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Superresolution microscopy tracks a walking biomolecule.

Superresolution microscopy tracks a walking biomolecule

23 March 2023

A clever optical fluorescence technique achieves nanometer and millisecond resolution simultaneously, even amid the complexity of a living cell.

Artist representation of a complex protein structure.
Credit: Tomoki Fukushima/CC BY-SA 4.0

In 2014 the Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their fluorescence imaging techniques that beat the optical diffraction limit. While imaging with visible light, with a wavelength of hundreds of nanometers, the laureates refined their resolution to just 20 nm or so—enough to produce still images of protein complexes, cell organelles, and other biological structures in stunning detail. (See Physics TodayDecember 2014, page 18.)

But the resolution revolution was just getting started.

One of the insights that made superresolution microscopy possible was that even though a fluorescing molecule appears on a camera as a blur hundreds of nanometers across, the center of the blur can be located much more precisely than that. In principle, it could be pinpointed to within a single nanometer—the size of the molecule itself. But to get such exquisite spatial precision in practice, the camera would have to collect tens of thousands of photons from each molecule. Not only does that take time, but fluorescent molecules can’t always reliably endure so many absorption–emission transitions. The demanding photon requirement left superresolution techniques far short of their potential for both spatial resolution and speed.

In 2017 Hell and colleagues found a way to make further progress. They devised a technique they called MINFLUX (not really an acronym, but an abbreviation for “minimal photon flux”) that achieved superresolution results with far fewer fluorescence photons. The technique uses a doughnut-shaped tube of light to zero in on a fluorescent molecule’s position. If the molecule is precisely centered inside the doughnut, it emits no fluorescence; even one emitted photon is a clear sign that the molecule is slightly off-center. Hell and colleagues developed an efficient algorithm to scan the doughnut’s position and quickly locate the molecule with close to nanometer precision. Furthermore, by training the doughnut on the molecule as it moved around, they could track molecular movements on time scales as short as a millisecond.

With a few years of refinement, MINFLUX has now matured to the point where it can follow the real-time movements of kinesin, a biological molecular motor. As shown in the image above, kinesin resembles a stick figure, with two feet (red) that walk along a microtubule in a cell and two hands (green) that grasp cargo and carry it around. The basic mechanism of kinesin walking—that the feet step alternately, one in front of the other, with a stride length of 16 nm—was first worked out two decades ago. But those early experiments required kinesin to be removed from the complexity of a living cell and starved of fuel to slow down its walking.

Researchers led by Jonas Ries, of the European Molecular Biology Laboratory in Heidelberg, Germany, have now used MINFLUX to observe kinesin molecules walking at full speed in living cells. As shown in panel a of the figure below, they attached a fluorescent molecule (yellow star) to one of the kinesin feet, and they observed that its position advanced by roughly 16 nm at a time, in agreement with previous research.

Chart of position vs time of a walking kinesin molecule.
Credit: Panel a adapted from T. Deguchi et al., Science 379, 1010 (2023); panel b adapted from J. O. Wolff et al., Science 379, 1004 (2023)

Meanwhile, Hell and colleagues—at the Max Planck Institute for Medical Research, also in Heidelberg—have dissected kinesin’s walking mechanism in more detail than ever before. To maximize their resolution, they looked at kinesin outside a cell, and they applied a new variant of MINFLUX that uses interference fringes rather than a doughnut. Among their many results, they found that the kinesin stalk rotates with each step. As shown in panel b, they attached a fluorescent molecule (yellow star) to the side of the stalk, and so finely resolved was their MINFLUX measurement that they could tell whether the molecule was hanging off the front of the stalk or the back. Rather than advancing by a steady 8 nm with each step, the molecule moved at a more uneven pace. The finding addresses, but doesn’t definitively resolve, a major open question about the kinesin mechanism: Does the molecule walk facing forward (perhaps while twisting from side to side), or does it pirouette along the microtubule like a ballerina? (T. Deguchi et al., Science 379, 1010, 2023; J. O. Wolff et al., Science 379, 1004, 2023.)

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