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.

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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.

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