St. Jude Children’s Research Hospital scientists have found a solution to the long-standing issue of accurate molecular scale distance measurements using single-molecule fluorescence resonance energy transfer (smFRET). smFRET measures the excitation and emission properties of fluorophores, a type of chemical.
Fluorophores emit light when an excited electron relaxes, causing the molecule to glow (fluoresce). The breakthrough by the scientists addresses this decades-long problem and will lead to more precise experiments.Horses do not always glow after being excited. Instead, they can enter long-lived triplet dark states that do not glow due to quantum mechanical processes related to the excited electron’s “spin” state. This can reduce the accuracy of smFRET measurements. St. Jude scientists have found a way to control the duration of these dark states through “self-healing” technologies, which mitigates the triplet dark states. This advancement significantly increases the resolution of the method and will contribute to the field of molecular imaging. The findings were published in Nature Methods.
Using specialized cameras with a high frame rate and careful lighting, it is possible to capture the rapid movement of a hummingbird’s wings without any blur. However, visualizing the flight of a hummingbird is nothing compared to the difficulty of capturing the actions of biomolecules in our bodies. Biomolecules are incredibly small, even smaller than the wavelength of light (about one billionth of an inch), and their functions are directly linked to their constant motion, constantly changing positions or shapes (conformation) hundreds to thousands of times per second. Being able to measure these rapid dynamics is crucial in order to truly understand how molecules carry out their functions and how these functions ultimately impact our bodies.In disease, scientists are interested in how biomolecules are affected and how drug treatments alter their functions. The molecular imaging technique known as smFRET allows for the direct observation of biomolecule movement in real-time and at the individual molecule level.
At St. Jude, Dr. Scott Blanchard, from the Departments of Structural Biology and Chemical Biology & Therapeutics, is working to advance smFRET imaging. The efforts of the Blanchard lab, in collaboration with the St. Jude Single-Molecule Imaging Center, have been crucial in developing and designing fluorophores that enable measurements at the molecular level.
Blanchard expressed that typical fluorophores were not adequate for measuring events at the molecular level, which motivated them to create their own. In the process, they discovered that the fundamental photophysics of fluorescence needed to be modified. In smFRET experiments, researchers position fluorophores on two locations of a biomolecule. When a laser is pointed at the first fluorophore (the donor), an electron within it becomes excited. If the second fluorophore (the acceptor) is nearby, the energy is transferred to it when the electron relaxes.The donor. By capturing and measuring bright flashes from both the donor and acceptor fluorophores, distances can be determined at a scale of one billionth of an inch. Each bit of data is crucial for understanding how biological processes work and when they go wrong. However, using this technique correctly requires a thorough understanding of the basic properties of fluorescence.
Electron spin flip locks in triplet state
The regulations governing a fluorophore’s release of light are centered on electron spin. When an excited electron relaxes, it is supposed to return to its original state, keeping it in a consistent position.The spin state of an electron, also known as its spin quantum number, can change when it is excited, according to Blanchard, the corresponding author of a study published in Nature Methods. Although this doesn’t happen all the time, there is a possibility that the electron could forget its spin and switch to an opposite spin state. If this occurs, the electron ends up in a much longer-lived triplet state, making the fluorophore much dimmer than it could be. This phenomenon has been a challenge in the field of fluorescence for many years.anchard explained that in the context of FRET, they have observed changes in triplet state accumulations with varying illumination intensity and for different fluorophores. FRET requires both the donor and acceptor fluorophores to behave in a similar manner. However, since the technique involves exciting one directly and not the other, increasing the laser intensity leads to different rates of occupancy of triplet states for the donor and the acceptor.
This results in a process where the donor and acceptor reach different plateau levels, causing them to lose performance to varying extents. According to Blanchard, this leads to experimental readouts becoming inconsistent.The variability of fluorophore triplet states can cause issues with the quality and consistency of imaging data, limiting the spatial and temporal resolution of smFRET measurements. A primary objective of fluorophore engineering research is to decrease the lifetime of triplet states as much as possible, which is the basis of ‘self-healing’ technologies. Currently, the accuracy of distance measurements in smFRET data relies on calibration steps that do not take triplet states into account, as explained by co-first author Zeliha Kilic, PhD, from the St. Jude Department of.Structural Biology. “Advancements in self-healing technologies are bringing us closer to ideal conditions where triplet states are eliminated, leading to more accurate calibration steps and distance measurements.”
Self-healing fluorophores leading the way
Chemicals known as triplet state quenchers, such as cyclooctatetraene, work against this phenomenon but also have the drawback of causing complications. “Cyclooctatetraene is oily, has inconsistent and poor solubilities, and is difficult to manage,” stated Blanchard.
Previous reports from Blanchard’s team detailed the creation of fluorophores with cycloctatetraene directly attached. This method addressed the problem of solubility and created “self-healing” fluorophores that reduced triplet state occupation by up to 1000 times. The researchers showed that using self-healing fluorophores in smFRET experiments improves data quality and reliability, and prevents loss in imaging quality as laser intensity increases. These advancements are pushing the boundaries of smFRET, and self-healing fluorophore technologies are being used in various applications around the world.
“The improved brightness and photostability of self-healing fluorophoreCo-first author Avik Pati, PhD, who was previously with the St. Jude Department of Structural Biology and is now with the Birla Institute of Technology and Science, stated, “s can significantly enhance the spatiotemporal resolution of smFRET imaging. We are now able to accurately measure the nanometer-scale conformational dynamics within individual biomolecules at sub-millisecond intervals and under physiological oxygen levels.”
Blanchard believes that these discoveries will benefit not only St. Jude researchers but also the wider scientific community. “Advancing the boundaries of imaging technologies at St. Jude is an integral part of the institution’s strategic plan, and we are confident that these advancements will have a positive impact.”The speaker emphasized the importance of using new fluorophores to achieve their goals. He also mentioned that many people stand to benefit from these advancements, as the self-healing approach has the potential to enhance most fluorescence applications.
