New technique brings very dilute samples into focus

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Scientists use a powerful technique called cryo-electron microscopy to see the 3D shapes of biological molecules, but the technique normally needs the molecules to be extremely concentrated. For rare or more delicate molecules this is hard to achieve. As a result scientists aren’t clear on the shapes of many important structures.

In a new study, researchers in the U.S. have created a workaround called Magnetic Isolation and Concentration cryo-electron microscopy (MagIC for short). It lets researchers sidestep the limitation and study samples roughly 100- to 1,000-times more dilute than before.

The findings were published in eLife on May 20.

The new method works by attaching molecules of interest in a sample to 50-nm beads, then clumping the beads together. The attachment was provided by a protein called a spacer. It held the target molecules far enough from the bead’s surface to keep the image from blurring.

Then, a magnet pulled the loaded beads onto a cryo-EM grid before freezing, sharply reducing the amount of sample lost in the blotting step. This way each micrograph also ended up with more than 100 usable particle images even when the solution had less than 0.0005 mg/ml of the molecules.

Because the beads were easy to spot even at low magnification, the scientists could quickly move the microscope to regions rich in particles, speeding up data collection.

Small particles often hide in background noise. To pull them out, the authors built a computer workflow called Duplicated Selection To Exclude Rubbish (DuSTER). It picked each particle twice, kept those that landed in the same place after two rounds of 2D or 3D classification, and threw the rest away.

Thus DuSTER rescued clear classes from images that initially may have looked hopeless.

The technique opens the door to studying many low-abundance molecules without laborious purification steps.

Manaswini Vijayakumar is interning with The Hindu.

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