Scientists invent a new type of microscope that can see through an intact skull

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 multiply scattered photons. The ballistic photons travel straight through the object without experiencing any deflection and hence are used to reconstruct the object image. On the other hand, the multiply scattered photons are generated via random deflections as the light passes through the material and show up as speckle noise in the reconstructed image. As the light propagates through increasing distances, the ratio between multiply scattered and ballistic photons increases drastically, thereby obscuring the image information. In addition to the noise generated by the multiply scattered light, optical aberration of ballistic light also causes contrast reduction and image blur during the image reconstruction process.

Bone tissues in particular have numerous complex internal structures, which cause severe multiple light scatterings and complex optical aberrations. When it comes to optical imaging of the mouse brain through an intact skull, the fine structures of the nervous system are hard to visualize due to strong speckle noise and image distortion. This is problematic in neuroscience research, where the mouse is widely used as a model organism. Due to the limitation of the currently used imaging techniques, the skull has to be removed or thinned to microscopically investigate the neural networks of brain tissues underneath.

Hence other solutions have been suggested to achieve deeper imaging of living tissues. For example, three-photon microscopy has been successfully used to image neurons beneath the mouse skull in recent years. However, three-photon microscopy is limited by a low laser repetition rate as it employs an excitation window in the infrared range, which can damage living tissue during in vivo imaging. It also has excessive excitation power, which means photobleaching is more extensive in comparison to the two-photon approach.

Recently, a research team made a major breakthrough in deep-tissue optical imaging. They developed a novel optical 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 termed a 'reflection matrix microscope,' and it combines the powers of both hardware and computational adaptive optics (AO), which is a technology originally developed for ground-based astronomy to correct optical aberrations. While a conventional confocal microscope measures reflection signal only at the focal point of illumination and discards all out of focus light, the reflection matrix microscope records all the scattered photons at positions other than the focal point. The scattered photons are then computationally corrected using a novel AO algorithm called closed-loop accumulation of single scattering (CLASS), which the team developed back in 2017. The algorithm exploits all scattered light to selectively extract ballistic light and correct severe optical aberration. Compared to most conventional AO microscopy systems, which require bright point-like reflectors or fluorescent objects as guide stars similarly to the use of AO in astronomy, the reflection matrix microscope works without any fluorescent labeling and without depending on the target's structures. In addition, the number of aberration modes that can be corrected is more than 10 times greater than that of conventional AO systems.

Seokchan Yoon et al, Laser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull, Nature Communications (2020). DOI: 10.1038/s41467-020-19550-x

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