NIH Researchers Merge Microscopy Techniques for Faster Images

To create sharper images more quickly, researchers blended instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM). The new technique, called instant TIRF-SIM, allows researchers to observe rapidly moving objects about 10 times faster than other microscopes at similar resolution.

TIRFM has been used in cell biology for decades, but it produces blurry images of small features within cells. In the past, superresolution microscopy techniques have been applied to TIRF microscopes to improve image resolution; but such attempts have compromised speed, making it difficult to use TIRFM to clearly image rapidly moving objects.

The rapid movements of Rab11 particles can be clearly imaged with the new instant TIRF-SIM microscope. Courtesy of Hari Shroff, National Institute of Biomedical Imaging and Bioengineering.

To resolve this issue, researchers at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) modified an iSIM microscope to perform like a TIRF microscope.

Developed by the NIBIB lab in 2013, iSIM, a high-speed, superresolution microscope, can capture video at 100 frames per second — more than three times faster than most movies or internet videos. However, iSIM does not have the contrast capabilities that TIRF microscopes do.

The team designed a simple mask that blocked most of the illumination from the iSIM, causing it to mimic the contrast features of a TIRF microscope. This step led to instant TIRF-SIM, a technique that improved the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, enabling frame rates up to 100 Hz over hundreds of time points.

Researchers applied instant TIRF-SIM to multiple live samples. They achieved rapid, high-contrast superresolution imaging close to the coverslip surface.

For example, researchers were able to follow rapidly moving Rab11 particles near the plasma membrane of human cells. Attached to molecular cargo that are transported around the cell, these particles move so fast that they are blurred when imaged by other microscopes.

“TIRF microscopy has been around for more than 30 years, and it is so useful that it will likely be around for at least the next 30,” said Hari Shroff, lab chief of NIBIB’s Section on High Resolution Optical Imaging.

“Our method improves the spatial resolution of TIRF microscopy without compromising speed — something that no other microscope can do,” Shroff said. “We hope it helps us clarify high-speed biology that might otherwise be hidden or blurred by other microscopes so that we can better understand how biological processes work.”

As with all of the microscopes developed by the Shroff team, researchers are welcome to contact the lab to try out the microscope or to acquire free schematics of the technology.

The research was published in Nature Methods (doi:10.1038/s41592-018-0004-4).