Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment

Abstract

Background Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells. Methods We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models. Results Ex vivo cleared mouse brain tissues had a > 10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma. Conclusions Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

“…Previously, tissue clearing methods were used to visualize the brain microvasculature and the routes of the invasion of glioblastoma cells. 33 Quantitative analyses of the topographical relationship between glioblastoma cells and their microenvironment were performed. It would be intriguing to combine our method with mouse models of ocular tumors, and it would be important to investigate the extent to which our method could be used for visualizing tissues in tumors.…”

Consensus guidelines for the use and interpretation of angiogenesis assays

“…For example, it is possible to quantify the abnormalities in structure and function that are hallmarks of tumor angiogenesis, and then assess how these abnormalities change when the vasculature is normalized with anti-angiogenic or anti-fibrotic therapies [478, 500, 508–513]. Intravital optical microscopy [514] has also been useful for calibrating clinical imaging modalities in patients [515]. …”

Control of optical transparency and infrared laser heating of costal cartilage via injection of iohexol

“…Optical clearing (OC) of biological tissues by injection of hyperosmotic agents enables to reduce the interstitial scattering of incoming laser radiation to achieve better tissue penetration for diagnostic imaging and light therapy . OC approach was successfully applied to enhance tissue visualization by different optical modalities, such as optical coherent tomography , confocal laser microscopy , fluorescent imaging including quantitative analysis , polarized photon scattering and others. While OC has a number of serious limitations in living systems associated with the rapid elution of agent and long times needed for accumulation of a proper concentration, ex vivo application is more easily controlled and can be carried out within acceptable time limits.…”