Volumetric digital histopathology of brain tissue using quantitative phase imaging and clearing technique using quantitative phase imaging and clearing technique

Abstract

The histological optical imaging is a gold standard method to observe the biological tissues, which follows routine process such as dissection, embedding, sectioning, staining, visualization and interpretation of specimens. This technique has a long history of development, and is used ubiquitously in pathology, despite being highly time and labour-intensive. Advanced optical imaging techniques developed over the last decade have enabled to provide high sensitivity, high resolution and non-invasive biological information. In particular, new optical imaging contrast rather than chemical staining has been presented to be utilized in histopathology while showing the strong potential. However, acquiring fast, high throughput, large volume tissue anatomy remains a difficult challenge. In this study, we propose novel protocol using staining-free digital histopathology combined with quantitative phase imaging (QPI) and tissue clearing. QPI has been evaluated to have outstanding performance to image transparent cells and tissue without staining. It reconstructs phase distribution of specimens using asymmetric illumination and regularized deconvolution method. Typically QPI has been utilized to image brain tissue within 20 micrometers, but it could be enhanced when it is combined with clearing technique. In order extend the imaging depth, we utilized near infrared QPI and OptiMuS solution which is uniquely developed to minimize size deformation and fast clearing. In validation work, one drop of OptiMuS solution was treated on the mouse brain sample slide glass and it made the tissue transparent in a few seconds. Whole process of clearing was monitored every 5 seconds followed by time series QPI images. Through preliminary experiment, we confirmed that our approach shows that the morphology of brain tissue between QPI and the stained histological section has a strong correlation. QPI images have even better contrast for the fiber bundles in the region of the corpus callosum (cc), caudoputamen (CP), and cerebral peduncle (cpd) due to phase and light scattering. QPI imaging with cleared brain presented 3D myelin fibers over 50 micrometer while identifying the distribution and orientation of each fiber without a contrast agent or any labeling process. Our system has been utilized to reconstruct single brain slices, but can be applied to multiple slices for the volumetric visualization of the brain. Thus, a new platform of QPI and clearing method is a very promising tool for use in neuroscience research, and is particularly well suited for systematic studies of brain anatomy for the understanding of mouse models of various cognitive disorders.