Using ultrafast spectroscopy, researchers at Michigan State University tracked the vibrational motions of single, unlabeled virus particles under ambient conditions across the megahertz to terahertz spectral range. The team’s methodology, called BioSonic spectroscopy, promises to provide insight into viral dynamics without the need for labeling and could serve as a means for viral fingerprinting. In the future, BioSonic spectroscopy could help speed the development of antiviral drugs for combating viral infections. Working with ultrafast spectroscopy, professor Elad Harel aims to reveal how microscopic phenomena impact large complex systems. Courtesy of Michigan State University. To observe and study the “sound” of a virus, the researchers tracked quantized acoustic vibrations in a single virus smaller than 100 nm. They placed the virus sample on a cover slip and interrogated the sample with a pair of ultrashort laser pulses inside a microscope. They used a nonresonant pump pulse of less than 100 femtoseconds (fs) at 1040 nm to excite collective vibrations in the single virus particle. A second, time-delayed probe pulse of less than 100 fs at 785 nm was used to detect changes in light scattering induced by the coherent vibrations. The researchers isolated the weak signal from the large background of backscattered light by using balanced detection and asynchronous optical sampling, a method in which the interpulse delays are rapidly scanned up to the laser pulse period in sub-milliseconds to reduce laser and environmental noise. “To initiate the sound, we use short pulses of light that generate coherent motion in the system,” professor Elad Harel said. “We then use a second pulse of light to probe that motion at different moments in time. By stringing together all the snapshots in time, we can produce a molecular movie that captures the vibrational motion of the object.” The team observed long-lived, coherent oscillations in a single virus that persisted for many nanoseconds. These coherent signals generated an acoustic spectrum that was highly sensitive to the virus morphology and the interactions between its glycoproteins and the environment, providing insight into viral mechanics not available through other single-particle methods. The time resolution was sufficient to examine a single virus particle through its life cycle, which occurs on the second-to-hour time scale. “It’s fascinating to experimentally observe the nanoscale motion of these tiny virus particles — they are actually ‘breathing’ under laser illumination,” researcher Yaqing Zhang said. A close-up of a beam-splitter cube among the Harel group’s laser instrumentation. Using short pulses of light, Harel can produce a molecular movie that captures the vibrational motion of a biological object based on its “sound.” Courtesy of Michigan State University. The team measured acoustic spectra in the virus in the 2 to 50 GHz range with sub-gigahertz resolution — a very low frequency for optical transitions, considering that visible light is in the hundreds of terahertz. “These are thousands to millions of times lower energy than what we typically think of in terms of optical spectroscopy,” Harel said. The researchers used the new spectroscopic method to listen to a virus rupture. “As the virus begins to break open and weaken, its acoustics start to change, going lower — almost like a deflating balloon,” Harel said. One of the goals of the team is to develop a spectroscopic approach that can achieve the resolution of electron microscopy for living systems. “Electron microscopy, or EM, itself is very powerful, but you’re really taking snapshots of life, and you’re doing it in an environment that’s quite different than what you find in living organisms,” Harel said. “EM is done in vacuum, and with cryo-EM, it is done at very low temperatures where life cannot be sustained.” The team also hopes that the BioSonic approach can be used as a powerful imaging probe without the need for labeling. “One of our goals is to show that this new methodology could use a virus’ or molecule’s natural labeling — basically, the sound of its own materials that distinguishes it from everything else in a system,” Harel said. The next step for the researchers will be to show that BioSonic spectroscopy can be used to dynamically track how a virus is moving. “If we want to watch a virus go into a cell now, the process is very, very challenging and slow via electron microscopy or utilizing complex fluorescence labeling,” Harel said. The sensitivity, high resolution, and speed of this approach could increase understanding of biological dynamics and early-stage diagnostics at the single microorganism level. “I am confident that this technique can be widely utilized for millions of viruses and other biological samples and will acquire more invaluable information from them,” Zhang said. “The more we know them, the better we can prepare for the next pandemic.” The research was published in the Proceedings of the National Academy of Sciences (www.doi.org/10.1073/pnas.2420428122).