Microstructural analysis of human white matter architecture using polarized light imaging: views from neuroanatomy

Abstract

To date, there are several methods for mapping connectivity, ranging from the macroscopic to molecular scales. However, it is difficult to integrate this multiply-scaled data into one concept. Polarized light imaging (PLI) is a method to quantify fiber orientation in gross histological brain sections based on the birefringent properties of the myelin sheaths. The method is capable of imaging fiber orientation of larger-scale architectural patterns with higher detail than diffusion MRI of the human brain. PLI analyses light transmission through a gross histological section of a human brain under rotation of a polarization filter combination. Estimates of the angle of fiber direction and the angle of fiber inclination are automatically calculated at every point of the imaged section. Multiple sections can be assembled into a 3D volume. We describe the principles of PLI and present several studies of fiber anatomy as a synopsis of PLI: six brainstems were serially sectioned, imaged with PLI, and 3D reconstructed. Pyramidal tract and lemniscus medialis were segmented in the PLI datasets. PLI data from the internal capsule was related to results from confocal laser scanning microscopy, which is a method of smaller scale fiber anatomy. PLI fiber architecture of the extreme capsule was compared to macroscopical dissection, which represents a method of larger-scale anatomy. The microstructure of the anterior human cingulum bundle was analyzed in serial sections of six human brains. PLI can generate highly resolved 3D datasets of fiber orientation of the human brain and has high comparability to diffusion MR. To get additional information regarding axon structure and density, PLI can also be combined with classical histological stains. It brings the directional aspects of diffusion MRI into the range of histology and may represent a promising tool to close the gap between larger-scale diffusion orientation and microstructural histological analysis of connectivity.

Keywords: brainstem; cingulum; extreme capsule; internal capsule; polarized light imaging; pyramidal tract.

Figure 1

Scales of anatomical structure and image modality. The image shows the different scales of anatomical structures of interest and the magnification of different image modalities, which can be used to study white matter architecture. Abbreviations: CLSM, confocal laser scanning microscopy; MOST, micro-optical sectioning tomography (Li et al., 2010).

Figure 2

Image acquisition and image processing of PLI. Equipment of the PLI system (A) consists of two rotatable perpendicularly oriented polarization filters and an insertable quarter-wave plate. For each section, nine images separated by 10° rotations of the filters using two crossed polars are acquired (B), which are used to calculate angles of fiber inclination (C). In addition, nine images separated by 20° rotations of the filters using an additionally introduced quarter-wave plate (D) are used to calculate angles of direction (E). Angles of inclination and angles of direction in every pixel of the section define a vector representing the major 3D fiber orientation at that point. Fiber orientation maps (FOMs) can be visualized using different color schemes (F,G).

Figure 3

Architectural parcellation of the anterior cingulum bundle. The cingulum bundle is marked by the white arrows in the Fiber Orientation Maps [FOMs (A)]. (E) Shows the cingulum bundle in a dissected human brain for comparison (black arrows). The anterior supracallosal and the pregenual part of the cingulum bundle is a single compact fiber bundle [white arrows in (A)]. In its midcingulate posterior part, the supracallosal cingulum bundle gets several inputs from fiber bundles coming from the adjacent white matter. This part is magnified in (B) and shows intermingling fiber bundles running into the cingulum bundle visualized in green and blue color (arrows). In the subgenual part, the fibers of the cingulum bundle curve and spread into the orbitofrontal regions [(C,D) arrows].

Figure 4

Fiber Orientation Maps of the human brainstem. (A) DTI color maps (1.5 T MRI with a voxel resolution of 2 mm × 2 mm × 2 mm) of comparable sections through the brainstem are able to show the larger fiber tracts which at least are at the scale of one voxel. However, the resolution is too low to distinguish smaller structures as seen in the medulla oblongata. Corresponding fibers are marked by the arrows. The advantage of PLI is its greater resolution, which also allows for visualization of smaller fiber bundles. Fiber orientation maps (FOMs) can be visualized using a color scheme similar to the DTI slices with the absolute X component of the vector shown in red, the absolute of the Y component in green, and the absolute of the Z component in blue (B). Another color scheme (C) is more beneficial for PLI data and codes in-plane orientation in color and out-of-plane rotation in intensity (inset key). Maximum intensity maps (D) show the highest intensities of each polarization sequence and give relative good anatomical contrast similar to histological myelin stains. Angles of direction (in-plane) and angles of inclination (out-of-plane) generate direction (E) and inclination maps (F). Note that the angle of inclination can be estimated between 0° and 90° only, not distinguishable from angles between 0° and (−90° (inclination ambiguity).

Figure 5

Three dimension reconstruction of the brainstem. Serial FOMs are three-dimensionally reconstructed and a 3D data set is produced with a vector in each voxel representing the 3D fiber orientation. The example shows one original axial (A) section, and a calculated sagittal (B), and horizontal (C) section through the 3D data set (D) shown as maximum intensity data. Pyramidal tract and lemniscus medialis were manually segmented, based on the orientation data. Volume models of the whole brainstem [(E) yellow], pyramidal tracts (G), and lemnisci mediales (F) were computed.

Figure 6

Volumes of pyramidal tract and lemniscus medialis. The volumes of the pyramidal tracts and the lemnisci mediales were normalized to the whole white matter volume of the individual brainstem. The measurements of the analyzed six brainstems are shown in (A). The normalized volumes (y-axis) of the pyramidal tracts revealed a negative correlation (B) to age (x-axis), while the normalized volumes of the medial lemniscus were not correlated to age (C). Abbreviations: PT, pyramidal tract; LM, lemniscus medialis.

Figure 7

Internal capsule. Maximum intensity map (A) and FOM (B) of a horizontal section through the internal capsule. (C,D) Show FOMs of sagittal sections through the internal capsule. Confocal images of different regions of the internal capsule demonstrate the fiber architecture at these points in high detail: (E) lateral anterior limb with parallel fibers and single nerve fiber crossing the internal capsule, (F) medial anterior limb with intermingling fiber bundles from the anterior thalamic radiation and the frontopontine fiber bundles (3D reconstruction), (G) posterior limb with intermingling fiber bundles from the pyramidal tract and superior thalamic radiation, (H) sublentiform part with larger intermingling compact bundles of fibers. Abbreviations: pl, posterior limb; g, genu; and al, anterior limb of the internal capsule; ca, anterior commissure; fo, fornix; gp, globus pallidus; pt, putamen; cn, caudate nucleus; tha, thalamus.

Figure 8

Extreme capsule. Macroscopical fiber dissection and PLI of the white matter in the deep of the limen insulae show comparable results. Different nerve fibers can be distinguished running from the temporal to the frontal lobe: fibers from the extreme capsule to the frontal operculum (a), fibers from the extreme capsule to the orbitofrontal cortex (b), and fibers from the amygdala to the orbitofrontal cortex (c, the uncinate fasciculus). The uncinate fasciculus is localized more deeply than the other two fiber systems and can be clearly distinguished from these. The figures at the bottom show fiber orientation maps through putamen (put) and claustrum (cl), where external (d) and extreme capsule (e) can clearly be distinguished from each other.