In vivo imaging of living tissue is usually performed by non-invasive microscopic techniques such as optical coherence microscopy and two-photon microscopy.
Two types of light – multiple scattering photons and ballistic photons – are produced when light passes through turbid materials like biological tissue. Ballistic photons tend to travel straight through the object without being deflected; thus, this type of light is used to reconstruct the image of the object.
In contrast, multi-scatter photons are produced due to random deviations as light passes through the material and appear as speckle noise in the reconstructed image. As light propagates over longer distances, the ratio of ballistic photons to multiple scattering increases dramatically, thereby obscuring image information.
Besides the noise produced by multi-scattered light, the optical aberration of ballistic light also causes image blurring and reduced contrast during the image reconstruction process.
Specifically, bone tissues comprise several complex internal structures, which cause complex optical aberration and severe multiple light scattering. When performing optical imaging of the mouse brain through an intact skull, it is very difficult to visualize the fine structures of the nervous system due to strong speckle noise and image distortion.
This poses problems in neuroscience research, which involves extensive use of the mouse as a model organism. The drawbacks of existing imaging techniques require removing or thinning the skull to analyze under the microscope the neural networks of brain tissue below.
Therefore, researchers have proposed other solutions to achieve more in-depth imaging of living tissue. For example, in recent years, three-photon microscopy has been successfully used for imaging neurons under the mouse skull.
However, three-photon microscopy is limited by a low laser repetition rate because it involves the use of an excitation window in the infrared domain, which can destroy living tissue during in vivo imaging. Additionally, it has a very high excitation power, which means that photobleaching is more prevalent than the two-photon approach.
A group of researchers led by Professor Wonshik Choi from the Center for Molecular Spectroscopy and Dynamics at the Institute of Basic Sciences (IBS) in Seoul, South Korea, recently achieved a significant breakthrough in optical deep tissue imaging. .
They created an innovative optical microscope capable of imaging through an intact mouse skull and developing a microscopic map of neural networks in brain tissue without loss of spatial resolution.
Dubbed a reflective array microscope, the new microscope incorporates the powers of hardware and computational adaptive optics (AO) – a technology originally created for ground-based astronomy to rectify optical aberrations.
The traditional confocal microscope quantifies the reflection signal only at the focal point of illumination and eliminates any out-of-focus light. In contrast, all scattered photons at positions other than the focal point are recorded by the reflection matrix microscope.
Then, the scattered photons are computationally rectified using an innovative AO algorithm known as single scattering closed-loop accumulation (CLASS), which was developed by the team in 2017. The algorithm harnesses all scattered light to selectively deflect ballistic light and rectify severe optical aberrations.
Unlike most traditional AO microscopy systems, which require fluorescent objects or bright point reflectors as guide stars quite similar to the use of AO in astronomy, the reflection array microscope operates without the need for marking fluorescent and without relying on the target. structures.
In addition, the number of rectifiable aberration modes is more than 10 times that of traditional AO systems. The reflection array microscope has a superior advantage because it can be integrated directly with a traditional two-photon microscope already widely used in the field of life sciences.
To eliminate the aberration experienced by the excitation beam of the two-photon microscope, the researchers added hardware-based adaptive optics in the reflective array microscope to compensate for the aberration of the mouse skull.
The capabilities of the new microscope were demonstrated by capturing two-photon fluorescence images of a dendritic spine of a neuron under the mouse skull, with a spatial resolution as close as the diffraction limit.
In general, a traditional two-photon microscope does not have the ability to resolve the delicate structure of the dendritic spine without completely removing brain tissue from the skull. This achievement is very crucial because the South Korean team presented the first high-resolution imaging of neural networks through an intact mouse skull. This implies that the mouse brain can now be studied in its most native states.
By correcting for wavefront distortion, we can focus light energy to the desired location within living tissue. Our microscope allows us to study fine internal structures deep within living tissue that cannot be resolved by any other means. This will greatly help us in the early diagnosis of diseases and accelerate research in neuroscience.
Research Professor Seokchan Yoon and Graduate Student Hojun Lee, Institute of Basic Sciences
For the team, the next step in the study is to reduce the form factor of the microscope and increase its imaging speed. Their goal is to develop a label-free reflecting array microscope with high imaging depth for application in clinics.
The reflection array microscope is the next generation technology that overcomes the limitations of conventional optical microscopes. This will allow us to broaden our understanding of the propagation of light through scattering media and expand the scope of applications that an optical microscope can explore..
Wonshik Choi, Vice Director, Center for Molecular Spectroscopy and Dynamics, Institute of Basic Sciences
Yoon, S. et al. (2020) Laser scanning reflection array microscopy for aberration-free imaging through an intact mouse skull. Nature Communication. doi.org/10.1038/s41467-020-19550-x.