Physical section microscopy has been spotlighted as a breakthrough technique for imaging large-scale biological tissue. Physical section microscopy therefore makes it possible for light microscopy to image entire organs, such as the brain. Although many physical section microscopies have been introduced over the past decade, there has been no technique to examine label-free whole mouse brains.
In this study, we proposed a new physical section microscopy based on optical coherence, which performs high-resolution tomographic imaging of label-free biological tissue, and we named it serial optical coherence tomography (SOCT). Our results indicate that SOCT has great potential to identify the morphological changes in the whole mouse brain in healthy and diseased subjects.
INTRODUCTION
- RESEARCH BACKGROUND
- MODERN LIGHT MICROSCOPY TECHNIQUES
- LIGHT MICROSCOPY USING TISSUE CLEARING
- PHYSICAL SECTIONING MICROSCOPY
- OPTICAL COHERENCE TOMOGRAPHY: BRAIN IMAGING
- OVERVIEW: SERIAL OPTIACL COHERENCE TOMOGRAPHY
In recent decades, modern techniques of physical section microscopy have been developed to overcome the inherent limitation of light microscopy. As the name suggests, a common feature of the physical section microscopy techniques is the use of tissue sections. Although the techniques have been successfully employed in the systematic imaging of whole mouse brains, they required extremely thin thickness of tissue sections.
Not only this, the techniques take chip images from tissue cutting so that the objects in the images are removed through image processing [17]. Since its invention by Fujimoto's group at the Massachusetts Institute of Technology [ 25 ], OCT has been rapidly translated into clinical studies in the peripheral nervous system, particularly the retina [ 26 ]. After demonstrating that OCT enables the differentiation between white and gray matter of the brain [27] as well as healthy and diseased brain tissue [28], it has begun to be applied to the study of the central nervous system, especially in the rodent brain.
Recently, OCT imaging of large-scale brain tissue was developed by the Akkin group at the University of Minnesota. Moreover, they have demonstrated the feasibility of high-resolution PSOCT in the study of fiber pathways [39].
THEORY OF OPTICAL COHERENCE TOMOGRAPHY
- INTRODUCTION
- OPTICAL COHERENCE TOMOGRAPHY VERSUS ULTRASOUND
- THE MICHELSON INTERFEROMETRY
- IMAGING CONDITION
- IMAGE RESOLUTION
- IMAGE ACQUISITION
- OPTICAL COHERENCE MICROSCOPY
Unlike conventional light microscopy, axial and lateral resolutions of OCT imaging are independent. The axial and lateral resolutions are controlled by the coherence length of the light source and the beam focusing conditions, respectively. For a source with a Gaussian spectral distribution, the axial and lateral resolution are respectively defined as
OCT imaging is obtained by performing successive axial measurements of back-reflected or back-scattered light at different positions. In fact, OCT implemented in single-mode fiber optics is confocal, because the sample path fiber also acts as a single-mode aperture for illumination and collection of light from the sample. Thus, in addition to the axial resolution that OCT obtains from low coherence interferometry, any OCT is also characterized by the axial and lateral resolutions that are purely a function of the beam focusing conditions.
In OCT, low NA focusing is used to provide a long depth of field to enable cross-sectional imaging using axial coherence gate scanning. The coherence gate overlaps with the focal volume to provide enhanced scattering of out-of-focus light while the sample is scanned in the xy or xz plane to create en-face or cross-sectional images, respectively.
MATERIALS AND METHODS
- SERIAL OPTICAL COHERENCE TOMOGRAPHY
- SERIAL OPTICAL COHERENCE TOMOGRAPHY SETUP
- SERIAL OPTICAL COHERENCE TOMOGRAPHY IMAGING
- OPTICAL COHERENCE MICROSCOPY
- CONTROL SOFTWARE
- IMAGE PROCESSING
- IMAGE PROCESSING FOR SOCT: VOLUMETRIC STITCHING
- IMAGE PROCESSING FOR OCM: EN-FACE STITCHING
- HIPPOCAMPUS SEGMENTATION
- VOLUME MEASUREMENT
- TISSUE PREPARATION
- VIBRATOME SECTIONING
At the bottom of the base part were three magnetic balls forming an equilateral triangle. The magnets helped us find the pre-set position and angle of the chamber as we moved it around several times during manual serial imaging of the block face. To begin, we created a block of brain embedded in agar and then glued the brain block to the bottom of the chamber.
Before serial imaging of the face of the block began, two important things had to be carefully and accurately controlled: the horizontality of the top surface of the brain block and the height of the water in the chamber. First, we slightly cut the brain block with a vibratome, and then adjusted the horizontality of the upper surface of the brain block with two goniometric stages. The water height was adjusted so that the focal plane of the OCT imaging was on the upper surface of the brain block.
Briefly, because the focal plane of OCT imaging must be located on the top surface of the brain block while no reflection and degradation occurred, we delicately adjusted the height of the immersing water. First, as shown in Figure 3.5A, we imaged the superficial volume of the mounted brain block. Second, as shown in Figure 3.5B, we poured out the water and disassembled the wall of the room.
Here, the imaging optics include a collimator, galvanometer scanners, and an objective lens so that the confocal parameter of the objective lens does not change during translation. This was because the vibratome incision increased the physical distance between the imaging optics and the top surface of the brain block. Finally, as shown in Figure 3.5D, we assembled the chamber wall and refilled the chamber with water, obtaining the same imaging conditions as the preset image.
The output beam diameter was aimed to fit the aperture of the objective lenses in use. In addition, the structure of the software was based on parallel computation of producer and consumer. Then we repeated the previous step of the atlas-based image segmentation in axial and sagittal planes.
Thus, by multiplying the voxel resolution and the number of voxels in an object, we can obtain the total physical volume of the object. Snapshots of the image processing: (C) the input SOCT datasets, (D-F) the alignment and merging process, and (G) the noise removal and intensity adjustment process.
RESULTS
- EX VIVO WHOLE MOUSE BRAIN OPTICAL IMAGING
- COMPARISON STUDY
- CONTRAST COMPARISON: MRI, OCM AND HISTOLOGY
- RESOLUTION COMPARISON: 10 × , 20 × AND 50 ×
- REGIONAL SEGMENTATION
- HIPPOCAMPUS SEGMENTATION
- OTHER BRAIN STRUCTURES SEGEMENTATION
- VOLUME MEASUREMENT
- ENRICHED ENVIRONMENT
Although MRI is a great technique for imaging in vivo brains, while SOCT is for ex vivo brains, the reason we compared them in this study is that there is no microscopic technique capable of imaging whole mouse brains. Here, three brain sections, prepared in coronal, axial and sagittal sections respectively, were imaged in order from MRI to OCM and histology. The MRI was taken at T2 and the histology was stained with Nissl and luxol fast blue (N&L) for myelinated neuronal fibers, as well as hematoxylin and eosin (H&E) for cell nucleus and cytoplasm.
For the hippocampus, MRI was limited in resolving hippocampal subfields, while OCM and histology clearly resolved this. On the other hand, MRI and H&E-stained histology for the cerebral peduncle could not visualize fiber tracts, while OCM and N&L-stained histology visualized them clearly. Through this study, it became clear that OCM has the unique contrast to study microscopic brain structures, especially in the hippocampal sulcus and fiber tracts.
From the 20× objective, visualization of hippocampal fibers and subfields became possible, but still insufficient. Magnified images were taken in (first and second columns) corpus callosum and (third column) hippocampus. Although the refractive indices of these brain regions are different, we approximated their refractive indices to that of the hippocampus.
Images were taken in the (top left) coronary, (bottom left) axial, and (top right) sagittal planes. 3D SOCT images of a regionally segmented whole mouse brain including the olfactory bulb, corpus callosum, hippocampus, spinal cord, and cerebellum. Here, mice from an enriched environment were raised in a special cage filled with various toys, while mice from a standard environment were raised in a relatively deprived cage filled only with litter, and the mice were littermates.
As a result, we found that the brain from the enriched environment had a larger volume than the brain from the standard environment. From this result, we found that SOCT can be used in morphological studies of the hippocampus under different conditions. 3D SOCT images of mouse brains from (A) standard environment, SOA, and (B) enriched environment, EE. C) Brain volume measurement.
DISCUSSION
EXTENDED APPLICATIONS
- MUSCLE, LIVER AND EMBRYONIC BODY
- TISSUE CLEARING
HIPPOCAMPAL SUBFIELDS
CONCLUSION
Cowey, "Imaging ex vivo and in vitro brain morphology in animal models with ultrahigh resolution optical coherence tomography," BIOMEDO. Srinivasan, "Volumetric imaging and quantification of cytoarchitecture and myeloarchitecture with intrinsic scattering contrast," says Biomedical Optics. Kabel, “Optical coherence microscopy for deep tissue imaging of the cerebral cortex with intrinsic contrast,” Opt.
Zhou, “Nondestructive assessment of progressive neuronal changes in organotypic mouse hippocampal slice cultures using ultrahigh-resolution optical coherence microscopy,” NEUROW. 34; Reconstruction of micrometer-scale fiber tracts in the brain: multicontrast optical coherence-based tractography," NeuroImage. Iseki, "Localization of nerve fiber bundles by polarization-sensitive optical coherence tomography," Journal of Neuroscience Methods.
Akkin, “Cross-validation of serial optical coherence scanning and diffusion tensor imaging: A study of nerve fiber maps in the human medulla oblongata,” NeuroImage.
