Biological tissues have natural light scattering properties that limit the depth at which high-resolution imaging can be achieved, even when using powerful laser scanning methods like confocal fluorescence microscopy. Chemical optical clearing methods are frequently used to render tissues transparent for imaging, but these methods can be highly time-consuming, require expensive reagents and are not compatible with live-cell imaging. A team led by researchers from the Daegu Gyeongbuk Institute of Science and Technology (DGIST) has now developed a new optical clearing method based on high-intensity pulsed ultrasound that greatly increases imaging depth and could be applied to living tissues.
The technique, called ultrasound-induced optical clearing microscopy (US-OCM), is based on the ability of high intensity ultrasound to cause micrometer-sized gas bubbles to form inside tissue; the research team first discovered the ability of these bubbles to preserve the propagation direction of incident light in 2017. The bubbles cause optical scattering in the same direction as the incident light, increasing its penetration depth; however, in previous experiments, the gas bubbles were too sparse to truly improve imaging depth. In their most recent work, the team improved the ultrasound technology to create a bubble layer in a desired area with dense gas bubbles (with a density of 90% or more) inside living tissue, and maintain this bubble layer during imaging using low-intensity continuous ultrasound.
The researchers wrote that inside the bubble “cloud” induced by the ultrasound, optical scattering and unwanted changes in the propagation direction of incident light were minimized, allowing the laser to be tightly focused deeper into the tissue. In ex vivo experiments on living tissue, the team reported that US-OCM increased imaging depth by a factor of six or more while maintaining similar resolution to conventional confocal fluorescence microscopy. Additionally, the researchers did not observe any damage to the tissue and noted that the gas bubbles disappeared after ultrasonic irradiation ceased, with the original optical properties of the tissue also returning. This suggests that the ultrasonic optical clearing method could be used for live-cell imaging and even in vivo applications in the future. This research was published in Nature Photonics.
“The technology secured through this study will be applied to various optical imaging techniques including multiphoton microscopy and photoacoustic microscopy in addition to several optical therapies including photothermal therapy and photodynamic therapy,” said co-corresponding author Jin Ho Chang, a professor in the Department of Electrical Engineering and Computer Science at DGIST. “This would enhance the application of existing technologies by increasing their image and treatment depth.”