From Mouse to Monkey: Tissue Clearing Scales Up 3D Imaging to Large Animals

Tissue optical clearing, a set of chemical techniques that render opaque biological organs transparent, has matured rapidly in rodents but now faces its toughest test: scaling up to large animals such as pigs and non-human primates. A review published in Frontiers of Optoelectronics by Yating Deng, Jianyi Xu, Tingting Yu and Dan Zhu of Huazhong University of Science and Technology systematically examines how clearing protocols developed for mouse brains and whole mouse bodies can be translated to specimens whose size, composition and architecture differ dramatically from the murine models where the technology was born. The authors argue that this translation is not a matter of simply adjusting incubation times, but of rethinking the chemistry, penetration dynamics and preservation strategies from the ground up.

The core problem the technology addresses is light scattering. Biological tissues are opaque because their internal components—proteins, lipids, water and minerals—have mismatched refractive indices, and microscopic structures such as collagen fibers and cellular membranes create countless scattering interfaces. Even powerful optical microscopes such as confocal, two-photon and light-sheet systems are limited to imaging depths of a few hundred micrometers in untreated tissue. Clearing methods work by removing or replacing highly scattering components, typically through delipidation, dehydration or decolorization, and then bathing the sample in a refractive-index-matching medium. An alternative strategy physically modifies the tissue microstructure, most notably through hydrogel embedding, in which proteins are crosslinked into a polymer network before lipids are extracted.

Over the past two decades, three principal methodological families have emerged. Organic solvent-based approaches, exemplified by 3DISCO and its successors uDISCO, iDISCO and FDISCO, achieve rapid transparency through gradient dehydration but tend to quench endogenous fluorescence and shrink tissue. Aqueous-based systems, led by CUBIC and later MACS, preserve fluorescence better but historically required long processing cycles and could cause tissue swelling. Hydrogel-embedding methods, pioneered by CLARITY and refined in derivatives such as SWITCH and SHIELD, transform tissue into a crosslinked hydrogel and remove lipids, often with electrophoretic assistance. The review traces how each scale expansion—from millimeter-scale brain slices to centimeter-scale whole organs and finally to whole cleared mice via techniques like uDISCO, vDISCO and wildDISCO—demanded new chemical formulations and faster penetration kinetics.

Large model animals matter because they bridge a persistent gap between rodents and humans. Non-human primates possess gyrencephalic, folded brains and cognitive and behavioral parallels to people, while pigs replicate many human cardiovascular and metabolic traits with high fidelity. Yet the review identifies what the authors call a paradoxical dilemma of scale-comparable but method-incompatible protocols: even when a pig organ matches a human organ in dimensions, rodent-derived clearing recipes often fail outright. The reasons are biological. Mouse brains contain roughly 86 percent gray matter with sparse white matter, whereas non-human primate and pig brains carry about 39 percent white matter and human brains about 45 percent, including highly specialized tracts. Myelin density climbs from rodents through non-human primates to humans, and pig brains closely resemble human myelin characteristics. Lipid composition diverges as well, with human brains richest in lipids and pig brains showing higher oxidative lipid modifications.

These compositional differences translate directly into clearing failures. The review notes that while a mouse brain may be fully penetrated by clearing reagents within days, comparable concentrations may need weeks or even months to permeate a pig brain or a non-human primate kidney. Prolonged processing frequently produces over-cleared peripheral zones surrounding inadequately cleared cores, and the extended exposure raises the risk of losing protein antigenicity and suffering structural collapse, undermining subsequent immunolabeling and three-dimensional reconstruction. Parenchymal organs compound the difficulty: pig and primate livers and hearts carry markedly denser extracellular matrices than rodent equivalents, with collagen content following a porcine-to-human-to-primate-to-rodent gradient, and conventional lipid-clearing agents effective in mice often cannot fully delipidate such tissue.

Early attempts to clear human and large-animal tissue by directly transferring mouse protocols met with limited success. Because of high myelin density, age-related lipofuscin accumulation, protein aggregation and autofluorescence from residual blood in unperfused tissue, many established methods could only be applied to thin sections of roughly 100 to 1000 micrometers. Combining CLARITY with alternating active and passive clearing and extended durations eventually achieved clearing of 8-millimeter-thick human brain sections. Purpose-built methods followed: MASH, OPTIClear, hFRUIT and ELAST can clear 5 to 10 millimeter human tissue blocks, and SHANEL, which uses small CHAPS detergent micelles for deep penetration, has achieved clearing of entire human organs. For primates specifically, ScaleSF enables multiscale light and electron microscopy in marmoset sections, and PuClear uses Triton X-100 permeabilization with high refractive-index matching to render thick macaque brain slices uniformly transparent.

Applied research now spans four major organ systems. In the central nervous system, a modified 3DISCO protocol with viral labeling produced high-resolution three-dimensional imaging of non-human primate spinal cords after injury, illuminating interactions between axonal regeneration and scar formation. An adapted iDISCO method delivered the first three-dimensional visualization of KNDy neurons in the sheep hypothalamus, revealing an unexpected subpopulation of kisspeptin neurons in the lateral hypothalamic area. Most strikingly, the SMART approach, which couples sequential sectioning with PuClear, enabled the first high-throughput three-dimensional reconstruction of an entire macaque brain, resolving fine-scale thalamocortical projection patterns, and subsequent work in cynomolgus monkeys achieved complete single-neuron morphological reconstructions revealing diversity among primary motor cortex neurons. SHANEL has cleared intact adult pig brains, while SHIELD combined with eFLASH electrophoretic labeling resolved laminar distributions of parvalbumin-positive and neuropeptide-Y-positive neurons in marmoset visual cortex blocks.

Sensory, cardiopulmonary and metabolic applications round out the picture. An optimized BoneClear technique achieved the first clearing of intact African green monkey cochleae and was later adapted to overcome the dense bone of pig cochleae from neonatal to adult stages, quantifying hair cell organization and frequency mapping for auditory regeneration research. CUBIC-based clearing with hydrogen peroxide bleaching rendered intact ferret eyeballs transparent for single-cell retinal imaging. In rhesus macaques, hybrid iDISCO-CUBIC protocols localized vaccine components within lymph nodes to guide lymph-targeting vaccine design, and a Visikol-based protocol visualized placental microvasculature in three dimensions. Pocket CLARITY applied to pig hearts documented degeneration of myocardial helical structures in heart failure and infarction, and 3DISCO with light-sheet microscopy delivered the first quantitative measurement of adipocyte volume and number across fat depots in obese pigs, revealing distinct growth patterns between visceral and subcutaneous fat. Visikol clearing also resolved the spatial relationship between hematopoietic stem cells and fat tissue in macaque bone marrow, and SHANEL imaged beta-cell distribution in transgenic pig pancreas.

The review’s authors emphasize that the field still lacks unified evaluation criteria for large-animal clearing, producing variability in permeation duration and reagent concentration across laboratories, and that imaging cleared large organs demands advanced light-sheet systems with greater penetration depth that are not yet widely accessible. Their proposed future directions include staged, tissue-responsive pretreatment protocols; small-micelle detergents and electric-field-assisted diffusion technologies such as SHIELD and eFLASH; perfusion, electromagnetic and ultrasonic strategies that exploit vascular networks for reagent delivery; species-specific parameter databases and machine-learning-based predictive models; and standardized multi-species validation platforms with quantitative clearing-efficiency metrics. On the labeling side, they point to nanobody fragments with zwitterionic modifications, ultrasound microbubble delivery and click-chemistry amplification to push antibodies into centimeter-scale sample cores. Light-sheet microscopy remains the workhorse for large cleared volumes, with multimodal fusion with optical coherence tomography and deep-learning-based segmentation of massive three-dimensional datasets identified as key accelerators.

The authors conclude that successful translation from rodents to larger species requires a fundamental rethinking of reagent chemistry, penetration dynamics and tissue preservation rather than simple parameter adjustment, and that while challenges of tissue heterogeneity, prolonged processing and imaging limits persist, recent innovations demonstrate feasibility across pig and non-human primate organs. Notably, in vivo clearing applications such as skull optical windows and skin clearing remain confined to murine systems, though reversible clearing combined with minimally invasive monitoring is anticipated to extend to large animals. If standardization, scalable imaging and interdisciplinary collaboration mature as the review urges, tissue optical clearing could bridge preclinical and clinical research at scales that approximate human physiology and pathology far more closely than the mouse models on which the technology was first built.

Subject of Research: Translating tissue optical clearing technology from rodent models to large animals for three-dimensional organ imaging

Article Title: Advances in tissue optical clearing for 3D imaging in large animal

Article References: Deng, Y., Xu, J., Yu, T., & Zhu, D. (2025). Advances in tissue optical clearing for 3D imaging in large animal. Frontiers of Optoelectronics, 18(1), Article 18. https://doi.org/10.1007/s12200-025-00162-6

Image Credits: AI Generated

DOI: 10.1007/s12200-025-00162-6

Keywords: tissue optical clearing, 3D imaging, large animal models, non-human primates, light-sheet microscopy, SHANEL, CLARITY, myelin density, organ clearing, biomedical research, refractive index matching, porcine models