Skip to content

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

Written by Tim Bushnell, PhD

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.

Tim Bushnell, PhD

BOOKS

Advanced Microscopy

Learn the best practices and advanced techniques across the diverse fields of microscopy, including instrumentation, experimental setup, image analysis, figure preparation, and more.
flow cytometry tablet eBook cover

Modern Flow Cytometry

Learn the best practices of flow cytometry experimentation, data analysis, figure preparation, antibody panel design, instrumentation and more. 

Advanced 4-10 Color Compensation

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.

Top 40 Networking Scripts For PhDs

If you want to get replies from top employers and recruiters, this ebook is for you. These networking scripts will show you the exact words ...

Informational Interviews For PhDs

If you want to learn how to set up and execute informational interviews with PhDs working in industry, this ebook is for you. Here, you ...

Industry Resume Guide For PhDs

If you have a PhD and want to create the perfect industry resume to attract employers, this ebook is for you. Here, you will get ...

Top 20 Industry Jobs For PhDs

If you want to learn about the top 20 industry careers for PhDs regardless of your PhD background, this ebook is for you. Here, you ...

Salary Negotiation For PhDs

If you have a PhD and want to learn advanced salary negotiation strategies, this ebook is for you. Here, you will learn how to set ...

Top 20 Transferable Skills For PhDs

If you want to learn the top 20 transferable skills the industry employers ranked as most important for PhDs to include on their resumes and ...

Related Posts You Might Like

5-Point Guide To Buying A New Microscope For Your Lab

Written By: Heather Brown-Harding, PhD Have you ever noticed how painful it can be to purchase a new microscope? It would be hard to miss ...
Read More

Ask These 7 Questions Before Purchasing A Flow Cytometer

Written By: Tim Bushnell, PhD Using funds to make a capital purchase can be an exciting time in a facility. If you don’t have the ...
Read More

6 Areas Of Consideration For Flow Cytometry Cell Cycle Analysis

Written by Tim Bushnell, PhD As discussed previously, cell cycle assays require optimization of fixation and dye concentrations, but that is just the beginning. There ...
Read More

Why Cell Cycle Analysis Details Are Critical In Flow Cytometry

Written by Tim Bushnell, PhD The lifecycle of a cell can be described in stages. In diploid cells, much of the time they exist in ...
Read More

Instrument Quality Control For Reproducible Flow Cytometry Experiments

Written by Tim Bushnell, PhD The NIH has released a series of reproducibility guidelines that scientists must address. These guidelines have been introduced because there's ...
Read More

How to Optimize Flow Cytometry Hardware For Rare Event Analysis

Written by Tim Bushnell, PhD “Not everything that can be counted counts and not everything that counts can be counted.” — William Bruce Cameron (but ...
Read More