Oxford University researchers have achieved remarkable strides in imaging proteins at the single-molecule level using holography. Their innovative process, which generates light-based holograms of proteins, improves the state-of-the-art sensitivity by five orders, enabling the measurement of biomolecule polarity. The imaging technique opens new avenues for studying biomolecular interactions and how they can be applied to nanoscience.

The research article, “Single-protein optical holography,” was published in Nature Photonics.

Single-molecule sensitivity with optical holography

Even though the primary purpose of microscopy is to provide qualitative assessments of cellular and subcellular properties, it can also perform a wide range of quantitative measurements when used with other tools like computers, lasers, photoelectric devices, and wavelength selectors. These quantitative measurements make it challenging to establish relationships between the structures and properties of biological material in all of its temporal and spatial complexities. When used together, these tools significantly improve the ability to study cells and subcellular structures at the macromolecular scale for their physical and chemical properties without causing any damage.

Although light scattering occurs in all materials to varying degrees, optical holographic methods—which create quantitative three-dimensional pictures by reassembling light waves that have diffracted from objects—have never detected the minute scattering from individual molecules. Holographic methods have numerous advantages over traditional intensity measurements regarding data content, tunability, and post-processing. However, label-free single-molecule research has been slow to advance due to the difficulty of capturing weak signals from individual molecules using previous methods.

Jan Christoph Thiele, Emanuel Pfitzner, and Philipp Kukura overcame these obstacles and showed that light-based holography could detect extremely weak light scattering from individual proteins. Researchers have improved the technique’s sensitivity by five orders of magnitude compared to the state-of-the-art in terms of interaction strength. The method works by splitting light emerging from the sample and coupling it with a reference before splitting the detection into four parallel channels. This enables the precise detection and measurement of individual proteins. The researchers successfully detected, resolved, and measured single proteins with masses under 100 kDa. This method measures both amplitude and phase separately, so it can give much information about the sample’s identity and determine if certain biomolecules are polarizable.

The team’s breakthrough makes it easier to measure molecular polarizability and sample identity (for example, particle size, material properties, or protein oligomerization) and opens up new ways to study biomolecular interactions and find uses for nanoscience in general.

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