Introduction
Fluorochromes are a critical component of flow cytometry experiments. Without them, we would not be able to detect specific antigens on the surface of a cell or particle, making characterizing cell phenotypes and functions impossible. Fluorochromes operate with the principles of fluorescence, and in this post I’ll explain how fluorescence works, and then dive into fluorochromes and flow cytometry.
What is Fluorescence?
Fluorescence is the emission of light after the absorption of energy, usually from a different source of light or radiation.
After absorbing energy, fluorescent light is emitted at specific wavelengths, and some of those wavelengths correspond with visible colors. For example, when a jellyfish fluoresces and it looks blue, the corresponding wavelength at which the light travels to your eye is approximately 488nm.
Light with the shortest wavelengths carry the most energy with them, in the form of a photon. A photon is a particle of light that carries energy and travels in a wave motion. Conversely, light with the longest wavelengths carry the least amount of energy with each photon. So, light emitted at different wavelengths carries different amounts of energy with it. This will become important as we discuss fluorescent brightness later on.
What are Fluorochromes?
A fluorochrome is a molecule that absorbs light from a specific wavelength and emits light, or fluoresces, in another wavelength. More specifically, when fluorochromes absorb light from a source they become temporarily excited (a state of increased energy), and then emit energy in the form of light to return back to its ground state. This emitted light can be collected and identified, enabling researchers to recognize what fluorochrome they are seeing by the wavelength of emitted light. You may have heard of popular fluorochromes such as FITC, PE, and APC. These fluorochromes are excited by blue light (488nm), yellow-green light (561nm), and red light (640nm), respectively. They emit light at different wavelengths, too. These differences makes it possible to distinguish the three different fluorochromes when looking at the light that they emit.
The above image highlights this process. High-energy blue light excites a fluorochrome so that the fluorochrome increases its energy to the S1’ state. From here, the fluorochrome releases a bit of energy in the form of heat to enter the S1 excited state. Subsequently, the fluorochrome emits lower-energy green light (in the form of photons) until it returns to its ground energy state.
You can see then that the excitation and emission wavelengths are different from each other. The difference in the peak of these wavelengths (there is some variation of wavelengths emitted around the peak) is called Stokes shift, shown here:
Using fluorochromes with a large Stokes shift is ideal in flow cytometry so that the emitted light collected is not confused with light generated from the source, usually from a laser.
A wide variety of fluorochromes are available that are excited by different wavelengths, and also emit light at different wavelengths from each other. It’s also possible to detect and distinguish fluorochromes that are excited by light traveling at the same wavelength, as long as they emit light in different wavelengths, shown below:
The best way to determine the excitation and emission spectra of a fluorochrome is to use a spectral viewer, such as the one shown below by BD. Many companies have spectral viewers on their websites that provide excitation and emission spectral profiles for the fluorochromes that they sell, such as this one for PE:
The dotted line represents the excitation spectra. This means that PE is excited primarily by light between the wavelengths of 450-600nm. The solid and filled line represents the emission spectra, showing the PE emits light between 550 and 650nm.
This graph also highlights the important fact that all fluorochromes are excited and emit light at a range of wavelengths, though often only their peak excitation and emissions are noted by companies (such as the fluorochrome in the above example, with peak excitation at 565nm and emission at 578nm).
Fluorochromes are not all created equal
If you’ve worked with fluorochromes, you know that some look brighter than others in the microscope or in a flow cytometry instrument. For example, PE and BV421 are two very bright fluorochromes. This is because when fluorochromes emit energy in the form of photons, some photons actually carry more energy with them than other photons, as a direct proportion to their wavelength. So, fluorochromes that are excited by the UV and Violet lasers tend to emit light at low wavelengths and can be quite bright.
However, other factors can also influence fluorochrome brightness, such as the efficiency of the excitation and the laser with which it is being excited. For example, PE can be excited by both the blue (488nm) and yellow-green (561nm) lasers. However, the excitation efficiency (or maximum) is highest when excited with the 561nm laser. So, if you have an instrument that only has a 488nm laser but not a 561nm laser, fewer PE molecules will be excited, and fewer photons will be emitted as a result. This means that the emitted light that you see will be dimmer than if you had used the 561nm laser.
Therefore, when testing new fluorochromes, don’t assume that a fluorochrome with a high wavelength will have less energy and be “dim”, or vice versa. It’s important to test all of your reagents and assess their brightness on each specific instrument.
Once you’ve established “dim” and “bright” fluorochromes on your instrument, use this information to decide which antigen you need to detect with which fluorochrome. Brighter fluorochromes are important to use when the antigen you’re detecting is rare or dimly expressed and cannot be otherwise resolved with using a dimmer fluorochrome.
One important note is that if two fluorochromes have very similar excitation and emission wavelengths, they’ll be indistinguishable from each other in a conventional cytometer. For example, FITC and Alexa Fluor 488 cannot be distinguished from each other because they are both excited by the 488nm laser, and the peak of emitted light is right around 520nm. To the computer (and to the human eye), the two colors look identical, and cannot be distinguished from each other.
One exception is when using spectral flow cytometers, where it’s possible to distinguish certain fluorochromes from each other even when they have overlapping excitation and emission profiles. This is because although their excitation and emission peaks are the same, their overall excitation and emission ranges, or their “spectral signature”, are unique enough that they can be teased apart.
Fluorochromes Conjugated to Antibodies Allow for Detection of Known and Specific Antigens
Of critical importance is that fluorochromes readily bind, or are conjugated to, antibodies:
Antibodies are specific to one and only one antigen. Thus, mixing conjugated antibodies with cells or particles of interest facilitates those antibodies to bind to specific antigens, allowing us to indirectly identify those antigens based on their emitted fluorescence, shown in the images below:
Mixing multiple different antibodies tagged with fluorochromes together with a cell or particle results in being able to identify several different antigens based on the different emission spectra of the different fluorochromes:
It is by using a mixture of different antibodies conjugated to different fluorochromes that allows researchers to deeply characterize cell phenotypes and functions.
Conclusions
Fluorochromes are key to identifying cells and their functions using flow cytometry. The ability for fluorochromes to fluoresce and emit light at defined wavelengths allows researchers to indirectly characterize a wide variety of particles and deeply advance knowledge of human, animal, and plant biology.