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Part 3: Familiarizing yourself with fluorophores, excitation/emission, and compensation

As you’re getting ready for your next big flow cytometry experiment, you’re going to need to figure out which fluorophores to use. There are a lot of pieces that go into learning about fluorophores, so I’ll break it up into the following sections:

1. What are lasers and what do they do?

2. What are filters/bandpasses?

3. What is compensation?

4. What are some good fluorophores that work well together in a panel?

What are lasers and what do they do?

Different flow cytometer models will have different lasers available to use. Here are a few examples of flow cytometers and their lasers:

BD FACSCanto: 633 nm, 488 nm, and 405 nm

BD FACSAria III: 633 nm, 561 nm, 445, nm, 405 nm, 375 nm

BD FACSSymphony: offers 20 different lasers

Throughout this blog, I will be using Biolegend's spectra analyzer for my examples (https://www.biolegend.com/spectraanalyzer). It’s a good tool to use as you’re planning your panels. Below is an example of how we will show different lasers throughout this blog; here I show lasers at 355 nm, 488 nm, 561 nm, and 633 nm.

Figure 1. Examples of different lasers that are used to excite fluorophores. Lasers shown: 355 nm, 488 nm, 561 nm, and 633 nm.

The more lasers that a flow cytometer has, the more flexibility you have to choose certain lasers for different fluors (i.e. you can obtain efficient energy transfer from the laser to the fluorophores). The lasers are used to excite the fluorophores linked to your antibodies. Let’s show FITC as an example:

Efficient excitation of FITC is between 480-500 nm, with lambda max* of 490 nm, The lambda max of FITC emission is at 520 nm,

*Lambda max is a wavelength of a spectrum where there is the strongest photon absorption (i.e. the peak of the spectrum).

Say we have a flow cytometer with three lasers: 405 nm (violet) , 488nmn (blue) , and 633 (red). Which laser should we use to excite FITC?

A 405 nm laser will excite FITC, but not very efficiently (~5% on excitation spectra).

A 633 nm laser does not fall within the excitation spectra of FITC, and therefore would not excite FITC.

A 488 nm laser works nicely for exciting FITC (~80% on excitation spectra).

Figure 2. Example of FITC excitation, emission, and excitation lasers.

Here are a few other examples of fluorophores and their excitation, emission, and laser used to excite them.

Table 1. Examples of other fluorophores and their excitation, emission, and excitation lasers.

What are filters/bandpasses?

As you are acquiring your data, you’ll need to collect light on certain wavelengths to get accurate data. For the FITC example above, there is a range of wavelengths that are emitted by FITC (~480-610 nm); we do not necessarily need to collect data from all of these emitted wavelengths. If you collect through large ranges of emissions, it will be difficult for you to acquire data on multiple fluorophores at one time; you want to look at one dye at a time per detector.

Instead, we can use filters to collect data from a constrained set of wavelengths. For example, the filter used for FITC data acquisition is 530/30; which means that it collects emission data for FITC from emitted wavelengths from 515-545; filter is 530, with a bandpass (range) of 30 nm. See pictorial representation below:

Figure 3. Example of FITC properties and filter/bandpass used for data acquisition.

Below is another example for APC. The lambda max for the excitation of this fluorophore is 650 nm, while the emission is 660 nm. A 633 laser can be used to excite this fluorphore with a filter/bandpass of 660/20 for data acquisition.

Figure 4. Example of APC properties and filter/bandpass used for data acquisition.

What is compensation?

Sometimes you will run into an issue where the emission spectra from two different fluorophores significantly overlap with one another, which could result in you observing a false positive in your data. We will use PE and FITC being excited with the same 488 laser in this example.

Figure 5. Compensation example for PE and FITC. On the top panel, emission and filters for FITC (yellow/blue) and PE (orange/green) are shown. In order to compensate samples, you need to run single stained samples to determine the degree of overlap between the fluorophores. The bottom panel shows FITC overlap into the PE channel. By single staining, you can perform a background subtraction of this signal to ensure that FITC does not spill over into the PE channel for your samples.

What you see from this plot is that the emission from FITC “spills over” into the filter use for PE data acquistion, such that your signal for PE would increase due to signal from FITC. This “spill over” is called spectral overlap. In order to properly compensate, you will need to run single stained samples of both PE and FITC. For your single stained FITC sample, you will need to see how much signal is being emitted in the PE channel and adjust it so that it does not spill over into the PE channel- i.e. compensation. Your flow cytometer program will have a compensation set-up that will easily allow you to modify your compensation values.

Below is another example between Alexa Fluor 700 and APC/Cy7 being excited with a 633 nm laser. In this example, Alexa Fluor 700 and APC/Cy7 both overlap each other’s filter.

Figure 6. Example of spectral overlap between Alexa Fluor 700 and APC/Cy7.

I want to give one other example of spectral overlap between APC/Cy7 and PE/Cy7. The emission spectra for APC/Cy7 and PE/Cy7 overlap almost completely, yet you can still use them in a panel together!

As your cells go through the flow cytometer, they are hit by the different lasers one at a time, not simultaneously. Therefore if you have emission spectral overlap using two different excitation lasers, you can still differentiate between the two fluorophores because detection for the two is spatially separated.

Figure 6. Example of spectral overlap between APC/Cy7 and PE/Cy7 with two different excitation lasers.

What are some good fluorophores that work well together in a panel?

There are many different combinations of fluorophores that you can use in panels. I will give an example 7 colors I have used together in my panels. I’ll show each laser that is used to detect the fluorophores used, and the bandpasses for each. I will not show excitation spectra for these plots, to keep it a little simpler. The panel consists of: BV421, FITC, PE, PE/Cy7, APC, AF700, and APC/Cy7.

Figure 7. Example of a 7 color panel of fluorophores that can be used together.

I know that was a bit of a ride! But hopefully there were some good lessons there. Leave a comment to discuss!

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