Freezing Time

You’ve probably seen the famous 19th century photos of a galloping horse, each photo freezing the horse’s motion and capturing the split second when all four hooves are suspended in the air. 

The photos proved a theory of horse locomotion—the idea that for at least an instant, all four hooves leave the ground—which was hotly debated among scientists and the public in the 1800s. But the images could not be made with the standard camera technology of the time. Only a new breakthrough shutter system—invented by photographer Eadweard Muybridge to capture the galloping horse mid-stride—could reveal a previously unseen reality to the human eye. 

Creating pictures of previously unseen realities is a specialty of Joachim Frank. His innovations in the field of cryo-electron microscopy (which earned him a Nobel Prize in Chemistry in 2017) have revolutionized the imaging of the molecules of life, bringing fuzzy images of individual molecules into sharp, three-dimensional focus. 

But even Frank says today’s cryo-EM technology is “clumsy,” somewhat like camera technology before Muybridge. Standard cryo-EM methods are great for getting sharp images of molecules in their stable states (like a horse standing still) but too slow to capture the fleeting intermediate configurations of a single molecule at work or the interplay of two molecules.  

These molecular moments only last for tens or hundreds of milliseconds, and just as Muybridge’s photos revealed the true motions of a galloping horse, capturing images of these intermediates will reveal how life’s molecules truly work. 

Various devices have been designed in the past decade to capture molecules in action but have either been unsuccessful or too expensive. Now a device created by Xiangsong Feng, a postdoc in Frank’s lab, has solved several key challenges and can capture molecules that change shape over a time range of 10 to 1,000 milliseconds (1/100th of a second to 1 second). 

A layered solution

Time-resolved cryo-EM was implemented earlier in Frank’s lab using a machine created by Howard White, a structural biologist at Eastern Virginia Medical School and the University of Leeds. White’s machine comes with pumps that push separate solutions of molecules into a microdevice, where the molecules can mix and react for a defined time before they are sprayed onto grids for freezing and later viewing under a cryogenic electron microscope. The idea is to freeze the molecules in the act. 

The microdevice, designed in a collaboration between the Wadsworth Center and Rensselaer Polytechnic Institute, combined all functions of mixing, reacting, and spraying on a single chip made of silicon, the same material used for computer chips. But fabrication of silicon components must be outsourced and is too expensive for academic laboratories.   

This is where Feng’s microdevice comes in. Feng created it by assembling three separate units: a micromixer, microreactor, and microsprayer. He turned to a polymer, polydimethylsiloxane (PDMS), to make his own micromixers and sprayers in Columbia’s Nanofabrication Facility. Inside the exceptionally clean room of the facility, Feng could ensure that no speck of dust would stray into the chambers. 

The only problem with switching from silicon to PDMS is the stickiness of the polymer’s hydrophobic surface, which tends to adsorb proteins. Feng tried dozens of different modifications over a year before discovering that a thin coating of silicon dioxide solved the problem. 

In the final assembly, two inlets bring millions of molecules into the micromixer, where the molecules begin to react with each other. The chemical reaction continues as the molecules travel through a narrow capillary tube before they are sprayed onto a small grid that is then plunged into a small cup of liquid ethane to freeze the action. The grid is put under an electron microscope later to visualize the molecules. 

Test run

To demonstrate the microdevice’s capabilities, Sayan Bhattacharjee, a postdoc in the Frank lab, used it to get a better understanding of how bacteria sometimes survive antibiotics. Antibiotics cause the bacterial ribosome to stall, but a bacterial molecule called HflX can split the two parts of the ribosome, freeing the pieces so they can reassemble and resume protein production. 

To learn how HflX splits apart the ribosome, Bhattacharjee and Feng worked together to pump solutions of HflX and the ribosome into the micromixer where HflX got to work on the ribosome. Because the reaction continues as the molecules travel through the microreactor tube, different reaction intermediates can be captured by repeating the process with different lengths of tubing.  

Bhattacharjee was able to capture reaction intermediates at three time points (10, 25, and 140 milliseconds). These intermediates reveal how HflX pries the ribosome halves apart like a clam shell, information that could potentially inform the design of better, broad-spectrum antibiotics. 

Capturing faster molecules?

Other structural biologists are now excited to use the device to explore other molecular intermediaries. 

“Many molecular machines—like the ribosome or RNA polymerase—have relatively large domains that take some time to change position for which our apparatus can be used to get new information,” Frank says.  

But to capture intermediates for molecules like enzymes or ion channels that move even faster, between 1 and 10 milliseconds, the device will need to be entirely redesigned. With a grant from the NIH, Qiao Lin in the Department of Mechanical Engineering is working on such a device. 

“A device that could freeze the action of molecules moving at this speed would open a new world of possibilities,” Frank says.