A tiny mirror could make a huge difference for scientists trying to understand what's happening in the micron-scale structures of living cells.
By growing cells on the mirrors and imaging them using super-resolution microscopy, a group of scientists from universities in Australia, China and the United States has addressed a problem that has long challenged scientists: seeing the structures of three dimensional cells with comparable resolution in each dimension.
"This simple technology is allowing us to see the details of cells that have never been seen before," said Professor Dayong Jin from the University of Technology Sydney.
"A single cell is about 10 micrometers; inside that is a nuclear core about 5 micrometers, and inside that are tiny holes, called the 'nuclear pore complex' that as a gate regulates the messenger bio-molecules, but measures between one fiftieth and one twentieth of a micrometer. With this super-resolution microscopy we are able to see the details of those tiny holes."
Reported in the Nature journal Light: Science & Applications, the technique was jointly invented and developed by scientists at UTS, Macquarie University, Peking University and the Georgia Institute of Technology.
Cells are normally grown on transparent glass slides for microscopy examination. The new technique uses the unique properties of light to create interference patterns as light waves pass through a cell on the way to the mirror and then back through the cell after being reflected.
The interference patterns provide, at a single plane within the cell, significantly improved resolution in the Z-axis – what scientists see as they look directly into a cell perpendicular to the slide. This improved view could help researchers differentiate between structures that appear close together with existing microscope technology, but are actually relatively far apart within the cells.
Microscope resolution in the X and Y axes is typically superior to resolution in the Z axis, regardless of the microscopy technique. The mirror approach works with super-resolution microscopy as well as with other technologies.
Being able to see these tiny structures may provide new information about the behaviour of cells, how they communicate and how diseases arise in them, said Professor Peng Xi, from Peking University.
"Previously, the vision of biologists was blurred by the large axial and lateral resolution," he said. "This was like reading newspapers printed on transparent plastic; many layers were overlapped. By placing a mirror beneath the specimen, we can generate a narrowed focal spot so there is only one layer of the newspaper to read so that every word becomes crystal clear."
The new system, he noted, allows scientists to see the ring structure of the nuclear pore complex, and the tubular structure of the human respiratory syncytial virus (hRSV). "With this simple, but powerful weapon, biologists can tackle many interesting phenomena that were invisible in the past because of poor resolution," Professor Xi added.
While changing the optical system was relatively simple, growing cells on the custom-made mirrors required adapting existing biological techniques, said Professor Phil Santangelo from Georgia Tech and Emory University.
Techniques for growing the cells on the mirrors were largely developed by Eric Alonas, a Georgia Tech graduate student, and Hao Xie, a graduate student shared by Georgia Tech and Peking University as part of a collaborative research program.
"Most people are not growing cells on mirrors, so it required some work to get the cell culture conditions correct," Santangelo said. "We had to make sure the mirror coating didn't affect cell growth, and staining the cells to make them fluoresce also required some adaption. Ultimately, growing cells on the mirrors became a simple process."
The new technique, known as mirror-enhanced, axial narrowing, super-resolution (MEANS) microscopy, begins with growing cells to be studied on a tiny mirrors custom made by a manufacturer in China. A glass cover slide is placed over the cells, and the mirror placed into a confocal or wide-field microscope in the place of a usual clear slide.
The time differences between Australia, China and the United States provided a challenge for the team's collaboration, but the researchers say the work was very worthwhile.
"The development of the mirror-enhanced super-resolution microscopy is a great example of what collaborative, international and multi-disciplinary research can achieve," said Professor Jin, who is the director of the Initiative for Biomedical Materials & Devices at UTS. "It is a significant achievement for the whole team, and the field, and one that we're proud to have been involved in."
CITATION: Xusan Yang, et al., "Mirror-enhanced super-resolution microscopy", Light: Science & Applications, 2016.