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.

If you’re serious about flow cytometry and want to be a part of our Mastery Class, click here to learn more.

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

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

By: Tim Bushnell, PhD

Cell cycle seems like such a straightforward assay. At its heart, it is a one-color assay and should be a simple protocol to follow. However, as discussed before, fixation and dye concentrations are critical. Once those are optimized, it becomes important to run the cells low and slow in order to get the best quality histograms for analysis — the topic of another blog. Adding the critical CEN and TEN controls will help standardize the assay, and ensure consistency and reproducibility between runs while helping identify non-standard (aneuploid, polyploid) populations from normal ploidy. Trying to isolate and focus on specific…

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

By: Tim Bushnell, PhD

Cell cycle analysis appears to be deceptively easy in concept, but details are absolutely critical. It is not possible to hide the data if there is poor sample preparation, incorrect dye ratios, too much (or too little) staining time, etc. Forgetting RNAse when using PI will doom your data to failure. Take these basics into account as you move into performing this simple, yet amazingly informative assay.

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

How To Choose The Correct Antibody For Accurate Flow Cytometry Results

By: Tim Bushnell, PhD

With the added emphasis on reproducibility, it is critical to look at every step where experiments can be improved. No single step makes an experiment more reproducible, rather it is a process, making changes at each stage that leads to reproducibility. Antibodies comprise a critical component that needs to be reviewed. As Bradbury et al. in a commentary in Nature pointed out, the global spending on antibodies is about $1.6 billion a year, and it is estimated about half of that money is spent on “bad” antibodies. This does not include the additional costs of wasted time and effort by…

5 Essential Beads For Flow Cytometry Experiments

5 Essential Beads For Flow Cytometry Experiments

By: Tim Bushnell, PhD

Flow cytometry is designed to measure physical and biochemical characteristics of cells and cell-like particles using fluorescence. Fundamentally, any single-particle suspension (within a defined size range) can pass through the flow cytometer. Beads, for better or worse, are a sine qua non for the flow cytometrist. From quality control,to standardization, to compensation, there is a bead for every job. They are important — critical, even — for flow cytometry.

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

How To Use Flow Cytometry To Measure Apoptosis, Necrosis, and Autophagy

By: Tim Bushnell, PhD

Using flow cytometry and a host of different reagents, it is possible to tease out how your cells may have died. Using these tools, you can readily eliminate the various suspects and come to your conclusion as to how your treatment may have killed your cells of interest. Here are some reagents to consider when measuring apoptosis, necrosis, and autophagy.

Flow Cytometry Protocols To Prevent Sample Clumping

Flow Cytometry Protocols To Prevent Sample Clumping

By: Tim Bushnell, PhD

Good flow cytometry depends on a high quality, single cell suspension. If the cells put through the instrument are not of high quality, the ensuing data will be difficult to analyze. Likewise, if the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Sample preparation becomes the critical first step in any flow cytometry experiment. To get high quality results, follow these 3 sample preparation steps.

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

How To Compensate A 4-Color Flow Cytometry Experiment Correctly

By: Tim Bushnell, PhD

Compensation in flow cytometry is a critical step to ensure accurate interpretation of data. It is also one of the areas that’s steeped in mystery, myths and misinformation. Manually adjusting the compensation values based on how the populations look, or so-called ‘Cowboy Compensation’, is not the correct way to determine proper compensation. The best practices for compensation involve following some very specific rules. Here are 4 steps to correctly compensating 4+ color flow cytometry experiments.

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

How To Differentiate T-Regulatory Cells (Tregs) By Flow Cytometry

By: Tim Bushnell, PhD

T regulatory cells (Tregs), formerly known as T suppressor cells, are a T cell subset with direct roles in both autoimmunity and responses to pathogens. Tregs decrease inflammation via the secretion of immunosuppressive cytokines (IL-10, TGF-b) and also through direct suppression of inflammatory effector T cells (such as Th1 and Th17 cells). Given the importance of this unique T cell subset in so many immune responses, many investigators feel remiss if they immunophenotype their cell populations of interest without including a Treg measurement in the mix. But quantifying Tregs can be complicated. This article will show you how to quantify…

How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment

How Cell Culture Medium Can Decrease Cell Viability During A Flow Cytometry Cell Sorting Experiment

By: Tim Bushnell, PhD

When setting up a cell sorting experiment, there are many things to consider. You must consider which controls you’re going to use, how you’re going to compensate the experiment, which instrument and which instrument settings are ideal, and how you plan to analyze, gate, and present your data. With so many things to consider, it’s easy to lose site of the small things that can drastically affect the viability of your cells, including the composition of your suspension buffer. The composition of the suspension buffer for preparation, staining, analyzing and sorting is perhaps the most important parameter for maintaining viability…

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.