A tiny fluorescent protein has been created by biomedical and genetic engineers at Duke University and the Albert Einstein College of Medicine that produces and absorbs light that deeply penetrates biological tissue. This protein, which is tailored to near-infrared (NIR) wavelengths, can assist researchers in obtaining clearer, more accurate, and deeper biomedical imaging.
It is difficult to image deep tissues with light. Researchers are frequently only able to observe a millimeter or two deep within a tissue due to the rapid absorption and scattering of visible light by bodily structures and chemicals. If they are successful in probing farther, chemicals like collagen or melanin frequently obstruct the image by fluorescing naturally, akin to background noise.
According to Junjie Yao, assistant professor of biomedical engineering at Duke, “Biological molecules naturally absorb and emit light in the visible spectrum, which is approximately 350 to 700 nanometers.” Therefore, utilizing it to photograph deep tissue is like attempting to see stars in broad daylight. “The signals are overwhelmed.”
Yao and his coauthor, Vladislav Verkhusha, a genetics professor at Albert Einstein College of Medicine in New York, created a protein that absorbs and emits longer wavelengths of light in the near-infrared (NIR) spectrum to help them navigate these murky waters.
According to Yao, in the 700–1300 nanometer range of NIR light, tissue is the most transparent. Since there is less background fluorescence from the environment to filter out at those wavelengths, it is possible to take longer exposures and produce sharper images.
In order to create these proteins, Verkhusha and his team employed a technique called directed molecular evolution, building on the structure of photoreceptors typically seen in bacteria. These photoreceptors can flip between an inactive and active state when exposed to a particular wavelength of light, making them suitable for imaging studies. They have the ability to bond with biliverdin, a biomolecule that is abundant in the tissues of mammals and people.
According to Verkhusha, the optimal way for the photoreceptor to attach to the biomolecule was determined by studying the biliverdin’s structure. “To increase the electron binding, which is required to produce the red-shifted fluorescence, we carefully incorporated replacements of important molecular components of the molecule linking to the biliverdin after we understood the binding process.” “The proteins were then subjected to random mutagenesis and high-throughput screenings in order to evolve and improve in brightness.”
The protein that shines the brightest, known as miRFP718nano, is easily made in cells and tissues and produces light that is just outside the visual spectrum. While the NIR activation is useful in and of itself, the subsequent period of activity offers much more promise for biological imaging.
Yao said, “We’ve discovered that the NIR spectrum may be divided into two main zones.” These proteins emit light in the first zone, which is between 700 and 900 nanometers, when NIR light first interacts with them. The wavelength, like the tail of a comet, gradually lengthens as they decompose.”At that point, they start to emit light in the second NIR zone, which ranges from 900 to 1300 nanometers.”
All the advantages of using zone one NIR light with a shorter wavelength are increased in this second zone: background fluorescence is significantly reduced, light can penetrate tissue twice as deeply, and image resolution can be two to three times higher, enabling detailed images of intricate structures.
Yao and his Duke team put the novel protein’s effectiveness to the test using the imaging method known as short-wavelength infrared (SWIR). In order to activate the fluorescent proteins, this technique emits NIR zone-one light deep into the tissue. Proteins produce NIR zone-two light as they degrade, which can be used to create high-resolution images by revealing details about the structure and makeup of the targeted tissues and cells.
After introducing them to their animal models, the scientists used altered miRFP718nano proteins to view cells in a mouse mammary gland, photograph bacteria in the mouse digestive system, and even track changes in inflammation in a mouse liver.Compared to images created using a typical NIR zone-one imaging protein, all of the images that were recorded were crisper and more detailed.
Yao and Verkhusha are certain that their continuous collaboration will benefit their work in protein engineering and biomedical imaging. Yao is eager to use the new technique to more carefully view the brain and possibly track the migration of cancer cells as Verkhusha continues to develop and perfect fluorescent proteins and biosensors.
According to Yao, because we can use imaging techniques to inform protein engineering decisions and improved protein engineering to enhance imaging capabilities, this is an intriguing new front in our decade-long relationship.
