured from the signal pulse. So when the signal is digitized, it is assigned one of these values.

For linear data, this is pretty self-explanatory. However, with log data, this binning gets a bit tricky, especially if you consider the difference between the older FCS2 data and the current FCS3 data.

Take, for example, the FACSCalibur, which has 10 bit resolution. Since this data, when log transformed, was being measured over 4 decades, the vendor decided to spread the 1024 bins equally across the log space.

So the first decade (1-10) had 256 values, as did the second decade (10-100), the third decade (100-1000) and the fourth decade (1000-10,000). One can see that in the case of the third and fourth decades, 256 bins did not give sufficient resolution, so that each bin contained more ‘values’ than a bin in the lower decades.

On the modern digital instruments, these values are spread out to better reflect the number of values available at the higher level ADCs. Therefore, there is more resolution at higher decades than at lower decades.

Flow cytometrists tend to use bins and channels interchangeably.

Photons are captured by the detector and converted from light to electrons

Conversion of photons to electrons:

The number of bins that a given system has is based on the resolution of the ADC.

With digital instruments and the FCS 3 format, the bins are spread out to better reflect the number of possible values in the log space.

In summary, a photon is emitted from a fluorochrome. The photon is captured by the detector (e.g. the photomultiplier tube, PMT), where it is converted from light to an electron. At the same time, the PMT amplifies the incoming signal in a linear, proportional manner.

The output is an electronic signal pulse. The electronic pulses coming off the PMT is sampled many millions of times a second, and the height of that sample is digitized into a discrete value that is placed in a bin. The resolution of the ADC dictates the number of possible bins available for use.

In the end, this value is stored in the FCS file, and ready for further analysis.

To learn more about how flow cytometry converts photons to digital data, 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

How To Extract Cells From Tissues Using Laser Capture Microscopy

How To Extract Cells From Tissues Using Laser Capture Microscopy

By: Tim Bushnell, PhD

Extracting specific cells still remains an important aspect of several emerging genomic techniques. Prior knowledge about the input cells helps to put the downstream results in context. The most common isolation technique is cell sorting, but it requires a single cell suspension and eliminates any spatial information about the microenvironment. Spatial transcriptomics is an emerging technique that can address some of these issues, but that is a topic for another blog.  So what does a researcher who needs to isolate a specific type of cell do? The answer lies in the technique of laser capture microdissection (LCM). Developed at the National…

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

The Importance Of Quality Control And Quality Assurance In Flow Cytometry (Part 4 Of 6)

By: Tim Bushnell, PhD

Incorporating quality control as a part of the optimization process in  your flow cytometry protocol is important. Take a step back and consider how to build quality control tracking into the experimental protocol.  When researchers hear about quality control, they immediately shift their attention to those operating and maintaining the instrument, as if the whole weight of QC should fall on their shoulders.   It is true that core facilities work hard to provide high-quality instruments and monitor performance over time so that the researchers can enjoy uniformity in their experiments. That, however, is just one level of QC.  As the experimental…

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

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

By: Tim Bushnell, PhD

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…

Optimizing Flow Cytometry Experiments - Part 2         How To Block Samples (Sample Blocking)

Optimizing Flow Cytometry Experiments - Part 2 How To Block Samples (Sample Blocking)

By: Tim Bushnell, PhD

In my previous blog on  experimental optimization, we discussed the idea of identifying the best antibody concentration for staining the cells. We did this through a process called titration, which  focuses on finding the best signal-to-noise ratio at the lowest antibody concentration. In this blog we will deal with sample blocking As a reminder, there are two other major binding concerns with antibodies. The first is the specific binding of the Fc fragment of the antibody to the Fc Receptor expressed on some cells. This protein is critical for the process of destroying microbes or other cells that have been…

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

How To Determine The Optimal Antibody Concentration For Your Flow Cytometry Experiment (Part 1 of 6)

By: Tim Bushnell, PhD

Over the next series of blog posts, we will explore the different aspects of optimizing a polychromatic flow cytometry panel. These steps range from figuring out the best voltage to use, which controls are critical for data interpretation, what quality control tools can be integrated into the assay; how to block cells, and more. This blog will focus on determining the optimal antibody concentration.  As a reminder about the antibody structure, a schematic of an antibody is shown below.  Figure 1: Schematic of an antibody. Figure from Wikipedia. The antibody is composed of two heavy chains and two light chains that…

2020 - A Year Turned Upside Down

2020 - A Year Turned Upside Down

By: Tim Bushnell, PhD

What an incredible year 2020 has been. It started off like any other year and bam SARS-CoV-2 (aka COVID 19) entered the equation, bringing chaos and havoc to the world. Things kept changing overnight as new rules and regulations popped up. Masking, quarantine, and flatten the curve became common words in the news. How we met, how we interacted changed almost overnight. Throughout all of this, as we look to 2021, there is hope and optimism. Multiple vaccines have been developed, building on years of research into the SARS-CoV virus, with some approved for human use, and others on the horizon.…

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

Brightness Is In The Eye Of The Detector - What To Consider When Designing Your Panel

By: Tim Bushnell, PhD

The heart and soul of the flow cytometry experiment is the ‘panel.’ The unique combinations of antibodies, antigens, fluorochromes, and other reagents are central to identifying the cells of interest and extracting the data necessary to answer the question at hand. Designing the right panel for flow cytometry is essential for detecting different modalities. The more parameters that can be interrogated will yield more information about the target cells. Current instruments can measure as many as 40 different parameters simultaneously. This is exciting, as it allows for more complex questions to be studied. Panel design is also valuable for precious samples,…

Tools to Improve Your Panel Design – Determining Antigen Density

Tools to Improve Your Panel Design – Determining Antigen Density

By: Tim Bushnell, PhD

When a researcher chooses to use flow cytometry to answer a scientific question, they first have to build a polychromatic panel that will take advantage of the power of the technology and experimental design. When we set up to use flow cytometry to answer a scientific question, we have to design a polychromatic panel that will allow us to identify the cells of interest – the target of the research.  To identify these cells, we need to build a panel that takes advantage of the relative brightness of the fluorochromes, the expression level of the different proteins on the cell,…

This Is How Full Spectrum Cytometry Works

This Is How Full Spectrum Cytometry Works

By: Tim Bushnell, PhD

There are 4 major ways to sort cells. The first way can use magnetic beads coupled to antibodies and pass the cells through a magnetic field. The labeled cells will stick, and the unlabeled cells will remain in the supernatant. The second way is to use some sort of mechanical force like a flapper or air stream that separates the target cells from the bulk population. The third way is the recently introduced microfluidics sorter, which uses microfluidics channels to isolate the target cells. The last method, which is the most common––based on Fuwyler’s work––is the electrostatic cell sorter. This…

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.