Picking The Right Functional Imaging Probe

As biologists, we study the process of life, however, it’s intricacies cannot be captured by a snapshot in time. Generally, the easiest imaging experiments are those where the samples are stained, fixed, and imaged within a few days of procurement, but that too doesn’t capture the dynamic processes common in cells and organisms. Live cell imaging when combined with reporters serves as a powerful tool to provide solid imaging data.

Cameleon —one of the first reporters— was developed in 1997 in Roger Tsien’s lab.  Cameleon is a green fluorescent protein (GFP) that undergoes a conformational change in the presence of Ca2+ providing an excellent reporter to image bursts of Ca2+ in neurons or muscles.

The tool chest of functional reporters has increased exponentially since 1999, so there is likely a reporter developed for your cellular process of interest. If you study popular areas such as ion flux or reactive oxygen species (ROS), there will be several options to select from. Here are the features that you should consider when selecting your probe.

Is The Process Reversible?

When selecting a function imaging probe it’s important to think about the biology of the process you are trying to study.  Often, the concentration of a signaling molecule will both increase and decrease.  If the probe is chemically changed (i.e. cleaved), then the imaging probe is not reversible. If this is the case, only an increase can be observed.

A good example is the H2O2 small molecule probe DCF, which enters the cell and increases in intensity in the presence of  H2O2. An alternative to DCF is the genetically encoded fluorescent protein “HyPer,” which is a circularly permutated YFP (cpYFP). In the presence of H2O2, a reversible disulfide bond is formed that shifts the emission spectrum.  HyPer is thus able to detect if there is a ROS burst due to a stimulus that diminishes with time. 

Figure 1:  Formation of fluorescent compound DCF by ROS (Biotek)

Is The Imaging Probe Ratiometric?

A ratiometric probe is one where you measure intensities over two or more wavelengths for excitation and emission, allowing the measurement to be a ratio of those intensities. Ratiometric probes are more resistant to environmental or experimental changes.  

Using the DCF vs HyPer example, we can examine the differences between the imaging probe types. DCF is not ratiometric and only has one excitation/emission while HyPer has two excitation peaks, one at 420 and another at 500. The change in intensity of emission between these two excitations provides a robust readout of H2O2 changes.  Why is it more reliable?  

Several factors other than the presence of your signaling molecule can affect signal intensity; permeability to a small molecule, expression of a fluorescent protein, and pH are just a few of them. The ratio of the two measurements normalizes for any variation that is affecting the imaging probe, which is not related to the phenomenon we are looking to quantify.  

Figure 2: Ratiometric measurement of ROS using genetically encoded sensor Hyper (Zhang, 2018)

What Is The Dynamic Range Of An Imaging Probe?

Ideally, you want an imaging probe with a broad dynamic range and for the probe to be responsive to the physiological conditions you will be measuring.

For example, you may be interested in the endocytic pathway so you choose to use pHrodo Red Dextran.  pHrodo has approximately a 10 fold difference in fluorescence when comparing pH 5 to pH 7.5, which makes it ideal for studying the endocytic pathway.

However, if you want to compare small changes in the cytosolic pH, pHrodo will only provide a 50% change.  BCECF is a ratiometric indicator and gives a much better dynamic range at neutral pH. Picking the right probe with sufficient dynamic range to measure your phenomenon is critical for robust experiments.

Figure 3: Fluorescent intensity of pHrodo increases as the pH of the environment decrease (ThermoFisher)

What Is The Specificity?

Specificity is a narrow range of substances that an imaging probe will react to. Preferably, you want the reaction to only occur when your molecule or ion of interest is present. SBFI and PBFI are very similar probes, but the size of the binding pocket creates the specificity for Na+ (SBFI) vs K+ (PBFI).  

DCF, the small molecule H2O2 probe, is touted for being specific for just H2O2.  Studies have shown that it likely reacts to other reactive oxygen species such as nitrate as well. If you are only trying to measure reactive oxygen species, then DCF is a good choice. However, if you are trying to determine which species is the major signaling element, then DCF might cause you to draw the wrong conclusion. 

There are many choices to consider when setting up your experiments. Before you begin, it’s important to think about the biological phenomenon you are trying to quantify. Once the data has been obtained, it’s critical to know the limitations of your probes. Understanding the experiment and the limitations will ensure that your conclusion is reproducible.

To learn more about important techniques for your flow microscopy lab, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Expert Microscopy wait list.

Join Expert Cytometry's Mastery Class
Heather Brown-Harding
Heather Brown-Harding

Heather Brown-Harding, PhD, is currently the assistant director of Wake Forest Microscopy and graduate teaching faculty.She also maintains a small research group that works on imaging of host-pathogen interactions. Heather is passionate about making science accessible to everyone.High-quality research shouldn’t be exclusive to elite institutions or made incomprehensible by unnecessary jargon. She created the modules for Excite Microscopy with this mission.

In her free time, she enjoys playing with her cat & dog, trying out new craft ciders and painting.You can find her on twitter (@microscopyEd) a few times of day discussing new imaging techniques with peers.

Similar Articles

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…

4 No Cost Ways To Improve Your Microscopy Image Quality

4 No Cost Ways To Improve Your Microscopy Image Quality

By: Heather Brown-Harding, PhD

Image quality is critical for accurate and reproducible data. Many people get stuck on the magnification of the objective or on using a confocal instead of a widefield microscope. There are several other factors that affect the image quality such as the numerical aperture of the objective, the signal-to-noise ratio of the system, or the brightness of the sample.  Numerical aperture is the ability of an objective to collect light from a sample, but it contributes to two key formulas that will affect your image quality. The first is the theoretical resolution of the objective. It is expressed with the…

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

What Is Total Internal Reflection Fluorescence (TIRF) Microscopy & Is It Right For You?

By: Heather Brown-Harding, PhD

TIRF is not as common as other microscopy based techniques due to certain restrictions. We will discuss these restrictions, then analyze why it might be perfect for your experiment.  TIRF relies on an evanescent wave, created through a critical angle of coherent light (i.e. laser) that reaches a refractive index mismatch.  What does it mean in practice?  A high angle laser reflects off the interface of the coverslip and the sample. Although the depth that this wave penetrates is dependent on the wavelength of the light, in practice it is approximately 50-300nm from the coverslip. Therefore, the cell membrane is…

5 Drool Worthy Imaging Advances Of 2020

5 Drool Worthy Imaging Advances Of 2020

By: Heather Brown-Harding, PhD

2020 was a difficult year for many, with their own research being interrupted- either by lab shutdowns or recruitment into the race against COVID-19. Despite the challenges, scientists have continued to be creative and have pushed the boundaries of what is possible. These are the techniques and technologies that every microscopist was envious of in 2020. Spatially Resolved Transcriptomics Nature Methods declared that spatially resolved transcriptomics was the 2020 method of the year. These are a  group of methods that combine gene expression with their physical location. Single-cell RNA sequencing (scRNAseq) was originally developed for cells that had been dissociated…

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.