If You Don’t Know This About GFP, FITC, And PE, You Might Publish False Flow Cytometry Data

When we learn about fluorescence, the first thing we are told is that fluorophores emit photons that are higher wavelength than the photons that they absorb.

What this specifically refers to is the stokes shift, which results from non-radiative energy transfer during the fluorescence process. When a photon is absorbed by a fluorophore molecule, some of the resultant energy is lost in molecular vibration and movement (among other things) so that the energy released after fluorescence is lower than the energy absorbed. Since wavelength is inversely proportional to energy, this lower output energy light is higher in wavelength than the input light.

It is important to examine a fluorophore in terms of its excitation and emission spectra, which essentially indicate the probability that a molecule will emit a photon of a certain wavelength of light given an excitation photon of a given wavelength. Figure 1 below illustrates the excitation and emission spectra of FITC under conditions of 488 nm excitation.

Figure 1

FITC’s emission maximum is around 530 nm. This means that if we excite this molecule, there is a very high probability that it will emit green photons. Thus, we choose bandpass filters on our flow cytometers that are centered on this region of the spectrum (e.g. 525/30) in order to capture as many photons from the fluorescence process as possible.

However, FITC’s emission is not restricted to green photons; it also emits yellow, orange, red, albeit at lower probabilities.

You may notice something curious about the spectrum if you look closely at the emission curve immediately around the excitation line. It appears that there is finite (but very low) probability that photons will be emitted below the laser excitation wavelength. This would mean that the photons we harvest from the molecule are higher in energy than the photons that we introduce.

But wouldn’t this mean that we are getting more energy out than we are putting in and defying the Law of Conservation of Energy?

The spectra of PE at two different excitation wavelengths, shown in Figure 2, illustrate this phenomenon even more compellingly.

Figure 2

PE has more complex excitation state than does FITC. Most strikingly, it has two excitation maxima: one at around 488 nm and one at around 561 nm. Interestingly, under conditions of 561 nm excitation, there is an appreciable probability that emitted photons will be higher in energy than absorbed photons.

How is this physically possible without violating some pretty well-established universal rules?

The answer is simple: the molecule provides some of the energy itself.

According to Howard Shapiro (p. 113 of Practical Flow Cytometry),

“Okay, you might say, but the excitation spectrum overlaps the emission spectrum. If the shape of the emission spectrum remains the same, no matter what the excitation wavelength is, wouldn’t that mean that we could get 500 nm emission from 5 10 nm excitation, seemingly violating the Law of Conservation of Energy? Well, we could get 500 nm emission from 510 nm excitation, if the molecule in question was already in a vibrational excited state when the 510 nm excitation photon arrived. The cost of electronic excitation remains the same, but the molecule itself is coming up with some of the money.”

There you have it – this odd and commonly misunderstood phenomenon is real and most importantly doesn’t defy the laws of physics.

You may notice this in your flow cytomtetry data when measuring FITC in the presence of PE, especially if using wide filters (e.g. 525/50 BP instead of 525/30 BP). Any PE signal you see in the FITC PMT is independent of the laser line. In other words, you would see this signal regardless of whether you excited PE with 488 laser light (the emission in this case would be higher in wavelength than the excitation line) or the 561 nm laser line (the emission would be lower in wavelength than the excitation line).

Note: This would only be observable on a 488/561 colinear system, where the emission light from both lasers are collected on the same optical path. Multi-spot or multi-pinhole instruments would not have a detector with FITC collection filters on it.

In fact, you would probably see more signal in the FITC detector when exciting at 561 than 488, as the PE molecule is more efficiently excited at 561 than at 488. Most critically, the shape of an emission curve is independent of the excitation wavelength.”

So, the statement “fluorophores emit photons that are higher in wavelength than the photons they absorb” is actually incorrect.

A more correct statement is “fluorophores emit photons based upon their emission spectrum, whose maximum is shifted to a higher wavelength than the maximum of the excitation wavelength.” These kinds of phenomena remind us that fluorescence is a lot more complicated than we usually give it credit for (and a lot more interesting, for that matter).

Additional ReferencesAll spectra from BD Fluorescence Spectrum Viewer.

Shapiro, H. M. Practical Flow Cytometry. Hoboken, New Jersey: John Wiley & Sons. 2003.

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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.

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