Laser-based Photoacoustic Method Provides Subcellular Insights

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City University of Hong Kong researchers developed a multiwavelength optical resolution photoacoustic microscopy (OR-PAM) system based on a single laser source. The system enables simultaneous multicontrast imaging of hemoglobin concentration, blood flow speed, blood oxygen saturation, and lymphatic concentration. Information, at the level the new system is able deliver, provides subcellular insights useful for the study of disease models, such as in cancer research.

Photoacoustic imaging works from the platform of the intrinsic absorption properties of the targeted biological tissue. When targeted by a laser beam, the tissue absorbs the light and generates heat, starting a process that causes thermal expansion and, from it, a mechanical ultrasonic wave, or photoacoustic wave. When an ultrasonic transducer receives that wave, it reconstructs it in a process that allows scientists to produce an image showing the light absorption distribution in the tissue.
Simultaneous multicontrast OR-PAM of hemoglobin concentration, oxygen saturation, blood flow speed, and lymphatic concentration. Courtesy of Liu et al.
Simultaneous multicontrast OR-PAM of hemoglobin concentration, oxygen saturation, blood flow speed, and lymphatic concentration. Courtesy of Liu et al.

OR-PAM, a relatively new hybrid imaging technique, provides high-resolution and high-contrast images, and has found utility in live, multicontrast functional imaging. The limited wavelength choice of most commercial lasers, combined with the limitations of existing scanning methods, means that the technique is able to obtain no more than two types of contrast in a single scan. Those factors make the method time-consuming and, to date, difficult to use in applications requiring the capture of the dynamic changes of functional information in biological tissues.

To improve the method, Lidai Wang and his team developed a five-wavelength fiber laser source based on a single wavelength nanosecond laser source. Because it is able to switch wavelengths at a submicrosecond timescale, the source provided a path toward the development of simultaneous, multifunctional OR-PAM. The team validated its system by measuring the energy fluctuation and drift, testing the imaging depth, as well as the lateral and axial resolution for OR-PAM imaging.

Wang said the system is based on the simulated Raman scattering (SRS) effect; in essence, the pumped laser source generated a scattered laser beam with a longer wavelength than the original incident beam through the optical fiber. Once the energy of the pumped laser source exceeds a threshold, the generated SRS wavelength sustains high directivity, high monochromaticity, and high coherence, making it a suitable source for OR-PAM, with the multiple scattered wavelengths enabling multicontrast photoacoustic imaging.

The team also developed a multiparameter image processing method for calculating the diameter, depth, and tortuosity in microvascular vessels. That development provides an image analysis basis for modeling disease.

The team further carried out multifunctional imaging of tumor development, lymphatic clearance, and brain imaging.

To test the independent system, team members performed multifunctional microscopic imaging of small animals in vivo evaluating hemoglobin concentration, blood flow speed, oxygen saturation, and lymphatic concentration. They also analyzed morphological and functional differences (including diameter, blood flow, blood oxygen level, etc.) of different blood vessels in the imaging area.

Traditional multifunctional OR-PAM requires multiple scanning and laser sources to achieve such results, meaning the researchers’ work addresses two significant problems. One is that the microenvironment of blood vessels within the tissue changes with time; multiple long-term scanning introduces inconsistencies in functional imaging.

The second problem is the asynchrony in the different laser sources. The resulting fluctuations create systematic calculation errors. By using a single source, the method realizes multifunctional imaging with single scanning, condensing imaging time and improving imaging accuracy.

The research was published in Advanced Photonics (

Published: March 2021
Research & TechnologyMicroscopyPhotoacoustic Microscopyphotoacoustic microscopephotoacoustic microscope laserLasersmultiwavelengthBiophotonicsmulticontrastImagingCity University of Hong KongLidai Wangsimultaneousbiomolecularbiomolecular imagingBioScan

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