Mirror-Enhanced Microscopy Captures Cell Behaviors

A novel technique for growing cells on miniature mirrors and imaging them using super-resolution microscopy may provide a way to view cell structures at a micron scale. The technique uses light waves to create interference patterns as light passes through the cell on the way to the mirror, which reflects the light back through the cell.

Researchers Phil Santangelo and Eric Alonas are shown with a spinning disk confocal microscope used to image cells. Santangelo is a professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University; Alonas is a Georgia Tech graduate student. Courtesy of John Toon, Georgia Tech.

Researchers at Peking University, the Georgia Institute of Technology, and University of Technology Sydney (UTS) found that when a mirror was placed behind a specimen instead of a microscopy slide, the interference between the excitation point spread function (PSF) and its reflection created an axially narrowed PSF away from the mirror surface, improving confocal excitation to approximately 110 nm. The researchers sectioned the axial by modulating wavelengths or by controlling the spacer between the mirror and the specimen.

The mirror-enhanced axial-narrowing super-resolution (MEANS) microscopy technique achieved an axial resolution that was more than six-fold higher than the optical diffraction limit. The lateral resolution was enhanced two-fold. The increase in lateral resolution and decrease in the thickness of an axial section were achieved without increasing the laser power directed on the biological specimen, thus avoiding the risk of photodamaging the specimen or the fluorescent dye.

The mirror-enhanced technique significantly improved resolution in the Z-axis, providing a way to view the 3D structure of a cell at a comparable resolution in each dimension. Microscope resolution in the X and Y axes is typically superior to resolution in the Z axis, regardless of the microscopy technique.

"Previously, the vision of biologists was blurred by the large axial and lateral resolution," said Peking University professor Peng Xi. "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."

This image shows a Vero cell that was grown on a first surface mirror and fluorescently stained to show the microtubules, which are part of the cell cytoskeleton. Courtesy of Eric Alonas, Georgia Tech.

While changing the optical system was relatively simple, growing cells on the custom-made mirrors required adapting existing biological techniques.

"Most people are not growing cells on mirrors, so it required some work to get the cell culture conditions correct," said professor Phil Santangelo, Georgia Institute of Technology. "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 technique allows scientists to see the ring structure of the nuclear pore complex and the tubular structure of the human respiratory syncytial virus (hRSV). The ability to see cell structures at the micron level may provide novel information about how cells communicate and how diseases develop.

"This simple technology is allowing us to see the details of cells that have never been seen before," said UTS professor Dayong Jin. "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 measure between one-fiftieth and one-twentieth of a micrometer. With this superresolution microscopy we are able to see the details of those tiny holes.”

The research was published in Light Science & Applications (doi: 10.1038/lsa.2016.134).

By growing cells on mirrors and imaging them using superresolution microscopy, scientists from Georgia Tech, Peking University and the University of Technology Sydney have addressed a problem that has long challenged scientists: Seeing 3D structures of cells with comparable resolution in each dimension. 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. Courtesy of Georgia Institute of Technology.