Quantitative Analysis of Tissue Clearing Based on Optical Coherence Tomography and Magnetic Resonance Imaging

Department of Biomedical EngineeringIn the past decades, many optical imaging modalities have played a key role to understand how neurons connect and mediate their function. Especially, deep brain imaging has been crucial in neural anatomy research by providing brain-wide structural information. Although the optical imaging renders the high resolution brain image, it has restriction to perform the deep brain imaging due to inherent scattering problem of light. To enhance the imaging depth, many optical imaging modalities have combined with serial sectioning. As name suggests, serial sectioning solves the penetration depth problem by successively sectioning the tissue and imaging the remained tissue. Although serial sectioning techniques enable us to visualize whole brain, these techniques still have remained challenging in terms of labor intensive technique as well as tissue damages due to physical sectioning. Therefore, it is very demanding the new approach for whole brain imaging while preserving the intact brain. In recent years, development of tissue clearing which renders biological sample transparent proposes a solution to solve the penetration depth issue. It reduces the problematic light scattering and thus extends the limited penetration depth by either matching the refractive index or removing the lipid. As mentioned above, many researchers have developed various optical clearing agents such as Scale, 3DISCO, SeeDB, CLARITY, and ClearT. Scale and CLARITY increase the imaging depth by removing the lipid which is scattering factor, whereas 3DISCO, SeeDB, and ClearT increase the imaging depth through the index matching. Because scattering is proportional to refractive index gap, index matching reduces the scattering. These clearing techniques open up the possibility of the deep brain imaging. With the help of this modern pioneering tissue clearing technique, fluorescence microscopy including confocal microscopy (CM), multi-photon microscopy (MPM), and single plane illumination microscopy (SPIM) now enables us to image brain much deeper than ever before. Although efforts to eliminate the problematic light scattering have been ongoing for past decades, previous research has rarely reported quantification of enhancement light penetration into the cleared brain. They have only focused on the capability of three-dimensional visualizationA few quantification studies end up in measurement of transmittance or depth profile. Limitation of these studies was not able to provide the analysis of tissue property change induced by tissue clearing and to compare the tissue clearing characteristics. That is, there have not been standardized techniques to measure the clearing efficiency of regional differences and to investigate the principle of various tissue clearing methods, despite its significant need for reliability and reproducibility. Here, we present optical coherence tomography (OCT) and magnetic resonance imaging (MRI) to quantitatively assess the tissue clearing technique. OCT can perform label-free, non-invasive optical imaging by using Michelson interferometer. Thanks to these strong characteristics, OCT is appropriate tool to validate increase of imaging depth through the analysis of A-line profile. Therefore, we quantitatively measured the effect of diverse clearing even each brain region by using OCT. On the other hands, MRI is also non-invasive imaging technique based on nuclear magnetic resonance (NMR). Because MRI signal is based on atomic characteristics, we can physically investigate the fundamental principle of tissue clearing by monitoring the tissue atomic properties change. Through this study, we can investigate the diverse tissue clearing characteristics and compare the existing clearing technique. Furthermore, we provide the standard to evaluate the various tissue clearing and it allows the choice of proper tissue clearing for experimental purpose. Therefore, this study is able to increase the reliability and reproducibility of experimental results.ope

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