Visualization of myelinated fiber bundles orientation during brain slice preparation by reflection polarized light microscopy

Abstract

Polarized light imaging (PLI) enables detecting the orientation of myelinated axon bundles in brain slices at microscopic resolution without histological staining. However, standard PLI requires labor‐intensive procedures such as mounting brain cryosections on slide glasses. We developed an optical system that does not require a mounting procedure for PLI. Specifically, we developed an optical system to perform PLI in reflection mode (rPLI) instead of employing transmitted light as in standard PLI. We integrated this rPLI system with a conventional vibratome slicer whose cutting blade surface is a mirror. This combination allowed PLI measurements directly during the slicing procedure at room temperature. Thus, mounting procedure for PLI is not necessary. As a proof‐of‐concept experiment, a perfusion‐fixed brain of a mouse was embedded in gelatin‐containing agar and cut serially at 40~200 μm intervals. The slicing procedure was temporarily halted after each cut to capture the PLI images of the slice on the reflecting blade surface while the slice was still held up by the agar block. The orientation of the fiber bundle estimated with this method agreed with the results obtained from previous reports. Combination of a popular vibratome slicer and our rPLI system that uses versatile and inexpensive optical components would increase popularity of PLI and facilitates connectome studies at microscopic resolution. Research Highlights Polarized light imaging (PLI) of brain slices was realized by using reflected light (rPLI) instead of transmitted light. The rPLI method allows detecting the myelinated fiber bundle orientation during slice preparation.

Using light and X-ray scattering to untangle complex neuronal orientations and validate diffusion MRI

“…A similar principle is used in polarization microscopy where polarized light is passed through thin brain sections and alterations in the polarization state are measured – a technique known for more than a century ( Brodmann, 1903 ; Fraher and MacConaill, 1970 ). Recent advances realized polarization microscopy also in reflection mode ( Takata et al, 2018 ), and three-dimensional polarized light imaging enabled the determination of 3D fiber orientations ( Axer et al, 2011a ; Axer et al, 2011b ; Menzel et al, 2015 ; Zeineh et al, 2017 ; Stacho et al, 2020 ; Takemura et al, 2020 ) using an advanced signal analysis ( Menzel et al, 2022 ) or a tiltable specimen stage ( Schmitz et al, 2018 ). However, in contrast to SLI, the techniques yield only a single fiber orientation for each measured tissue voxel, and voxels with multiple crossing fibers may yield erroneous fiber orientations ( Dohmen et al, 2015 ), while retrieving the out-of-plane angle is also challenging.…”

Quantum optical tomography based on time-resolved and mode-selective single-photon detection by femtosecond up-conversion

“…However, MRI devices are expensive, and its spatial resolution is not as high as that of optical microscopes. Optical anisotropy microscopy can image the trajectory of myelinated nerve fiber bundles without staining by exploiting the optical anisotropy of myelin molecules [38]. However, obtaining three-dimensional information is difficult without continuous brain sectioning, as the polarization characteristics disappear due to scattering.…”

Using light and X-ray scattering to untangle complex neuronal orientations and validate diffusion MRI

“…A similar principle is used in polarization microscopy where polarized light is passed through thin brain sections and alterations in the polarization state are measured – a technique known for more than a century ( Brodman, 1903; Fraher et al, 1970 ). Recent advances realized polarization microscopy also in reflection mode ( Takata et al, 2018 ); but just as PS-OCT, the techniques only derive 2D fiber orientations. In contrast, threedimensional polarized light imaging determines the 3D-orientations of the nerve fibers ( Axer et al, 2011a; Axer et al, 2011b; Menzel et al, 2015; Zeineh et al, 2017; Stacho et al, 2020; Takemura et al, 2020 ) using an advanced signal analysis ( Menzel et al, 2022 ) or a tiltable specimen stage ( Schmitz et al, 2018 ).…”