Researchers have developed a small fluorescent protein that can penetrate deep into biological tissue. The study, published in Nature Methods, describes the design and development of the protein, which has the potential to vastly improve biomedical imaging.
“Observing stars in daylight”
Biomedical imaging techniques based on visible light do not capture detailed images of the complex structures in the body. This is because visible light is absorbed quickly and gets scattered. As a result, imaging deep tissues is challenging.
Junjie Yao, Assistant Professor of Biomedical Engineering at Duke University said, “Biological molecules naturally absorb and emit light in the visible spectrum, which is about 350 to 700 nanometres. So when using it to image deep tissue, it’s like trying to observe the stars in daylight. The signals get flooded out.”
Assistant Professor Yao and his collaborator Vladislav Verkhusha, Professor of Genetics at the Albert Einstein College of Medicine, developed a fluorescent protein, termed miRFP718nano. The protein emits longer wavelengths of light than visible light in the near-infrared spectrum (NIR). The longer wavelengths emitted by the protein scatter less than shorter wavelengths. This means that clearer and more detailed biomedical images can be captured.
“Tissue is the most transparent in the 700-1300 nanometre window of NIR light. At those wavelengths, light can penetrate deeper into a tissue, and because there is less natural background fluorescence to filter out, we can take longer exposures and capture clearer images,” said Assistant Professor Yao.
The brightest of the bunch
The researchers developed the structure of the fluorescent protein from a bacterial phytochrome receptor. These usually contain two domains. The protein developed in this study is the first single-domain fluorescent protein that can emit the longer wavelengths. The smaller size means that it can penetrate further into tissues and has more stability. The protein interacts with biliverdin, which is an abundant and endogenous chromophore found in mammalian cells (figure 1).
Professor Verkhusha and his lab used rational structure-based design and directed molecular evolution to introduced mutations into the protein. This improved binding between miRFP718nano and biliverdin. High-throughput screening was then used to identify the brightest protein.
The researchers also found that the wavelength of light emitted by miRFP718nano was in two main zones.
“When NIR light first hit these proteins, they emit light in the first zone, which is about 700-900 nanometres. But as they decay, the wavelength gradually gets longer, like the tail behind a comet. That’s when they begin to emit light in the second NIR zone, which is from 900-1300 nanometres,” said Assistant Professor Yao.
This second zone gives miRFP718nano its image enhancing qualities. The light penetrates deeper and background fluorescence is lower. This means that images are clearer and more detailed, allowing complex structures to be captured.
The study highlights the potential of rational protein engineering to improve biomedical imaging techniques. The researchers aim to further improve the properties of the fluorescent protein and will apply the tool to capture more detailed images of the brain.
Assistant Professor Yao said, “This is an exciting new front of our decade-long collaboration because we can use the imaging tools to guide the protein engineering decisions, and we can use the advanced protein engineering to improve the imaging capabilities.”