Sharper, Deeper, Faster: Interdisciplinary Team Develops Advanced Live-Imaging Approach
For modern biologists, the ability to capture high-quality, three-dimensional (3D) images of living tissues or organisms over time is necessary to answer problems in areas ranging from genomics to neurobiology and developmental biology. The better the image, the more detailed the information that can be drawn from it. Looking to improve upon current methods of imaging, researchers from the California Institute of Technology (Caltech) have developed a novel approach that could redefine optical imaging of live biological samples by simultaneously achieving high resolution, high penetration depth (for seeing deep inside 3D samples), and high imaging speed.
The imaging technique is explained in a paper in the advance online publication of the journal Nature Methods, released on July 14. It will also appear in an upcoming print version of the journal.
“Before our work, the state-of-the-art imaging techniques typically excelled in only one of three key parameters: resolution, depth, or speed. With our technique, it’s possible to do well in all three and, critically, without killing, damaging, or adversely affecting the live biological samples,” says biologist Scott Fraser, director of the Biological Imaging Center at Caltech’s Beckman Institute and senior author of the study.
The research team achieved this imaging hat trick by first employing an unconventional imaging method called 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. This allows millions of image pixels to be captured simultaneously, reducing the light intensity that needs to be used for each pixel. This not only enables fast imaging speed but also decreases the light-induced damage to the living samples, which the teams demonstrated 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. This process 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.