Researchers have developed a novel set of chemical probes to improve real-time imaging of the activity that takes place inside individual cells.
Described in the journal eLife, the probes, called FLAREs (FLuorescence Anisotropy REporters), can also be used alongside other optical methods, such as optogenetics, to provide more in-depth analysis of living tissue. They could ultimately help with studies on how cell signalling pathways are regulated in space and time and how different pathways ‘crosstalk’ with one another.
FLAREs were designed by researchers at UC San Diego School of Medicine and Johns Hopkins University to accurately detect the activity of enzymes, called kinases, and second messengers in cells. Second messengers are molecules that take signals received by the cell surface, such as the arrival of a drug or hormone, to molecules in other areas of the cell.
“Genetically encoded biosensors have revolutionised the study of cell signalling by allowing the real-time monitoring of signalling activities in live cells,” says first author Brian Ross, PhD Candidate in Biomedical Engineering at Johns Hopkins University. Existing biosensors can be divided into two broad classes: single-colour, where the readout is a change in fluorescence intensity, and ratiometric, where the readout is a ratio between two different fluorescence intensities. When creating a fluorescence image, light within a defined range in the colour spectrum is collected – called a colour channel.
“As they only require one colour channel, single-colour sensors allow for more flexibility in imaging experiments,” Ross explains. “However, they are sensitive to variations in the concentration of the probes, which can be caused by changes in the cell shape or illumination intensity. On the other hand, sensors with a ratiometric readout cancel out many of these variations, but the requirement for two distinct colour channels limits their ability to monitor multiple signalling activities at the same time within a cell.”
Taking these limitations into account, Ross and his team created a panel of probes that occupy only a single channel, while still cancelling out the effects of varying imaging conditions and probe concentrations. They based their probes on homo-FRET imaging – a single-colour method that is often used to study the clustering of proteins in cells.
“Our FLAREs combine the ability of homo-FRET to track multiple elements of a cell with the ability to provide quantitative ratiometric readouts,” explains senior author Jin Zhang, Professor of Pharmacology at UC San Diego School of Medicine. “Unlike hetero-FRET sensors that are often used one at a time, FLAREs involve the use of a variety of colour sensors to demonstrate imaging of three activity reporters working simultaneously in the same cell.”
The team also demonstrated the probes’ compatibility with other imaging techniques, including optogenetics. Zhang suggests further development and optimisation could improve the probes’ performance, expand the colour palette and extend the design to include other signalling activities, potentially making them a popular tool in future research on neurons and other cells.
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