Combining polarized light methods reveals hidden molecular orientations with precision

Image quality often makes the difference between an amazing multimedia experience, like feeling immersed in a high-definition movie, and a visual letdown. When it comes to biomolecular imaging, the details matter even more. When scientists increase resolution in quantitative imaging, they improve accuracy and confidence in results, ultimately facilitating discoveries in studies of proteins, cells and other biomedical applications.

Scientists have long been able to look at single molecules to study their nanoscale structures and dynamics in biological systems. However, distinguishing between two closely spaced dipole emitters, which are fluorescent molecules that can emit light in specific directions and intensities, has remained a major challenge, especially when such molecules emit light at the same time and are spatially coincident, or located at nearly the same point in space.

This limitation has hindered researchers’ ability to measure the orientation and angular separation of dipoles accurately, which is vital to understanding their rotational dynamics in crowded cellular environments.

New research published in Physical Review Letters from Matthew Lew, an associate professor of electrical and systems engineering at the McKelvey School of Engineering at Washington University in St. Louis, and first author Yiyang Chen, a graduate student in WashU’s imaging science doctoral program, proves it is impossible to distinguish two coincident dipole molecules from one single molecule using existing polarization imaging techniques.

To solve this problem, Lew and Chen combined two methods, manipulating the polarization of the illumination laser and measuring the polarization of the collected fluorescence, to discriminate between pairs versus single molecules. Their combined technique also improves precision in measuring the relative orientation between pairs of molecules.

“Structure always determines function,” Chen said. “Structures of proteins and other biomolecules are the underlying reasons behind the behaviors of cells. For example, when antibodies recognize viral antigens, they need to find a way to ‘meet’ and interact with each other, which depends on their relative orientations. These nanoscale details are always hidden in complex biological structure, but they have large-scale impacts on the function of the whole system.”

At the onset of the project, Chen and Lew had assumed that polarization microscopes could resolve two nearby fluorescent molecules by measuring the polarization of the light they emit, but this proved mathematically impossible, even with state-of-the-art imaging techniques.

They worked out the mathematical details to demonstrate that dipole pairs always produce images identical to those of a single rotating dipole. In addition, the team discovered that combining polarized illumination and polarized fluorescence detection into a single new technique could overcome the confusion and produce unique images for one versus two molecules.

The team’s method improves the precision of measuring a dipole molecule’s orientation by 50% and boosts angular separation measurement precision by twofold to fourfold compared with traditional methods.

This dramatic improvement is remarkable in the well-established field of orientation microscopy and holds the potential to transform how molecular dynamics are studied, especially in live biological systems where real-time observation is critical, Lew said.

“To push science forward, details matter,” Lew said. “In the past, it’s been convenient to think about fluorescent molecules as points because that’s simpler, but at the nanoscale, thinking of molecules as dipoles is essential to correctly measuring the direction and intensity of the light they emit.

“Likewise, biomolecules aren’t spheres, so by using our technology, fluorescent dipoles allow us to measure biomolecular orientations and protein conformations that have a huge impact on biological processes,” Lew added. “By resolving molecular structures and dynamics with greater precision, our imaging method could eventually support applications ranging from the study of protein interactions to drug development and disease research.”