Why Understanding The Jablonski Diagram Will Help You Publish Your Flow Cytometry Data

We are all used to interruptions during our working day, from the ping of an email notification to the knock of a fellow researcher who wants to troubleshoot their experiment.

Fortunately, most of these interruptions only last a few minutes. Some past researchers were not so lucky.

Imagine your work being interrupted by a war. Imagine it being interrupted by two wars that you had to fight in.

Alexander Jablonski often had his studies interrupted, not by emails or colleagues, but by war. Jablonski’s work was held up for years due to military service in two wars. First, he served in the war for Polish independence in 1916, then he served again in the Polish-Bolshevik war in 1920.

In 1930, after the wars were over, Jablonski earned his doctorate. His dissertation, entitled “On the influence of the change of wavelengths of excitation light on the fluorescence spectra” laid the foundation for the rest of his career in physics.

A few years later, in 1935, he created what we flow cytometrists call the Jablonski Diagram.

jablonski diagram explanation | Expert Cytometry | jablonski diagram fluorescence

The Jablonski Diagram Explained 

Flow cytometrists use the Jablonski diagram to aid in understanding and explaining the kinetic events of fluorescence.

Fluorescent compounds start at the ground state (S0) until they are excited by interacting with a photon of light (Step 1). This photon excites the compound, promoting an electon to a higher energy state (S1’).

As shown in Step 2, some of this energy is lost by emission of heat and other non-radiative processes, leading to the S1 state.

The final step in the process (Step 3) shows an electron falling back to the ground state while releasing a photon of light. This photon has a lower energy (higher wavelength) than the exciting photon of light.

3 Practical Takeaways From The Jablonski Diagram 

During a flow cytometry experiment, we capture this photon using a photo-multipler tube (PMT).

With knowledge of the filters in front of that PMT, we can assign the signal to a specific fluorochrome in our panel, leading to the identification of the cell of interest.

The Jablonski diagram helps researchers understand several critical factors about the physics of fluorescence, which is critical to designing higher quality experiments and collecting higher quality data that has a better chance of being published.

Here are 3 practical takeaways from the Jablonski diagram…

1. Quantum Yield Helps Determine “Brightness” 

Looking at the Jablonski diagram, you can see that some energy is lost without generating light. In other words, not all of the photons absorbed are released again and therefore will not be measured by your flow cytometer or cell sorter.

The difference between the number of photons absorbed versus the number of photons released for the instrument’s detectors to pick up is called the quantum yield. 

This yield, in part, is what makes some of your fluorochromes “bright” and therefore best used for dim cell markers, or “not-so-bright” and thus better suited for highly expressed cell surface markers.

For example, Phycoerythrin (PE) has a quantum yield of 0.84, meaning that for every 100 photons absorbed, 84 are released just a few femtoseconds later at a longer wavelength. In this case, it’s easy to see why PE is a favorite fluorochrome for use with low-expression markers in multicolor panels.

Everything else being equal, a marker stained with PE will be seen as brighter than one stained with a lower quantum yield dye, such as Cy3, which has a quantum yield of 0.15. 

But, of course, everything else is not equal. Cy3 is in fact excited by a different range of light than PE and in some conditions will absorb more photons than other dyes on the same laser line.

2. The Extinction Coefficient Helps Determine Fluorescence Intensity

Quantum yield is not everything in terms of the brightness of a fluorochrome.

Output is also a component of input. A compound that can absorb more energy at a particular wavelength than another dye, can still be “brighter” and therefore more easily detected by a flow cytometer. This is true even if the compound has a lower quantum yield than the other dye.

Fluorescence intensity at a given wavelength is thought to be proportional to the product of quantum yield and extinction coefficient. 

Compared to fluorescein, with an extinction coefficient of approximately 80,000 cm-1M-1, PE and other phycobiliproteins have very large extinction coefficients, some on the order of 2.4 million cm-1M-1. These large extinction coefficients and high quantum yield values make phycobiliproteins very attractive fluorochromes.

Quantum dots also have very high extinction coefficients (~2×106 cm-1M-1), though they utilize light sources in the violet range and below.

3. The Stokes’ Shift Allows The Use Of All Available Excitation Sources. 

To efficiently use all available excitation sources, flow cytometrists have learned to place multiple dyes on the same laser line. However, this is only possible because of a key characteristic of the Jablonski diagram, the Stokes’ shift.

The Stokes’ shift is routinely visualized in excitation and emission spectra diagrams, such as the diagram below from Life Technologies. This shift is the difference in energy and wavelength represented by (hνEX – hνEM) in the Jablonski diagram.

As the diagram shows, the Stokes’ shift, in concert with your flow cytometer’s optical filters, allows you to separate distinct signals for 4 different fluorochromes using the 488nm blue laser line.

fluorescence jablonski diagram | Expert Cytometry | jablonski diagram in photochemistry

The Jablonski diagram is simple in nature, but powerful in terms of its practical takeaways. Understanding the various characteristics of the diagram, including the quantum yield, extinction coefficient, and Stokes’ shift, will help you design better flow cytometry experiments. Consider these three characteristics when determining your fluorescent dyes and markers for your next experiment. By understanding the fundamentals of fluorescence that Alexander Jablonski laid out after warring many years ago, you’ll increase the quality of your flow cytometry data.

To learn more about key flow cytometry processes 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.

Join Expert Cytometry's Mastery Class

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.

Tim Bushnell, PhD

Similar Articles

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

The Power Of Spectral Viewers And Their Use In Full Spectrum Flow Cytometry

By: Tim Bushnell, PhD

What photon from yonder fluorochrome breaks?  It is … umm… hmmm. Let me see. Excitation off a 561 nm laser, with an emission maximum of 692 nm. I’m sure if Shakespeare was a flow cytometrist, he might have written that very scene. But the play is lost in time. However, since the protagonist had difficulty determining what fluorochrome was emitting photons, let’s consider how this could be figured out. In my opinion, one of the handiest flow cytometry tools is the spectral viewer. This tool helps visualize the excitation and emission profile of different fluorochromes, as well as allowing you…

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

Fickle Markers: Solutions For Antibody Binding Specificity Challenges

By: Tim Bushnell, PhD

Reproducibility has been an ongoing, and important, concept in the sciences for years.  In the area of biomedical research, the alarm was sounded by several papers published in the early 2010’s.  Authors like Begley and Ellis, Prinz and coworkers, and Vasilevsky and colleagues, among others reported an alarming trend in the reproducibility of pre-clinical data.  These reports indicated between 50% to almost 90% of published pre-clinical data were not reproducible.  This was further highlighted in the article by Freedman and coworkers, who tried to identify and quantify the different sources of error that could be causing this crisis.  Figure 1,…

3 Must-Have High-Dimensional Flow Cytometry Controls

3 Must-Have High-Dimensional Flow Cytometry Controls

By: Tim Bushnell, PhD

Developments such as the recent upgrade to the Cytobank analysis platform and the creation of new packages such as Immunocluster are reducing the computational expertise needed to work with high-dimensional flow cytometry datasets. Whether you are a researcher in academia, industry, or government, you may want to take advantage of the reduced barrier to entry to apply high-dimensional flow cytometry in your work. However, you’ll need the right experimental design to access the new transformative insights available through these approaches and avoid wasting the considerable time and money required for performing them. As with all experiments, a good design begins…

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

The Fluorochrome Less Excited: How To Build A Flow Cytometry Antibody Panel

By: Tim Bushnell, PhD

Fluorochrome, antibodies and detectors are important. The journey of a thousand cells starts with a good fluorescent panel. The polychromatic panel is the combination of antibodies and fluorochromes. These will be used during the experiment to answer the biological question of interest. When you only need a few targets, the creation of the panel is relatively straightforward. It’s only when you start to get into more complex panels with multiple fluorochromes that overlap in excitation and emission gets more interesting.  FLUOROCHROMES Both full spectrum and traditional fluorescent flow cytometry rely on measuring the emission of the fluorochromes that are attached…

Flow Cytometry Year in Review: Key Changes To Know

Flow Cytometry Year in Review: Key Changes To Know

By: Meerambika Mishra

Here we are, at the end of an eventful year 2021. But with the promise of a new year 2022 to come. It has been a long year, filled with ups and downs. It is always good to reflect on the past year as we move to the future.  In Memoriam Sir Isaac Newton wrote “If I have seen further, it is by standing upon the shoulders of giants.” In the past year, we have lost some giants of our field including Zbigniew Darzynkiwicz, who contributed much in the areas of cell cycle analysis and apoptosis. Howard Shapiro, known for…

What Star Trek Taught Me About Flow Cytometry

What Star Trek Taught Me About Flow Cytometry

By: Tim Bushnell, PhD

It is no secret that I am a very big fan of the Star Trek franchise. There are many good episodes and lessons explored in the 813+ episodes, 12 movies (and counting). Don’t worry, this blog is not going to review all 813, or even 5 of them. Instead, some of the lessons I have taken away from the show that have applicability to science and flow cytometry.  “Darmok and Jalad at Tanagra.”  (ST:TNG season 5, episode 2) This is probably one of my favorite episodes, which involves Picard and an alien trying to establish a common ground and learn…

5 Flow Cytometry Strategies That Sun Tzu Taught Me

5 Flow Cytometry Strategies That Sun Tzu Taught Me

By: Tim Bushnell, PhD

Sun Tzu was a Chinese general and philosopher. His most famous writing is ‘The Art of War’, and has been studied by generals and CEOs, to glean ideas and strategies to help their missions. I was recently rereading this work and thought to myself if any of Sun Tzu’s lessons could apply to flow cytometry.  So I have identified 5 points that I think lend themselves to thinking about flow cytometry.  “Quickness is the essence of the war.” In flow cytometry, speed is of the essence. The longer the cells are out of their natural environment, the less happy they…

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

A Basic Guide To Flow Cytometry (3 Foundational Concepts)

By: Meerambika Mishra

Mastering foundational concepts are imperative for successfully using any technique or system.  Robert Heinlein introduced the term ‘Grok’  in his novel Stranger in a Strange Land. Ever since then it has made its way into popular culture. To Grok something is to understand it intuitively, fully. As a cytometrist, there are several key concepts that you must grok to be successful in your career. These foundational concepts are the key tools that we use day in and day out to identify and characterize our cells of interest.  Cells Flow cytometry measures biological processes at the whole cell level. To do…

Which Fluorophores To Use For Your Microscopy Experiment

Which Fluorophores To Use For Your Microscopy Experiment

By: Heather Brown-Harding, PhD

Fluorophore selection is important. I have often been asked by my facility users which fluorophore is best suited for their experiments. The answer to this is mostly dependent on whether they are using a widefield microscope with set excitation/emission cubes or a laser based system that lets you select the laser and the emission window. Once you have narrowed down which fluorophores you can excite and collect the correct emission, you can further refine the specific fluorophore that is best for your experiment.  In this blog  we will discuss how to determine what can work with your microscope, and how…

Top Industry Career 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.