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.
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.
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.
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:
|Accuri||BD Biosciences||80 MHz||24|
|FACSCanto II||BD Biosciences||20 MHz||14|
|FACSDiva (LSR-II, Aria, Fortessa)||BD Biosciences||10 MHz||14|
|FACSVerse||BD Biosciences||25 MHz||14|
|MoFlo XDP||Beckman-Coulter||100 MHz||16|
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.
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.
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