CALTECH (US) — New technology could enhance in triplicate optical imaging of live biological samples by simultaneously improving resolution, penetration depth, and speed.
“Before our work, the state-of-the-art imaging techniques typically excelled in only one of three key parameters: resolution, depth, or speed,” says Scott Fraser, professor of biology and bioengineering at California Institute of Technology (Caltech).
“With our technique, it’s possible to do well in all three and, critically, without killing, damaging, or adversely affecting the live biological samples.”
3-D live imaging of zebrafish (upper panel) and fruit fly (lower panel) embryos with two-photon light sheet microscopy. (Credit: Willy Supatto, Seth Ruffins, Thai Truong)
In developing the technology, reported in the journal Nature Methods, researchers first used light-sheet microscopy, where a thin, flat sheet of light is used to illuminate a biological sample from the side, creating a single illuminated optical section through the sample.
The light given off by the sample is then captured with a camera oriented perpendicularly to the light sheet, harvesting data from the entire illuminated plane at once, allowing millions of image pixels to be captured simultaneously, reducing the light intensity that needs to be used for each pixel.
This not only allows fast imaging speed but also decreases the light-induced damage to the living samples, which the teams demonstrated by using the embryos of fruit fly and zebrafish.
To achieve sharper image resolution with light-sheet microscopy deep inside the biological samples, the team used a process called two-photon excitation for the illumination, a process that has been used previously to allow deeper imaging of biological samples; however, it usually is used to collect the image one pixel at a time by focusing the exciting light to a single small spot.
“The conceptual leap for us was to realize that two-photon excitation could also be carried out in sheet-illumination mode,” says Thai Truong, a postdoctoral fellow in Fraser’s laboratory and first author of the paper.
“With this approach, we believe that we can make a contribution to advancing biological imaging in a meaningful way,” Truong says. “We did not want to develop a fanciful optical imaging technique that excels only in one niche area, or that places constraints on the sample so severe that the applications will be limited.
“With a balanced high performance in resolution, depth, and speed, all achieved without photo-damage, two-photon light-sheet microscopy should be applicable to a wide variety of in vivo imaging applications.”
“We believe the performance of this imaging technique will open up many applications in life sciences and biomedical research—wherever it is useful to observe, non-invasively, dynamic biological process in 3-D and with cellular or subcellular resolution,” says Willy Supatto, co–author of the paper and a former postdoctoral fellow in Fraser’s lab and now at the Centre National de la Recherche Scientifique, in France.
One example of such an application would be to make 3-D movies of the entire embryonic development of an organism, covering the entire embryo in space and time that could capture what individual cells are doing, as well as important genes’ spatiotemporal expression patterns—elucidating the activation of those genes within specific tissues at specific times during development.
“The goal is to create ‘digital embryos,’ providing insights into how embryos are built, which is critical not only for basic understanding of how biology works but also for future medical applications such as robotic surgery, tissue engineering,”
The research was supported by the Beckman Institute and the National Institutes of Health.
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