Fluorescence microscopy method harnesses photobleaching weakness

The technique takes photobleaching and turns it into a strength to improve fluorescence microscopy imaging output by up to 3X, with no additional hardware required.

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Seeking to improve the quality and quantity of information that can be extracted in fluorescence microscopy, researchers at the Australian Research Council (ARC) Centre of Excellence for Nanoscale BioPhotonics (CNBP) developed a technique that takes photobleaching and turns it into a strength that improves imaging output by up to 3X, with no additional hardware required.

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The technique, which they call bleaching-assisted multichannel microscopy (BAMM), will help researchers gain biological insights into the intricate processes taking place within living cells. This includes the interplay between proteins and molecules, which have the potential to impact a wide range of health areas, including fertility, pain, and heart disease.

"When the fluorophore is excited by light from the microscope, it reacts by emitting a specific color signature. Seeing that color signature under the microscope helps us view, track, and understand the cellular target that the fluorophore has been bound to," explains Antony Orth, CNBP Research Fellow at RMIT University and lead author of the research paper. Notably, he says, you can attach different colored fluorophores to different cell targets, all in the one sample, to maximize the data and imaging information that is received.

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Antony Orth, CNBP Research Fellow at RMIT University.

This traditional approach to fluorescence microscopy is versatile, but there is a major limitation: the visible (or color) spectrum, where most fluorophores operate, can get crowded. In an ideal experiment, each target should be chosen to have a distinct color emission, but this becomes increasingly difficult to arrange as the number of targets increases.

"The visible color spectrum spans a range of 400 nm to 700 nm and only about 200 nm of this range is available for fluorescence color emission," Orth explains. "A typical fluorophore emits over a 50 nm range of the color spectrum. Dividing 200 nm of the visible spectrum into 50 nm segments means that the colors of the fluorescent emitters begin to blend together when you attempt to squeeze in more than four colors." This generally limits researchers to four or fewer fluorescent targets in a sample, he says.

To help overcome this limitation, Orth and his team developed BAMM to increase their imaging output. "Instead of using color to differentiate between fluorophores, we use the fourth dimension of time and exploit a phenomenon called photobleaching—the dimming of a collection of fluorophores or pigments under repeated exposure to light," Orth says. "Because each type of fluorophore photobleaches at a different rate, we can differentiate between fluorophores without using any color information. We use the rate of photobleaching as the identifier."

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This figure shows the information-rich cellular images made possible by using the newly reported BAMM technique. The original image (left) shows cells containing multiple fluorescent targets, all having similar colors. This results in a monochrome image. With BAMM, photobleaching rates are color-coded red, green, and blue for visualization so that each fluorescently labeled structure can be identified, even though the fluorophore's native color information was never used.

"When paired with traditional color information, this added dimension of photobleaching enables scientists to use 2-3X more types of fluorescent molecules, all in one sample. This lets us extract far more information from a single investigation," Orth says. "Researchers will be able to design more informative tests—for example, highlighting five targets when only two were previously practical. They will no longer have to avoid using two fluorophores with the same color, since a difference in photostability alone is enough to distinguish between the two targets." Traditionally, the phenomenon of photobleaching (or fading) has been detrimental to the fluorescent microscopy process. This is where high-intensity and ongoing illumination from the microscope permanently destroys a fluorophore’s ability to fluoresce so that imaging of the cell target becomes impossible.

"BAMM doesn't require any additional hardware, it’s comparatively simple to do, and doesn't require any specialized sample preparation," Orth says. "It's an extremely exciting new approach which has the potential to benefit all fluorescence microscopy users and their exploratory science."

Researchers involved with the BAMM project were affiliated with CNBP (RMIT University and the University of Adelaide) and Thermo Fisher Scientific.

Full details of the work appear in the journal Biomedical Optics Express.

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