MBIC scientists develop novel method of cell imaging
Experimentalists are often faced with the task of staining cells for visualization under a microscope.
There are a variety of techniques to approach this problem, but they all pose the similar threat of yielding smeared or unclear results. However, thanks to groundbreaking research at Carnegie Mellon’s Molecular Biosensor and Imaging Center (MBIC), the process of cell imaging may have just taken one giant leap for biologists.
The visualization of proteins and biomolecules within a cell is traditionally accomplished in two ways. The first technique involves dyes, like malachite green and coomassie blue, which bind to a protein of interest and change the color of the solution.
This provides quantitative and spatial information about where the protein is located and how much of it is present in the sample. The information is directly proportional to the amount of dye that is added.
A second imaging technique involves the use of fluorescence molecules — most commonly the green fluorescent protein, or GFP. This protein is extracted from the jellyfish Aequorea victoria and emits a brilliant green color when exposed to blue light.
The fluorescence can be seen under a fluorescent microscope. GFP is genetically expressed in various organisms using viral vectors, making them useful illuminators of specific biomolecules within cells.
However, both dyes and fluorescent molecules pose a nagging problem: background disturbance. Unbound dye molecules are visible in a sample, making the resulting image ambiguous and muddy. The fluorescence given off by fluorescent molecules like GFP can sometimes wash out the entire image, obscuring the molecule of interest.
“There are huge issues of signal to noise,” said Chris Szent-Gyorgyi, a researcher at the MBIC who headed the effort to isolate fluorogen activating proteins (FAPs), in reference to cell imaging. FAPs are fragments of antibody molecules that can be genetically expressed on an experimenter’s molecule of choice; they create a binding pocket for free-floating dye molecules. When a dye molecule binds to the FAP, the binding event causes it to fluoresce.
“If you’re going to detect 10 or 20 molecules inside a cell and visualize them and localize them, that is not a small task,” Szent-Gyorgyi said.
FAPs are a type of single-chain variable fragments (scFv) composed of a full-size antibody. The scFvs were generated and isolated from a yeast cell library expressing 50,000 to 100,000 scFvs. This allowed scientists to specifically extract fragments that induced fluorescent properties in otherwise non-fluorescent dyes.
The team was able to isolate two non-fluorescent dyes that exhibit this property of induced fluorescence when bound to an scFv: thiazole orange and malachite green.
“What you want, in this case, is a situation where you have a dye that finds a target in a highly specific way and fluoresces,” Szent-Gyorgyi said.
“If the dye is initially dark, and it does not interact adventitiously [randomly] with other cell components and provide background fluorescence, then you have a system whereby you can simply add your dye to a living cell that contains a genetically engineered component,” he added.
FAP technology is a vast improvement from traditional methods of cell imaging because light is given off only when a dye is bound to the FAP. This eliminates background interference from free-floating dye molecules. The mechanism behind FAP technology can be understood by visualizing the FAP as a plastic cup bound to a string.
The string can be pinned onto any number of biomolecules — from cell-surface receptors for scientists studying the cell membrane to intracellular signaling molecules.
Whether a FAP is pinned to one molecule or hundreds of them, it will not give off light if the cup is empty. Fluorescence only occurs only when a derivative of malachite green or thiazole orange enters the cup and binds there.
Malachite green and thiazole orange can be viewed as colorless balls with the potential to emit green or orange fluorescence, respectively, when combined with FAPs. The balls float around in solution, but are invisible unless they land in the binding pocket (or “cup”) of a FAP.
Once combined, the dyes fluoresce. The unbound dye molecules remain dormant, resulting in a clear, disturbance-free image of the molecule of interest.
“One of the tricks of the trade here is working with dyes that do not light up adventitiously,” said Szent-Gyorgyi.
“There are a lot of dyes that are dark and will bind a lot of things non-specifically. We want things that will light up only when bound specifically to the scFv,” he said.
Conventionally, malachine green and thiazole orange do not fluoresce.
The breakthrough in this technique comes from the fact that derivatives of these dyes found by Szent-Gyorgyi and his team fluoresce when bound to a FAP.
Fluorescence is achieved when the absorbance of one photon by a molecule triggers the release of another photon with a higher wavelength.
Scientists at the MBIC screened billions of scFvs before finding eight that were capable of binding malachine green and thiazole orange and making them fluoresce.
“These FAPs are the essential first step in developing molecular biosensors that will monitor dynamic changes occurring within cells,” said Alan Waggoner, director of the MBIC and a biology professor at Carnegie Mellon.
“The ultimate goal is to put molecular biosensors based on FAP technology inside cells. [We] have used the FAPs in conjunction with several fluorogens to visualize proteins at the cell surface and are now using the technology to image proteins inside cells,” Waggoner said.