Optical scattering is a real problem for biological imaging. By preventing light from being focused deeply into biological tissue, scattering effects limit imaging depths to around 100 microns, producing only blurred images beyond. A new technique called ultrasound-induced optical clearing microscopy could increase this distance by more than a factor of six, thanks to the somewhat counterintuitive step of inserting a layer of gaseous bubbles in the area being imaged. Adding this bubble layer ensures that the photons do not deviate as they propagate through the sample.
Optical scattering occurs when light interacts with structures smaller than its wavelength. The incident light perturbs electrons in the structure, forming oscillating dipole moments that re-emit the light in many different directions.
“Techniques like confocal microscopy are widely employed in life science research such as cancer and brain tissue imaging, but they are limited because of this problem,” explains Jin Ho Chang at the DGIST (Daegu Gyeongbuk Institute of Science and Technology) in Korea. “The imaging-depth limitation is mainly due to incident photons being severely deflected from their original propagation directions as a result of optical scattering. Indeed, the number of non-scattered photons decreases exponentially with the distance travelled by the photons, so light cannot be tightly focused after a depth of about 100 microns.”
While researchers have developed various types of light wavefront-shaping techniques to address this limitation, none of them can be used to take three-dimensional images. These other techniques also require high-performance optical modules and sophisticated optics systems.
No optical scattering in the bubble cloud
In the latest work, Chang and colleagues developed a new approach in which they use high-intensity ultrasound to generate gas bubbles in the volume of tissue located in front of the imaging plane. To prevent the bubbles from collapsing and possibly damaging the tissue, the researchers transmitted low-intensity ultrasound continuously during the laser scanning microscope imaging process, maintained a continuous flux of bubbles throughout. They found that when the concentration of gas bubbles in the volume is higher than 90%, photons from the imaging laser experience hardly any optical scattering inside the gas bubble region (dubbed the “bubble cloud”). This is because the temporarily-created gas bubbles reduce optical scattering in the same direction as the propagation of the incident light, thus increasing its penetration depth.
“As a result, the laser can be tightly focused on the imaging plane, beyond which conventional laser scanning microscopy cannot acquire sharp images,” Chang tells Physics World. “This phenomenon is analogous to optical clearing based on chemical agents, so we named our approach ultrasound-induced optical clearing microscopy (US-OCM).”
Unlike conventional optical clearing methods, UC-OCM can localize the optical clearing in the region of interest and restore the original optical properties to the region once the bubble flux is switched off. This implies that the technique should be harmless to living tissue.
According to the researchers, who detail their work in Nature Photonics, the main advantage of US-OCM are: an increase in the imaging depth by a factor of more than six with a resolution that is similar to that of conventional laser microscopy; fast image data acquisition and image reconstruction (just 125 milliseconds is required for one frame image consisting of 403 x 403 pixels); and easy-to-obtain 3D images.
And that is not all: the team point out that implementing the new method requires only a relatively simple acoustic module (a single ultrasound transducer and a transducer-driving system) to be added to a conventional laser scanning microscopy setup. The technique could also be extended to other laser scanning microscopy techniques such as multiphoton and photoacoustic microscopy.
Ultrasound and light easy to combine
“I personally believe that the development of hybrid technology is one of the new research directions, and ultrasound and light are relatively easy to combine to maximize their advantages while complementing each other’s disadvantages,” Chang says. “Researchers working in the field of ultrasound have known for a long time that strong ultrasound can create gas bubbles in biological tissue and that they can disappear completely without damaging tissue.”
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The idea for the experiment came up during discussions with team member Jae Youn Hwang, an optics specialist at the DGIST. The thought was that ultrasound-induced gas bubbles could be used as an optical clearing agent if they could somehow create densely packed bubbles in the area of interest. “Conventional optical clearing relies on the fact that optical scattering is minimal when the refractive indices of light scatterers in tissue are similar to each other,” Chang explains. “Chemical agents are employed to reduce the high refractive index of scatterers so that it approaches that of the tissue itself.”
According to the DGIST team, the technique might be used for high-resolution brain tissue imaging, early diagnosis of Alzheimer’s disease and precise diagnosis of cancer tissue in combination with endoscope technology. “I also believe that the basic concept of this study can be applied to optical therapies, such as photothermal and photodynamic therapies to improve their efficacy because they also suffer from limited light penetration,” Chang says.