SUMIC: A Simple Ultrafast Multicolor Immunolabelling and Clearing Approach for Whole-Organ and Large Tissue 3D Imaging

Abstract

High-resolution whole-organ imaging of cleared tissues captures cellular and molecular insights within the intact tissue and tumour microenvironments. However, current immunolabelling and clearing methods are complicated and time-consuming; extending to several weeks. Here, we developed Simple Ultrafast Multicolor Immunolabelling and Clearing or SUMIC, a method that enables multicolor immunolabelling and clearing of whole murine organs and human tissues within 2 to 2.5 days. Moreover, SUMIC is simple, robust, non-hazardous and versatile comprising antigen retrieval, permeabilization, collagenase-based digestion, immunolabelling, dehydration, and clearing. SUMIC permits quantitative and singlecell resolution analysis and detection of rare cells in whole organs, for example, round αSMA positive cells in the thymus. Upon volumetric imaging, SUMIC-processed samples retain normal tissue architecture and can be used for paraffin-embedding and histology. We employed the SUMIC method for whole-organ mapping of lymphatic vessels across different ages and organs. This analysis revealed the expansion of lymphatic vessels in endocrine tissues but not in any other organs with aging. Hence, SUMIC will accelerate discoveries compared to other whole organ imaging pipelines.

Enhancing cardiovascular research with whole-organ imaging

“…The ability to image the intact vasculature in adult organs at cellular resolution promises to enable new discoveries on how the cardiovascular system functions during homeostasis and in response to injury. Exciting advances over current protocols are frequently published and more are on the horizon [30,[50][51][52]. These new methods address current limitations as well as introduce new features that expand functionality.…”

“…For example FLASH [52] has an antigen retrieval component not found in earlier methods, which could expand the list of compatible antibodies. SUMIC [30] and ELAST [51] promise substantially shorter antibody incubation times, which can currently last as long as a month. Lastly, advances in LSMs have made room for methods that can use three-dimensional printing to transform an imaged vascular bed and into a perfusable microfluidic device [50].…”

“…The rehydration step, missing from previous methods, allows for immunolabeling, whereas DBE stabilizes the fluorescent signals. Moreover, hundreds of antibodies have been tested and several validated to be compatible with the iDISCO protocol and its modified derivatives [5,29 ▪▪ ,30]. Most relevant to the vascular biology field is the fact that several of the validated antibodies detect cell types within the vasculature, allowing for specific labeling of veins, arteries, lymphatics, and capillaries.…”

Influence of Age and Breed on Bovine Ovarian Capillary Blood Supply, Ovarian Mitochondria and Telomere Length

“…Particular attention should be paid to the spatial distribution of the blood vessels (superficial and deep cortical stroma). To better understand the spatial distribution of vessels and their connection with functional structures in the cow s ovary, a promising approach could be the imaging of whole organs or bigger blocks of cleared tissues, which makes intact tissue transparent and enables the generation of detailed 3D structures of organs [31,32]. This approach would also allow a detailed analysis of angiogenic and angioregressive figures (e.g., capillary sprouting, dilation, intussusception, thinning, and resorption) that previously required scanning electron microscopy and the preparation of corrosion specimens [33].…”

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

“…It should be added, however, that such unrestricted labeling required two presumably important (as these were not quantified directly) steps, namely cholesterol extraction via addition of methyl-β-cyclodextrin and loosening of the collagen network with trans-1-acetyl-4hydroxy-L-proline. A similar step of loosening the extracellular matrix via collagenase digestion was recently proposed in a preprint by Biswas et al [94] who described a rapid, 3-day long approach for TOC and immunolabeling of entire murine organs. A similar treatment aimed at the digestion of the extracellular matrix with hyaluronidase is a prominent feature of EMOVI (efficient tissue clearing and multiorgan volumetric imaging), a recent TOC pipeline focused on multiplexed antibody-based immunolabeling of immune cells [95].…”