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Laser scanning reflection-matrix microscopy sees inside intact mouse skull

08 Dec 2020

South Korea team maps neurons in the brain without losing spatial resolution.

Visualizing tissues and neurons inside an animal's skull has long been a challenge for biophotonics techniques, thanks to the skull's thickness and the complex structures inside.

Approaches to this problem have included the use of endoscopic platforms in brain imaging, two-photon and three-photon microscopy of small animals, and for surgical purposes the creation of a transparent window in the skull itself.

A team at the Institute for Basic Science (IBS) in Daejeon, South Korea, has now developed a new approach to tackling the optical aberrations involved in imaging through an intact skull, and tested the system on a mouse brain.

Reported in Nature Communications, the technique is christened laser scanning reflection-matrix microscopy (LS-RMM) and points towards a new way of creating label-free in vivo images of axons in living systems.

The fine microstructures in an animal skull induce severe optical aberrations as well as strong multiple-scattering noise, so some form of adaptive optics (AO) is needed to remove them.

To date only AO two-photon or three-photon fluorescence imaging with excitation wavelengths greater than 1300 nanometers have been able to visualize mouse brain structures through the skull, according to the IBS team, but these approaches can suffer from low repetition rates and high excitation powers. Label-free reflectance imaging has not been achieved at all.

"Reflection matrix microscope is the next-generation technology that goes beyond the limitations of conventional optical microscopes," commented Wonshik Choi of the IBS Center for Molecular Spectroscopy and Dynamics (CMSD).

"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."

Diagnosis of disease

The new approach builds on conventional optical coherence microscopy (OCM), in which optical coherence tomography measures a subset of the reflection matrix from a confocal microscopy operation.

While conventional confocal microscopy measures reflection signals only at the focal point of illumination and discards all out of focus light, a reflection matrix microscope records all the scattered photons at positions other than the focal point.

A key difference in LS-RMM relates to the detection scheme. According to the team's published paper "a camera was placed on a plane conjugate to the image plane, instead of using a confocal pinhole and a photodetector, and a reference wave was introduced to the camera to form off-axis low-coherence interferometry."

The scattered photons are then computationally corrected using a novel AO algorithm called CLASS, developed by the team in 2017, which extracts ballistic light and corrects severe optical aberrations.

"In LS-RMM, the identification of wavefront aberrations is based on the intrinsic reflectance contrast of targets," noted the team. "It does not require fluorescent labeling and high excitation power, contrary to existing AO modalities that rely upon multi-photon fluorescence feedback signals."

The platform was tested on the imaging of a mouse brain through an intact skull, and used to take two-photon fluorescence images of a dendritic spine of a neuron behind the mouse skull. An ideal diffraction-limited spatial resolution of 450 nanometers was achieved, along with fluorescence imaging of neuronal dendrites and their spines by correcting the aberrations identified from the reflection matrix.

The next steps will include minimizing the form factor of the microscope and increasing its imaging speed, so as to ultimately make a label-free reflective matrix microscope suitable for clinical use.

"Our microscope allows us to investigate fine internal structures deep within living tissues that cannot be resolved by any other means," said IBS's Seokchan Yoon. "This will greatly aid us in early disease diagnosis and expedite neuroscience research."

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