Team develops optical microscope using ultrasound for navigation

Ultrasound and optical microscopy combined for high-resolution deep-tissue observations

Skeletal muscle fibers in live whole-body zebrafish visualized

▲ From left: Professor Wonshik Choi (corresponding author), Professor Mooseok Jang (co-first author), 

Hakseok Ko, doctoral student in Department of Physics at Korea University (co-first author)

A local research team successfully developed a microscope capable of visualizing skeletal muscle fibers. Led by Associate Director Wonshik Choi of the Center for Molecular Spectroscopy and Dynamics at the Institute for Basic Science (IBS) and Professor Mooseok Jang of the Department of Bio and Brain Engineering at KAIST (Brain Dynamics Laboratory), the team devised an ultrasound-based technique for observing the microstructures of organisms, which was not possible with existing microscopes.

The results were published in the online version of Nature Communications (IF 11.878) on February 5.

* Title of article: Deep tissue space-gated microscopy via acousto-optic interaction / Nature Communications

* Author information: Mooseok Jang, Hakseok Ko, Jin Hee Hong, Won Kyu Lee, Jae-Seung Lee & Wonshik Choi

The human eye can distinguish objects placed 250 mm away and 70 mm apart. An optical microscope, which provides a magnified view of microstructures, is required for microstructural observations. However, observing the microstructures of live organisms is more complex and challenging.

Light passing through an organism is comprised of ballistic waves and scattered waves. Ballistic waves propagate through without being influenced by the organism, while scattered waves are components of light scattered in random directions due to cells within the organism or cell structures. A major disadvantage of using optical microscopes for deep tissue observations is that the stronger intensity of scattered waves compared to ballistic waves results in blurred images. The countless cells and structures within the organism induce light scattering, which causes blurring of images. Ultrasound imaging, on the other hand, is suited for deep tissue observations but comes at a poor resolution.

Combining the advantages of optical microscopy and ultrasound imaging, the team developed an ultrasound-based optical microscope capable of observing deep tissues within organisms at high resolution. The ultrasound-based microscope lowers the intensity of scattered waves by measuring only the light passing through the focal point of ultrasound. In other words, ultrasound is employed as a navigation device for the optical microscope.

Ultrasound influences the progression of light by condensing or expanding biological tissues to modify the refractive index. The team proposed a technique called space-gating, which involves the selective measurement of light passing through the focal point of ultrasound. Ultrasound acts as a light filter within the organism, and blocks the randomly scattered light. The space-gating technique resolved the issue of image blurring by lowering the intensity of scattered waves by at least 100 times.

Using the proposed microscope, the team obtained muscle tissue images of the spinal cord of whole-body zebrafish at 30 days post fertilization. With existing technology, muscle fibers could only be observed using sliced samples because of scattering within the organs and spinal cords of zebrafish. The team’s study holds significance in enabling deep tissue observations of zebrafish in an intact state.

The team plans to develop the space-gating technique for human body applications. Space-gating, with the miniaturization of microscopes and enhanced imaging speed, is expected to be useful in real-time medical diagnosis.

Wonshik Choi (professor, Department of Physics, Korea University), the associate director of the Center for Molecular Spectroscopy and Dynamics, said, “The ultrasound-based optical microscope addresses the shallow imaging depth problem of existing optical microscopes. Our space-gating technology can be further developed to enhance understanding of light scattering and to see more applications in biomedical optics.”

[Figure description]

[Fig. 1] Space-gating microscope

The space-gating microscope developed by researchers of the Center for Molecular Spectroscopy and Dynamics of IBS. The image shows the imaging objective lens (left, right) and the ultrasound transducer (bottom). The device enables the visualization of microstructures, including muscle fibers, of small organisms in an intact state.

[Fig. 2] Principle of the space-gating microscope

The team developed a microscope capable of deep tissue observations by combining ultrasound imaging and optical microscopy. The space-gating technique measures only the light passing through the focal point of ultrasound (green lines), and lowers the intensity of scattered waves by 100 times. The decreased intensity of scattered waves allows clearer images to be obtained.

[Fig. 3] Red blood cells visualized through space-gating

Wide-field imaging of red blood cells with a general microscope (a) and space-gated microscope (b). Red blood cells become more prominent with reduced scattering.

[Fig. 4] Change in small organism imaging paradigm

With existing optical microscopes, deep tissue observations required slicing and staining of samples (a). The team succeeded in employing ultrasound for deep tissue observations of zebrafish in an intact state (b).

[Fig. 5] Results of muscular observations of adult zebrafish

The team observed the inner structure of zebrafish at 30 days post fertilization with a general microscope (a) and space-gated microscope (b) Space-gating was successful in observing the myosepta (dotted yellow lines), the muscle-bone junction (dotted red line), and direction of muscle fibers (dotted white arrow).