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Microscopy

Scattered light weighs single biomolecules

Microscopy technique monitors protein aggregation and molecular binding in real time, without fluorescent tags

by Mark Peplow
April 26, 2018 | APPEARED IN VOLUME 96, ISSUE 18

09618-scicon1-monomerrxn.jpg
Credit: Science
As a protein complex assembles from its monomers, it scatters more light, which iSCAMS can analyze to measure the complex’s growing mass.

A microscopy technique that tracks individual biomolecules in solution—and generates an accurate readout of their mass—could offer biologists a powerful new tool to investigate processes such as the assembly of protein complexes or to study how drugs bind to their targets (Science 2018, DOI: 10.1126/science.aar5839).

The technique, called interferometric scattering mass spectrometry (iSCAMS), should offer a useful complement to existing methods for studying proteins, says Niek van Hulst at the Institute of Photonic Sciences, who uses similar microscopy techniques but was not involved in the work.

Credit: Science
iSCAMS produced this real-time video of actin filament assembly (growing dark spots). Scale bar = 1 μm.

Researchers sometimes tag biomolecules with fluorescent proteins, dyes, or quantum dots in order to image them. But these fluorescent probes can cause complications, by affecting protein-protein interactions, for example. Methods for measuring the mass of biomolecules also have drawbacks. Mass spectrometry inevitably destroys the molecule, and its high-vacuum conditions may erase important details about how a protein forms complexes in solution.

iSCAMS offers an inexpensive alternative that delivers real-time imaging of single, unlabeled biomolecules in their natural environment, says Philipp Kukura of the University of Oxford, who developed the new technique with his colleague Justin L. P. Benesch.

The iSCAMS microscope bounces laser light off a droplet of biomolecule solution on a glass slide. Proteins have a different refractive index than water, so they scatter light in a different way than their surroundings. When a photodetector captures the light, it reveals a contrast between the protein and its surroundings, visible as a dark spot wherever a protein is stuck to the glass.

Larger proteins scatter more light in a very predictable way, so measurements of the light also reveal the mass of each individual protein in the spotlight. The team could weigh biomolecules ranging from about 50 to 800 kDa, accurate to within 2% of the protein’s true mass, and with a precision of 1 kDa. The technique is the first to both image unlabeled proteins in real time and weigh them, van Hulst says. “I think biologists will jump on this.”

The researchers used their microscope to watch the aggregation of α-synuclein, a protein associated with Parkinson’s disease, allowing them to determine the reaction kinetics of the process; they also tracked the assembly of actin filaments in real time.

Stretching the technique to its limits, the team monitored the protein streptavidin as it bound four molecules of biotin, with each binding event showing up as an increase in the mass of the complex. Biotin has a molecular mass of 244 Da, but Kukura thinks they could soon detect the binding of molecules as small as 50 Da, which may prove useful in drug discovery assays. “Biotin was at the extreme of what we could do when we submitted the paper,” he says. “But we’re already better than we were back then. We’re feeling pretty confident that seeing small molecule binding will be quite possible with this technique.”

Although Kukura has spent a decade refining the technique, he and van Hulst both say that it is no more difficult to use than the fluorescence microscopy techniques that have become mainstays of biology labs. The Oxford team recently launched a company, Arago Biosciences, to commercialize the technology in a more compact and user-friendly form. “It’s actually pretty straightforward once everything is packaged,” Kukura says.

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