Merged Microscope Illuminates Biological Processes In Vivo

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National Institutes of Health (NIH) researchers combine two microscope technologies to create sharper, faster images.

A new microscope that combines two different microscope technologies creates sharper images of rapidly moving processes inside a living cell, according to scientists at the National Institute of Biomedical Imaging and Bioengineering (NIBIB), a part of the National Institutes of Health (NIH).

In a May 2018 published paper in the peer-reviewed journal, Nature Methods, Hari Shroff, PhD, chief of NIBIB’s lab section on high resolution optical imaging (HROI), described his new improvements to traditional total internal reflection fluorescence (TIRF) microscopy. TIRF microscopy illuminates a sample at a sharp angle so that light reflects back. This illuminates only a thin section of the sample that is extremely close to the coverslip. This process creates very high contrast images because it eliminates much of the background, out-of-focus, light that conventional microscopes pick up.

TIRF microscopy has been used in cell biology for decades, but it produces blurry images of small features within cells. Super-resolution microscopy techniques applied to TIRF microscopes have traditionally been able to improve the resolution, but such attempts have always compromised speed. This has made it impossible to create clear images of rapidly moving objects. As a result, many cellular processes remain too small or fast to observe.

Shroff and his team realized that if they could take a high-speed, super-resolution microscope and modify it to act like a TIRF microscope, they could obtain the benefits of both. Instant structured illumination microscopy (iSIM), developed by the Shroff lab in 2013, can capture video at 100 frames per second, more than three times faster than most movies or internet videos. iSIM does not have the contrast that TIRF microscopes do, however. The team designed a simple “mask” that blocked most of the illumination from the iSIM-mimicking a TIRF microscope. Combining the strengths of both types of microscopy enabled the researchers to observe rapidly moving objects about 10 times faster than other microscopes at similar resolution.


“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 Shroff in a NIH press release. “Our method improves the spatial resolution of TIRF microscopy without compromising speed-something that no other microscope can do. 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.”

With the new microscope, Shroff and his team 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 very fast and are blurred when imaged by other microscopes. They also used their technique to reveal the dynamics and spatial distribution of HRas, a protein that has been implicated in facilitating the growth of cancerous tumors. The new combined microscope is available to researchers to try out at Shroff’s lab, and the schematics of the technology are available for free, as stated by Schroff.

Source: National Institutes of Health