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Non-invasive microscopic techniques such as optical coherence microscopy and two-photon microscopy are commonly used for in vivo imaging of living tissues. When light passes through turbid materials such as biological tissues, two types of light are generated: ballistic photons and multiplied scattered photons. Ballistic photons travel directly through the object without undergoing any deviation and then are used to reconstruct the image of the object. On the other hand, the multiply scattered photons are generated via random deflections as light passes through the material and appears as speckled noise in the reconstructed image. As light travels across increasing distances, the ratio of scattered to ballistic photons increases dramatically, thus obscuring image information. In addition to the noise generated by the multiplied scattered light, the optical aberration of the ballistic light also causes contrast reduction and image blurring during the image reconstruction process.
Bone tissues in particular have numerous complex internal structures, which cause severe multiple light scattering and complex optical aberration. When it comes to optical imaging of the mouse brain through an intact skull, the subtle structures of the nervous system are difficult to visualize due to the loud speckled noise and image distortion. This is problematic in neuroscientific research, where the mouse is widely used as a model organism. Due to the limitation of the imaging techniques currently used, the skull must be removed or thinned to investigate the neural networks of the underlying brain tissues under a microscope.
Then other solutions were suggested to obtain deeper images of living tissues. For example, three-photon microscopy has been used successfully in recent years to visualize neurons under the mouse skull. However, three-photon microscopy is limited by a low repetition rate of the laser since it uses an excitation window in the infrared range, which can damage living tissue during in vivo imaging. It also has excessive excitation power, which means that photobleaching is more extensive than the two-photon approach.
Recently, a research team led by Prof. CHOI Wonshik at the Center for Molecular Spectroscopy and Dynamics at the Institute of Basic Science (IBS) in Seoul, South Korea, took a major step forward in optical deep tissue imaging. . They developed a novel light microscope that can image through an intact mouse skull and acquire a microscopic map of neural networks in brain tissues without losing spatial resolution.
This new microscope is defined as a reflection matrix microscope and combines the powers of hardware and computational adaptive optics (AO), a technology originally developed for terrestrial astronomy to correct optical aberrations. While the conventional confocal microscope measures the reflection signal only at the focal point of illumination and eliminates all out-of-focus light, the reflection matrix microscope records all scattered photons at locations other than the focal point. The scattered photons are then computationally corrected using a new AO algorithm called Single Dispersion Closed Loop Accumulation (CLASS), which the team developed in 2017. The algorithm leverages all scattered light to selectively extract ballistic light. and correct severe optical aberration. Compared to more conventional AO microscopy systems, which require bright point reflectors or fluorescent objects as guide stars similar to the use of AO in astronomy, the reflection matrix microscope works without any fluorescent labeling and without depending on the target structures. Furthermore, the number of aberration modes that can be corrected is more than 10 times that of conventional AO systems.
The reflection matrix microscope has a great advantage as it can be directly combined with a conventional two-photon microscope which is already widely used in the life sciences. To remove the aberration experienced by the excitation beam of the two-photon microscope, the team implemented hardware-based adaptive optics inside the reflection matrix microscope to counteract the aberration of the mouse skull. They demonstrated the capabilities of the new microscope by acquiring two-photon fluorescence images of a neuronal dendritic spine behind the mouse skull, with spatial resolution close to the diffraction limit. Normally a conventional two-photon microscope cannot resolve the delicate structure of the dendritic spine without completely removing the brain tissue from the skull. This is a very significant result, as the South Korean team demonstrated the first high-resolution image of neural networks through an intact mouse skull. This means that it is now possible to investigate the mouse brain in its most native states.
Research professor YOON Seokchan and graduate student LEE Hojun, who led the study, said, “By correcting the wavefront distortion, we can concentrate light energy in the desired location within living tissue.” “Our microscope allows us to investigate subtle internal structures deep in living tissues that cannot be resolved by any other means. This will greatly help us in the early diagnosis of disease and accelerate neuroscientific research.”
The researchers set their next search direction to minimize the microscope’s form factor and increase its imaging speed. The goal is to develop an unlabeled reflective matrix microscope with a high imaging depth for use in clinics.
Deputy Director CHOI Wonshik said: “The reflection matrix microscope is the next generation technology that goes beyond the limitations of conventional optical microscopes. This will allow us to broaden our understanding of the propagation of light through diffusion media and expand the reach. of the applications that an optician microscope can explore “.
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Materials provided by Institute of Basic Sciences. Note: The content can be changed by style and length.
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