How can we ascertain precisely what occurs at a molecular level during exceptionally rapid events, such as the burning in combustion? In less time than it takes for a blink, one chemical compound may appear in a flame, only to dissolve and yield to others. Recognizing which molecules are present offers researchers a means to comprehend the underlying mechanisms of the chemical reactions in progress.
However, conventional techniques for assessing molecule dimensions often fail to capture such swift, fleeting phenomena. Additionally, traditional optical microscopy not only lags in speed but also lacks the capability to resolve molecules spatially, which are typically no bigger than a few nanometers, or billionths of a meter, in size.
Now a team from Caltech, headed by Lihong Wang, the Bren Professor of Medical Engineering and Electrical Engineering, has created a novel tool known as Compressed Ultrafast Planar Polarization Anisotropy Imaging (CUP2AI). This development offers insights into dynamic occurrences, with promising applications not only in the study of combustion but also in fields such as drug development and nanoparticle creation. In a publication that recently emerged in the journal Nature Communications, Wang and his team present the CUP2AI method and detail its successful application in visualizing carcinogenic substances in flames and illuminating, in water, a common fluorescent compound employed in biomedical uses.
“When an event occurs rapidly, one wishes to observe the entire occurrence dynamically,” remarks Wang, who also holds the Andrew and Peggy Cherng Medical Engineering Leadership Chair and serves as the executive officer for medical engineering at Caltech. “You aim to grasp the physical process in both spatial and temporal contexts, and that is what our new light-speed imaging technique allows.”
Consider the instance of combustion flames, suggests Peng Wang, a former postdoctoral researcher from Lihong Wang’s group and a lead author of the new study, alongside Yogeshwar Nath Mishra of Caltech, JPL, and the Indian Institute of Technology in Jodhpur, as well as Florian J. Bauer from the Friedrich-Alexander University of Erlangen-Nuremberg in Germany. To thoroughly comprehend the phenomena, scientists must examine flames utilizing various fuels, leading to distinct chemical reactions. Furthermore, altered conditions must be factored in, and in each scenario, modified chemical reactions will occur, generating a unique array of molecules.
“If we can investigate the size of the molecules, we can comprehend how these reactions transpire for different fuels under varying conditions. Since combustion is employed in vehicles, aircraft, and even rockets, it is crucial to understand these chemical reactions. This knowledge may lead to the development of more efficient combustion engines and potentially mitigate the pollutants produced through combustion.”
A Novel Technique Grounded in the Innovations of the World’s Fastest Camera
CUP2AI builds on earlier research conducted in Wang’s lab that led to the creation of the world’s fastest camera and the development of a series of ultra-fast imaging approaches via a technique referred to as compressed ultrafast photography (CUP).
This new method depends on the polarization of light. Alongside wavelength and intensity, polarization is a key characteristic of light, representing the orientation of the electric component of a light wave concerning the wave’s general trajectory. Interestingly, the polarization of fluorescent light correlates with the orientation of the molecule that radiates it.
To grasp this, first consider that molecules are perpetually in motion. They exhibit translational movement—essentially moving from one location to another—as well as rotational movement, involving spinning around one or more of three axes.
“The attributes of this second type of motion is dictated by the size of the molecule,” explains Peng Wang. “As one might presume, the larger the molecule, the more challenging it is to rotate.” Conversely, smaller molecules can rotate more easily and thus can do so at greater speeds.
When a laser beam engages with a molecule, the electrons within it are excited to a higher energy level. As they revert to a lower energy state, they emit photons, generating fluorescence emissions that diminish over time. For molecules excited by a linearly polarized laser, this fluorescence will exhibit a certain level of polarization. However, that polarization will evolve swiftly over timescales measured in billionths or trillionths of a second, and this is what CUP2AI detects.
Specifically, CUP2AI conducts two distinct polarization measurements—one aligned parallel to the laser beam’s polarization and the other perpendicular to it. Initially, most of the fluorescence is derived from the parallel polarization, yet over time, rotational movement increases the perpendicular polarization, reducing the difference, or anisotropy, progressively. The rate at which this anisotropy shifts is influenced by the size of the molecule.
“The larger the molecule, the slower this difference will diminish, which enables us to ascertain the size of the molecule,” comments Peng Wang. He notes that other techniques previously employed to measure molecular size in dynamic settings have only been capable of determining sizes at a single point or averaging the entire sample. “Our tool is the first able to produce a 2D map of fluorescence all in a single exposure,” he adds.
Lihong Wang further mentions that the connection between molecular size and the decay of polarization anisotropy is rooted in an equation originally formulated by eminent scientists Albert Einstein, George Stokes, and Peter Debye. “Thus, we are merging classical physics with contemporary technology and applying it to a very pressing issue—combustion efficiency—that pertains to energy,” he states. “That truly excites me.”
The publication is entitled “Single-shot two-dimensional nano-size mapping of fluorescent molecules by ultrafast polarization anisotropy imaging.” Murthy S. Gudipati of JPL, which is managed by Caltech for NASA, is also a collaborator on the study. This research was supported by the National Institutes of Health, the Swedish Research Council, and JPL/NASA. Lihong Wang serves as an affiliated faculty member with the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech.