Efficient Tissue Clearing and Multi-Organ Volumetric Imaging Enable Quantitative Visualization of Sparse Immune Cell Populations During Inflammation

Abstract

Spatial information of cells in their tissue microenvironment is necessary to understand the complexity of pathophysiological processes. Volumetric imaging of cleared organs provides this information; however, current protocols are often elaborate, expensive, and organ specific. We developed a simplified, cost-effective, non-hazardous approach for efficient tissue clearing and multi-organ volumetric imaging (EMOVI). EMOVI enabled multiplexed antibody-based immunolabeling, provided adequate tissue transparency, maintained cellular morphology and preserved fluorochromes. Exemplarily, EMOVI allowed the detection and quantification of scarce cell populations during pneumonitis. EMOVI also permitted histo-cytometric analysis of MHC-II expressing cells, revealing distinct populations surrounding or infiltrating glomeruli of nephritic kidneys. Using EMOVI, we found widefield microscopy with real-time computational clearing as a valuable option for rapid image acquisition and detection of rare cellular events in cleared organs. EMOVI has the potential to make tissue clearing and volumetric imaging of immune cells applicable for a broad audience by facilitating flexibility in organ, fluorochrome and microscopy usage.

The Wanderings of Gut-Derived IgA Plasma Cells: Impact on Systemic Immune Responses

“…Advances in single-cell and high-throughput techniques provide valuable tools; however, spatial information of ASC in their unique niche will also be necessary to understand these reciprocal interactions in their complexity. Novel imaging techniques such as volumetric imaging of tissue sections or whole mount organs (53,56,82) might help to detect rare IgA ASC and determine the specific microenvironment in order to better understand ASC function and local production of potentially harmful or protective IgA. A better understanding of the migration, function and survival of long-lived IgA PC in their specific niches is needed in order to modulate immune responses for therapeutic intervention.…”

B/T cell crosstalk and aberrant inflammatory IgG exacerbate autoimmune intestinal inflammation

“…Slides were washed in PBS, mounted with Fluoromount-G (Southern Biotech) and sealed with a clear nail polish. Colon pieces were prepared and cleared as previously described (37). In short, colon was harvested, the muscle layer removed and 1 cm pieces were fixed for 1 hour at 4°C; blocked; stained with antibodies as outlined in Table S3, for 72 hours at 37°C; washed; dehydrated using ascending dilutions of isopropanol (pH∼9) (Sigma-Aldrich) and cleared using undiluted ethyl cinnamate (#W243000, Sigma-Aldrich).…”

Spatial determinates of effector and memory CD8+ T cell fates*

“…Light scattering is reduced by minimizing the refractive index mismatches between the immersion media and various tissue components, such as lipids and proteins. 30,31 We and others have used the Ce3D clearing method to conserve endogenously expressed fluorophores and enable additional epitope staining using directly labeled antibodies. 30,32 These features were essential to allow tracking of antigen-specific CD8 + T cells and identify essential lymph node landmarks to identify B220 + B cell follicles and the CD31 + endothelial cell network.…”

“…Optical tissue clearing maintains tissue and cellular morphology, while reducing the lateral scattering of light upon illumination. Light scattering is reduced by minimizing the refractive index mismatches between the immersion media and various tissue components, such as lipids and proteins 30,31 . We and others have used the Ce3D clearing method to conserve endogenously expressed fluorophores and enable additional epitope staining using directly labeled antibodies 30,32 .…”