Ghost Imaging Speeds Up Superresolution Microscopy

Scientists from the Chinese Academy of Sciences have developed a new imaging technique that produces nm-scale resolution using significantly fewer images than traditional nanoscopy techniques. The scientists used ghost imaging to enhance the imaging speed of their technique. The new approach could be useful for live cell imaging.

The technique is based on stochastic optical reconstruction microscopy (STORM), a wide-field microscopy method that uses fluorescent labels that switch between light-emitting (on) and dark (off) states. By acquiring hundreds or even thousands of images, each capturing the subset of fluorescent labels that are “on” at a given time, STORM allows the user to determine the location of each molecule and use this information to reconstruct a fluorescence image.

To speed up the STORM imaging process, the researchers turned to ghost imaging, a technique that forms a picture of an object by correlating a light pattern that interacts with the object with a reference pattern that does not. The researchers also used compressive imaging, a computational approach that enables image reconstruction with fewer exposures because it uses an algorithm to fill in the missing information.

A Chinese research team has developed an advanced imaging technique to achieve superresolution microscopy faster and with many fewer images. The new method could make it possible to capture processes in living cells at speeds not previously possible. Courtesy of Wang Zhongyang.

Using a random phase modulator, the researchers turned the fluorescence from the object into a random speckle pattern. Coding the fluorescence in this way allowed each pixel of a very fast CMOS camera to collect light intensity from the entire object in a single frame.

To form the image via ghost imaging and compressive imaging, the light intensity was correlated with a reference light pattern in a single-step process. The result was more efficient image acquisition and a reduction in the number of frames required to form a high-resolution image.

“While STORM requires a low density of fluorescent labels and many image frames, our approach can create a high-resolution image using very few frames and a high density of fluorophores,” researcher Han Shensheng said. “It also doesn’t need any complex illumination, which helps reduce photobleaching and phototoxicity that could harm dynamic biological processes and living cells.”

The researchers tested the technique by using it to image a 60-nm ring. The new nanoscopy approach resolved the ring using just 10 images, while traditional STORM approaches would have needed up to 4000 frames to achieve the same result, according to the team. The new approach resolved a 40-nm ruler using 100 images.

This is the nanoscopy setup used to image a 60-nm ring (inset). Courtesy of Wang Zhongyang.

The researchers showed that their nanoscopy technique can reduce the number of sampling frames by one order of magnitude compared to previous superresolution imaging methods based on single-molecule localization.

“Our imaging method can potentially probe dynamics occurring on millisecond timescales in subcellular structures with spatial resolution of tens of nanometers — the spatial and temporal resolution at which biological processes take place,” researcher Wang Zhongyang said.

The researchers hope to make the technique fast enough to achieve video-rate imaging with a large field of view in order to acquire 3D and color images. The new method could make it possible to capture the details of processes occurring in living cells at speeds not previously possible.

“We hope this method can be applied to a variety of fluorescent samples, including those that exhibit weaker fluorescence than those used in this research,” Wang said.

The research was published in Optica, a publication of The Optical Society (OSA) (www.doi.org/10.1364/OPTICA.6.001515).

The new microscopy approach uses a random phase modulator, which turns fluorescence from the sample into a random speckle pattern. Coding the fluorescence in this way allows each pixel of a very fast CMOS camera to collect light intensity from the whole object in a single frame. To form the image via ghost imaging and compressive imaging, this light intensity is correlated with a reference light pattern. Courtesy of Wang Zhongyang, Chinese Academy of Sciences.