Nanotechnology

New imaging techniques are not a last resort


June 17, 2023

(Nanowerk News) There are various ways to image a biological sample at the microscopic level, and each has its pros and cons. For the first time, a research team, including from the University of Tokyo, combined aspects of two leading imaging techniques to create a new method of imaging and analyzing biological samples. The concept, known as RESORT, paved the way for observing living systems in unprecedented detail.

This research has been published in Science Advances (“Super-resolution vibrational imaging based on photo-switchable Raman probes”). Living cells under a microscope. Comparison of RESORT and an earlier imaging technique known as stimulated Raman scattering (SRS). While the differences may seem subtle at first glance, the sharper images offered by RESORT can be of great benefit to researchers identifying and labeling cell components, such as mitochondria. (Image: Ozeki et al. CC-BY)

For as long as humanity has been able to manipulate glass, we have used optical devices to peer into microscopic worlds in increasing detail. The more we can see, the more we can understand, hence the pressure to improve the tools we use to explore the world around, and within us. Contemporary microscopic imaging techniques go far beyond what traditional microscopes can offer. Two leading technologies are super-resolution fluorescence imaging, which offers good spatial resolution, and vibrational imaging, which compromises spatial resolution but can use a wide range of colors to help label many types of constituents in cells.

“We were motivated by the limitations of this kind of imaging technique to try and create something better, and with RESORT we believe we have achieved this,” said Professor Yasuyuki Ozeki of the University of Tokyo’s Center for Advanced Science and Technology Research. “RESORT stands for saturable optical Raman transition, and it combines the benefits of super-resolution fluorescence and vibrational imaging without inheriting the disadvantages of either. This is a laser-based technique that uses something known as Raman scattering, a special interaction between molecules and light that helps identify what’s in a sample under a microscope. We successfully performed mitochondrial RESORT imaging in cells to validate the technique.”

There are several steps to RESORT imaging, and while it may seem complicated, the setup is not nearly as difficult as the technique with which it is intended to change. First, the specific components of the sample to be imaged need to be labeled, or stained, with special chemicals called photo-exchangeable Raman probes, whose Raman scattering can be controlled by the different types of laser beams used by RESORT.

Next, the sample is placed in an optical device that is used to properly illuminate the sample and create its image. For that to happen, the sample is then irradiated with a bi-color infrared laser pulse to detect Raman scattering, ultraviolet light and a special donut-shaped beam of visible light. Together, this limits the area where Raman scattering can occur, meaning the final stage, imaging, can detect the probe at very precise points, which results in high spatial resolution.

“It’s not just about getting higher-resolution images of microscopic samples; after all, an electron microscope can image these things in much greater detail,” said Ozeki. “However, electron microscopes inevitably damage or block the samples they observe. Through future developments by adding more colors to the Raman probe palette, RESORT will be able to image multiple components in live samples to analyze complex interactions like never before. This will contribute to a deeper understanding of fundamental biological processes, disease mechanisms, and potential therapeutic interventions.”

The team’s main goal is to improve microscopic imaging for use in medical research and related fields. However, the advances he made in laser design can be used in other laser applications as well, those requiring high power or precision control, such as materials science.





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