Dec 3 2020
In vivo imaging of living tissues are usually performed through non-invasive microscopic techniques like optical coherence microscopy and two-photon microscopy.
The schematic of the reflection-matrix microscope that was developed by researchers at the IBS Molecular Spectroscopy and Dynamics Research Center. The system makes use of confocal scanning and a Mach-Zehnder interferometer, similar to optical coherence microscopy. However, instead of confocal detection, interferometric images of reflected waves from the sample are measured using a camera. In addition, a spatial light modulator (SLM) is introduced to physically correct sample-induced wavefront distortion. (BS: Beam splitter, GMx/y: Galvo mirror, DG: Diffraction grating, sDM: Spectral dichroic mirror, OL: Objective lens). Image Credit: Institute for Basic Science.
Two types of light—multiply scattered photons and ballistic photons—are produced when light travels through turbid materials like biological tissues. The ballistic photons tend to move directly through the object without undergoing any deflection; thus, this type of light is used to reconstruct the image of the object.
By contrast, the multiply scattered photons are produced due to random deflections when the light travels through the material and appear as speckle noise in the reconstructed image. With the propagation of light through longer distances, the ratio between ballistic photons and multiply scattered increases dramatically, thus cloaking the image information.
Apart from the noise produced by the multiply scattered light, optical aberration of ballistic light also leads to image blur and reduction in contrast during the image reconstruction process.
Specifically, bone tissues include several complex internal structures, which cause complex optical aberration and severe multiple light scattering. While performing optical imaging of the mouse brain through an intact skull, it is very difficult to visualize the fine structures of the nervous system because of the 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 the existing imaging techniques necessitate the skull to be removed or thinned to microscopically analyze the neural networks of brain tissues underneath.
Therefore, researchers have proposed other solutions to realize more in-depth imaging of living tissues. For instance, in recent years, three-photon microscopy has been successfully employed for imaging neurons underneath the mouse skull.
However, three-photon microscopy is restricted by a low laser repetition rate as it involves using an excitation window in the infrared range, which can destroy the living tissue during in vivo imaging. Moreover, it has a very high excitation power, meaning that photobleaching is more widespread than the two-photon approach.
A group of researchers led by Prof. Wonshik Choi from the Center for Molecular Spectroscopy and Dynamics within the Institute of Basic Science (IBS) in Seoul, South Korea recently achieved a significant advancement in deep-tissue optical imaging.
They created an innovative optical microscope with the ability to image through an intact mouse skull and develop a microscopic map of neural networks in the brain tissues without the loss of spatial resolution.
Dubbed as a reflection matrix microscope, the new microscope integrates the powers of both hardware and computational adaptive optics (AO)—a technology originally created for ground-based astronomy to rectify optical aberrations.
Traditional confocal microscope quantifies reflection signal only at the focal point of illumination and discards all out-of-focus light. By contrast, all the scattered photons at positions other than the focal point are recorded by the reflection matrix microscope.
Then, the scattered photons are computationally rectified with the help of an innovative AO algorithm known as closed-loop accumulation of single scattering (CLASS), which was developed by the team in 2017. The algorithm leverages all scattered light to selectively derive ballistic light and rectify severe optical aberration.
In contrast to the most traditional AO microscopy systems, which necessitate fluorescent objects or bright point-like reflectors as guide stars quite similar to the use of AO in astronomy, the reflection matrix microscope operates without the need for fluorescent labeling and without relying on the target’s structures.
Moreover, the number of rectifiable aberration modes is over 10 times more compared to those of the traditional AO systems. The reflection matrix microscope has a higher benefit since it can be integrated directly with a traditional two-photon microscope that is extensively used already in the field of life sciences.
To eliminate the aberration undergone by the excitation beam of the two-photon microscope, the researchers added hardware-based adaptive optics into the reflection matrix microscope to offset the aberration of the mouse skull.
The new microscope’s capabilities were demonstrated by capturing two-photon fluorescence images of a dendritic spine of a neuron beneath the mouse skull, with a spatial resolution as close as the diffraction limit.
In general, a traditional two-photon microscope lacks the ability to resolve the dendrite spine’s delicate structure without completely eliminating the brain tissue from the skull. This achievement is a highly crucial one as the South Korean team showcased the first high-resolution imaging of neural networks through an intact mouse skull. This implies that the mouse brain can now be investigated in its most native states.
By correcting the wavefront distortion, we can focus light energy on the desired location inside the living tissue. Our microscope allows us to investigate fine internal structures deep within living tissues that cannot be resolved by any other means. This will greatly aid us in early disease diagnosis and expedite neuroscience research.
Research Professor Seokchan Yoon and Graduate Student Hojun Lee, Institute for Basic Science
For the team, the next step in the study is to reduce the form factor of the microscope and increase its imaging speed. Their aim is to develop a label-free reflective matrix microscope with high imaging depth for application in clinics.
Reflection matrix microscope is the next-generation technology that goes beyond the limitations of conventional optical microscopes. This will allow us to widen our understanding of the light propagation 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 for Basic Science
Journal Reference:
Yoon, S., et al. (2020) Laser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull. Nature Communications. doi.org/10.1038/s41467-020-19550-x.