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Sub-wavelength Bessel-beam Photoacoustic Microscopy

20 Mar | By Byullee Park
Sub-wavelength Bessel-beam Photoacoustic Microscopy
In vivo mouse ear photoacoustic (PA) images by (a, b) conventional sub-wavelength (SW) PA microscopy (PAM) and (b, d) newly developed SW Bessel-beam PAM.
Image source: Journal of Biophotonics
By: Byullee Park et al.

A research team at Pohang University of Science and Technology, Republic of Korea has developed a sub-wavelength (SW) resolution Bessel-beam photoacoustic microscopy (BB-PAM) system in a reflection-mode. The BB-PAM has the potential for observing in vivo microvascular structures with red blood cells (RBCs) and real-time changes, such as cell migration and cell culture in three-dimensional spaces. SW resolution and extended depth-of-field (DOF) of the system have been demonstrated through in vitro and in vivo imaging experiments in the article published in Journal of Biophotonics in 2018.

 

Optical-resolution photoacoustic microscopy (OR-PAM), which combines abundant light absorption contrast and sensitive ultrasound signal detection, has been a promising imaging system for biological research from single cells to microvessels. SW photoacoustic microscope (SW-PAM) refers OR-PAM with a lateral resolution to reach the optical diffraction limit. The SW-PAM achieves the high resolution because the spot size of the light is very small. However, as the focus of the light becomes smaller, the DOF becomes very short. Thus, its imaging speed may be limited because multiple sectioning is required to obtain information at various depths. Based on this perception, "We tried to overcome the short DOF problem of SW-PAM by using the non-diffracted characteristic of Bessel-beam (BB)," said Chulhong Kim, Mueunjae Chaired Associate Professor at Creative IT Engineering Department, POSTECH, and Republic of Korea. 

 

The research team has successfully developed a BB-PAM system that can simultaneously obtain both SW resolution and extended DOF using an axicon and a high numerical aperture objective lens. “We used an axicon lens to create a BB and combined it with a high numerical aperture objective lens. We also conducted in vitro experiment to measure the performance of the system, and it was confirmed that the system had a SW resolution and that the DOF was 7 times better than the conventional one,” said Byullee Park, a PhD candidate working in Dr. Kim’s research group.

 

BBs have the advantage of a diffractive-free nature because of their inherent characteristics, but they have the disadvantage that undesired signals as an artifact can occur due to the presence of side-lobes. To tackle the challenge of the side-lobes, the team used an image processing method by using MATLAB. “We applied the blind-deconvolution algorithm to photoacoustic (PA) images to effectively remove the artifacts caused by the side-lobe of the BB. In particular, to maximize the performance of the algorithm, an actual BB distribution was obtained and used as an initial point spread function,” explained Kim.

 

The research team designed the reflection-mode SW resolution BB-PAM so that light illumination and PA signal detection can be performed on the same side. “To obtain a PA image regardless of the sample thickness, the system must be designed in a reflection mode. We were able to develop a SW resolution BB-PAM in reflection-mode by attaching a miniature ultrasonic detector in front of the objective lens,” explained Kim.


The team demonstrated the possibility that the SW resolution BB-PAM in the reflection-mode can be applied to a variety of biological studies by performing in vivo mouse imaging experiments with the system. “Because our BB-PAM maintains high resolution at the extended DOF, compared to the existing photoacoustic microscopy, we expect this system to be useful for various biological studies such as monitoring of microvascular structure with RBCs, cell migration and cell culture in 3D space,” said Kim.

 


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