Switching off the light to see better

Researchers from Osaka University used structured light and switchable fluorescent molecules to reduce the background light from the out-of-plane regions of microscope samples. This method allowed for the acquisition of images that surpassed the conventional resolution limit, and it may be useful for further study of cell clusters and other biological systems.

Fluorescence images of nuclei in a cell spheroid labeled by rsGamillus-S with a diameter of 100 µm observed with widefield, SPA-SIM, and 3DSIM at a depth of 43 µm.

Researchers at Osaka University employ fluorescent molecules that can switch on and off, along with selective illumination in a plane, to create sharper microscope images that can be used to visualize internal cell structures or cell clusters

Osaka, Japan – To study living organisms at ever smaller length scales, scientists must devise new techniques to overcome the so-called diffraction limit. This is the intrinsic limitation on a microscope’s ability to focus on objects smaller than the wavelength of light being used. Structured illumination microscopy is one of the super-resolution techniques that can help, by illuminating sinusoidal-patterned light on a sample to obtain a sharper image. However, background light from out-of-focus regions can still smear out the final picture.  

Configuration and imaging schemes of SPA-SIM (selective plane activation structured illumination microscopy). Simulated distribution of effective excitation pattern in SPA-SIM

In a study recently published in the journal Nature Methods, researchers from Osaka University demonstrated a new approach for super-resolution microscopy capable of observing structures inside a single cell or a cell cluster. This was accomplished by selecting only a desired plane to image using thin “light sheet” illumination, projected perpendicular to the lens, to switch on fluorophores. “We show that selective-plane activation allows us to image dense microstructures inside cells with excellent sharpness not readily available previously,” lead author of the study, Kenta Temma says. That is, sinusoidal “structured” light selectively excited only a thin plane where on-state fluorophores were localized, which allowed for background-free super-resolution imaging.

3D projection images of a living HeLa cell expressing Skyla-NS on mitochondria observed with SPA-SIM(proposed method) and conventional 3DSIM.

While some previous methods utilized random fluorescence emission from single molecules, or “donut” shaped second light source to deactivate or deplete fluorescent sources outside of a desired area, this new method can be gentler on cells that might be damaged by intense or long exposure to light. The researchers believe that their approach is especially effective when trying to understand what is happening in living systems with spatial structure, which can often exhibit background light outside the desired focal plane. This includes organoids, which are artificial assemblies of different cell types meant to reproduce the behavior of actual body organs much better compared with collections of cells cultured on a flat petri dish. “We anticipate that our technique will be useful for future biological studies of 3D cell clusters, including organoids,” says senior author, Katsumasa Fujita. The same could apply to other complex biological systems.

The article, “Selective plane activation structured illumination microscopy,” was published in Nature Methods at DOI: https://doi.org/10.1038/s41592-024-02236-3


About Osaka University
Osaka University was founded in 1931 as one of the seven imperial universities of Japan and is now one of Japan's leading comprehensive universities with a broad disciplinary spectrum. This strength is coupled with a singular drive for innovation that extends throughout the scientific process, from fundamental research to the creation of applied technology with positive economic impacts. Its commitment to innovation has been recognized in Japan and around the world, being named Japan's most innovative university in 2015 (Reuters 2015 Top 100) and one of the most innovative institutions in the world in 2017 (Innovative Universities and the Nature Index Innovation 2017). Now, Osaka University is leveraging its role as a Designated National University Corporation selected by the Ministry of Education, Culture, Sports, Science and Technology to contribute to innovation for human welfare, sustainable development of society, and social transformation.
Website: https://resou.osaka-u.ac.jp/en


Published: 22 Apr 2024


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Ministry of Education, Culture, Sports, Science and Technology,
Japan Science and Technology Agency