(Nanowerk News) A new way of seeing details smaller than half the wavelength of light has revealed how nanoscale scaffolding within cells bridges to the macroscale during cell division. Unlike previous super-resolution techniques, the one developed and tested at the University of Michigan does not rely on molecules being worn down by long-term use.
Superresolution can reveal structures down to 10 nanometers, or roughly the area of 100 atoms. This opened up a new world in biology, and a technique that first allowed him to receive the Nobel Prize in 2014. However, the drawback is that it can only take snapshots of tens of seconds. This makes it impossible to observe the evolution of the cell machinery over long periods of time.
“We wondered—as the entire system divides, how do the nanometer-scale structures interact with their neighbors at the nanometer scale, and how do these interactions extend throughout the cell?” said Somin Lee, a UM assistant professor of electrical and computer engineering, who led the research published in Nature Communications (“Phase intensity nanoscope (PINE) opens a long window of investigation of living matter”).
To answer that question, Lee and colleagues needed a new kind of superresolution. Using their new method, they were able to continuously monitor cells for 250 hours.
“The living cell is a busy place with busy proteins here and there. Our superresolution is very interesting to see this dynamic activity,” said Guangjie Cui, Ph.D. student in electrical and computer engineering and co-author of the first study with Yunbo Liu, a Ph.D. electrical and computer engineering graduate.
Like the original method, this new technique uses probes near nanoscale objects of interest to elucidate them. Superresolution 1.0 uses fluorophores for this, fluorescent molecules that will send back light after being irradiated. If the fluorophore is closer than whatever size was imaged, the image can be reconstructed from the light bursts produced by the fluorophore.
The new technique uses gold nanorods, which are not damaged by repeated exposure to light, but making use of the light they interact with is more challenging. The nanorod responds to the phases of light, or where it is in the oscillating oscillations of the electric and magnetic fields that compose it. This interaction depends on how the nanorod is tilted to the incoming light.
Like fluorophores, nanorods can attach to specific cell structures by targeting molecules on their surface. In this case, the nanorods are looking for actin, a protein that adds structure to soft cells. Actin is shaped like branching filaments, each about 7 nanometers (millionths of a millimeter) in diameter, though they are linked together by thousands of nanometers. Although the nanorods are often more than twice the diameter of actin, the data they provide as a group can illuminate the tiny details.
To find the nanorods, the team created a filter made of a thin layer of polymer and liquid crystals. This filter enabled the detection of light with a specific phase, allowing the team to select the nanorods at a certain angle to the incoming light. By taking 10–30 images — each looking at a different subset of nanorods — and combining them into a single image, the team was able to deduce nanometer-scale details of the filaments inside the cell. These details would be obscured in a conventional microscope.
Using this technique, the team discovered three rules governing how actin regulates itself during cell division:
Actin behavior is connected to cell behavior — but cells contract when actin expands, and cells expand when actin contracts. The team wanted to explore this further, discovering why movement is opposite at different scales. They also wanted to investigate the consequences of dysregulation of this molecular process: Is it at the root of some diseases?
More broadly, they hope to use superresolution to understand how self-organization is built into biological structures, without the need for central control.
“Our genetic code doesn’t actually include enough information to encode every detail of organizational processes,” says Lee. “We wanted to explore mechanisms of collective behavior without central coordination that are like birds flying in formation—where systems are driven by interactions between individual parts.”