Tissue clearing and its applications in human tissues: A review

In three‐dimensional (3D) space, an unbiased and systemic view of human specimens between structures and functions is required. However, conventional histological sections from specimens have made only limited progress in exploring intact information about 3D biological tissues. With significant advances in optical physics and chemical engineering, state‐of‐the‐art tissue‐clearing methods can revolutionize the intact subcellular level of human tissue histological analysis, from thick human tissues to intact human organs. The present review summarizes the principle of tissue clearing so that a trainee researcher can implement effective human tissue‐clearing protocols. Furthermore, this review highlights existing tissue‐clearing methods in specific human tissue applications, describes imaging strategies, and presents various efficient computational approaches for processing and visualizing large image data. Finally, potential future directions for developing tissue‐clearing methods for human tissues are also discussed.


INTRODUCTION
A fundamental medical application is to create reference maps of human organs across all ages and diseases at a resolution of the cellular scale. [1]It is also crucial to compile a comprehensive atlas of three-dimensional (3D) imaging of human organs to serve as a preoperative tool for clinical doctors to decide on the best course of treatment.However, there are limitations to obtaining deciphering complex 3D anatomy as reference images from a large volume of human tissues or mapping intact human organs. [2]he study of historical human anatomy is primarily macroscopical.For many decades, modern pathology has relied on histopathologic analysis of two-dimensional (2D) tissue dissections, a typical method for examining tissue structures.With a growing need for 3D structural information, many studies have attempted to create 3D reconstructions of sections, which has been extremely time-consuming and labor-intensive.Furthermore, the 2D histological sections technique destructs human tissues to achieve a conventional 3D reconstruction, resulting in the loss of some valuable information.The need for transferring new pathology techniques from 2D to 3D space could allow researchers to investigate biological mechanisms in health and disease conditions. [3]Thus, there is an urgent need for 3D scalable and unbiased cell-level technologies, enabling complete 3D examination of large volumes of human tissues and entire human organs.
Developing an efficient technology can only bridge the gap between large-scale intact human tissue labeling and high-resolution histological imaging.In this context, noninvasive imaging techniques that can image anatomical structures in multiple organisms in 3D space include computed tomography, [4] magnetic resonance imaging (MRI), [5] and positron emission tomography.These techniques can be repeatedly used on the same organism to investigate spatiotemporal changes.However, the cellular resolution of these techniques is insufficient to obtain extra information. [6]A tissue-clearing technique has been introduced to allow rapid 3D cellular resolution visualization of human tissues without causing any damage to human organs. [7]The advantages of this tissue-clearing technique render human tissue optically transparent, allowing full penetration of light deep into the cleared tissue, thus accelerating progress in human tissue anatomy and pathology detection.Combining tissue-clearing techniques with newly developed light-sheet microscopy [8,9] revolutionizes laboratory animal research, human anatomy, and disease diagnosis.
In this review, we presented mechanisms and fundamental principles of tissue-clearing techniques, described existing tissue-clearing techniques for human tissues, discussed their biomedical applications for various human tissues, and then presented microscopic imaging techniques, along with data handling strategies.

PRINCIPLES OF OPTICAL TISSUE CLEARING
Most tissues in the human body have heterogeneous properties and structural compositions comprised of different refractive indices (RIs).For instance, it was found that the values of RI ∼1.34 for interstitial fluid and cytoplasm >1.44 for proteins and lipids. [10]Besides, the inorganic matrix of bone contains minerals with RI >1. 6.In addition, biomolecules are characterized by specific RI values: proteins RI ∼1.43; lipids RI ∼1.44; water RI ∼1.33, where the overall tissue RI is generally set equal to 1.4-1.59. [11]uman tissues are nontransparent and impenetrable to light owing to the disproportion of nonuniform amounts of RI at interfaces between different substances in a large volume that causes light scattering.Notably, tissueclearing technology overcomes the problems associated with the detrimental effects of scattering by reducing optical inhomogeneities.Various strategies of tissue-clearing techniques have been implemented, including chemical treatment steps of dehydration, delipidation, and decalcification, which can remove multiple organ RI components and homogenize the organ's final RI.Thus, the implantation of an expandable chemical increases the overall volume of the organ, which ultimately reduces RI and achieves a homogeneous RI for the organ. [12]Therefore, the primary goal of tissue clearing is to match the RI of different tissue components and minimize light scattering.
Tissue-clearing techniques can be classified into three types: hydrophobic, hydrophilic, and hydrogel embedding-based techniques. [8]In these, hydrophobic and hydrophilic tissue-clearing methods are also known as "organic solvent-based" and "aqueous-based" tissue-clearing methods, respectively, shown in Figure 1.

Organic solvent-based tissue clearing
Organic solvent-based methods for tissue clearing have the advantage of achieving rapid and complete transparency.Tissue clearing involves removing lipids and hydrating the tissue with organic solvents such as ethanol and dichloromethane.The Spalteholz technique was the first organic solvent-based method developed in 1911, using a mixture of benzyl benzoate and methyl salicylate, but it preserves fluorescence only to a limited extent due to dehydration. [8]To improve the Spalteholz technique, the ethyl cinnamate (ECi) tissue clearing [13] and 2ECi [14] methods were developed, which use ethyl cinnamate as an RI-matching solution to reduce the toxicity of the organic solvent.Poly-ethyleneglycol-associated solvent system [15] is another organic solvent-based method that uses polyethyleneglycol (PEG) and a clearing agent to dissolve lipids and improve tissue transparency, making it suitable for imaging both hard and soft tissue.Many organic solventbased tissue-clearing methods have evolved from the 3D imaging of solvent-tissue cleared organs (3DISCO) developed by Erturk et al., which was the first to render an adult mouse brain optically transparent in 1-2 days. [16]Methods based on DISCO include steps to remove the primary light scattering in the tissue, such as dehydration to remove water and delipidation to remove lipids (the RI of water is 1.33, whereas that of the lipid tissue is approximately 1.44).This was followed by an immersion step in an organic solvent solution to increase the value of RI, matching with biological tissue.Organic solvents and shrunken brain tissue have a RI of about 1.56.3DISCO and its variants were improved to address the issue that solvent chemicals preserve fluorescent proteins (FPs) for only a few days.Pan et al. developed the ultimate DISCO (uDISCO) method that allowed for the clearing and imaging of whole mice, preserved endogenous FPs for months, and shrank mouse bodies to approximately one-third of their original size. [17]lkaline solutions pH adjustment tissue-clearing methods, such as alkaline pH-based uDISCO (a-uDISCO) [18] and DISCO with superior fluorescence preserving capability (fDISCO), [19] involves the use of a basic solution to alter the pH of the cleared tissue.The a-uDISCO, a modified version of uDISCO, improved the intensity and stability of FPs by adjusting pHs in the clearing solutions. [20]DISCO effectively preserves FPs and fluorescent chemical tracers for months by controlling temperature and pH settings, has been extensively used in imaging human biopsy specimens. [19]The recently developed stabilized DISCO (sDISCO) to better preserves fluorescent signals for over a year by stabilizing the immersion solution by adding antioxidants. [21]Apart from FPs and fluorescence chemical probes-relied DISCO methods, immunological deep labeling methods were used to study mouse organs, human embryos, and human cancer biopsy samples.Renier et al. developed immunolabeling-enabled DISCO (iDISCO ), combining different chemicals in the pretreatment solutions with permeabilization to successfully immunolabel whole mouse organs for labeling large tissues with specific antibodies. [22]Cai et al. recently developed nanobody (VHH)-boosted DISCO (vDISCO), [23] a whole mouse immunolabeling technology that introduces nanobodies targeting FPs into the entire mouse body under high pressure.Thus, the low signal intensity of endogenous FPs can be overcome using enhanced fluorescent signals. [23]HANEL (small-micelle-mediated human organ efficient clearing and labeling), utilizes small micelles that enables efficient clearing and labeling of human organs for 3D imaging [24] (Table 1).

Aqueous-based tissue clearing
In order to overcome the disadvantages of toxicity, aqueous-based tissue-clearing techniques that are relatively simple, biologically safe, and friendly to FPs have been developed. [25]Here, hydrophilic reagents, which contain hydrogen bonds that link proteins and surrounding water molecules, are designed to be dissolved in water at high concentrations and used as RI-matching media. [26]hese hydrogen bonds can help maintain the 3D structure of the tissue and preserve the signal of FPs.
There are two types of aqueous-based tissue-clearing methods: simple immersion and delipidation categories.In simple immersion, high osmotic pressure solutions such as fructose, 2,2′-thiodiethanol (TDE), for example, FLASH F I G U R E 1 Summary of different tissue-clearing methods.(A) Organic solvent tissue-clearing methods and their primary features.The major step in organic solvent tissue-clearing methods is the complete dehydration of the tissue and lipid extraction, followed by the immersion of refractive index (RI)-matching organic solvents.(B) The primary features of hydrophilic tissue-clearing methods.In each step, the hydrophilic tissue-clearing methods rely on water-soluble regents.Typically, these methods include decolorization and delipidation steps, followed by matching RI in the final step.Some hydrophilic tissue-clearing methods cause tissue enlargement.(C) Hydrogel-based tissue-clearing methods and their primary features.The hydrogel-based tissue-clearing methods use monomers and polyepoxide to create a synthetic gel and are generally associated with delipidation, staining, expanded tissue size, and RI matching.
(fast light-microscopic analysis of antibody-stained whole organs), [27] and formamide are gradually converted before being used to directly impregnate samples using the corresponding aqueous RI-matching solutions.The biological samples are neither stretched nor shrunk due to the absence of detergents, preserving the morphology.Imai and coworkers developed the see deep brain (SeeDB) protocol using fructose immersion as the RI-matching component. [28]Still, the SeeDB-based method was inap-propriate for whole organ tissue clearing and limited to mouse brain slices at a thickness of several millimeters.Hou et al. found a modified SeeDB2 protocol that uses a higher RI aqueous solution, allowing for super-resolution imaging of neural circuits in mouse brains. [29]In 2015, the SeeDB-derived tissue-clearing method, FRUIT (SeeDBderived optical-clearing method), was modified by adding urea into SeeDB and clearing the entire brain of adult rabbits. [30]Subsequently, the ScaleS (sorbitol-based Scale tissue-clearing) method was developed by combining urea and sorbitol in RI-aqueous solutions to achieve higher transparency in tissue-clearing procedures. [31]There are other aqueous-based tissue-clearing methods, for example, ClearT/T2 (detergent-and solvent-free clearing method) uses formamide and PEG as RI-matching agents and increases its transparency [32] (Table 2).
The chemical can be easily applied by simple immersion, but it does not achieve ideal optical transparency after tissue clearing.Therefore, Ueda and co-workers conceived and developed a more powerful aqueous-based tissueclearing method called CUBIC (clear, unobstructed brain or body imaging cocktails and computational analysis). [33]hey used computationally selected clear unobstructed imaging cocktails and amino alcohols with delipidizing and decolorizing functions.Additionally, they found more than 1600 hydrophilic chemicals that could be used as RImatching reagents in the CUBIC method.This method has been used in many applications, such as wholebrain imaging of light-related gene expression and drug delivery and whole-brain imaging of cancer metastases.Combining the most advanced delipidation CUBIC-L and RI-adapting reagents CUBIC (CUBIC-RI), which offers higher performance in terms of biocompatibility, biosafety, and protein function preservation, an improved tissueclearing method is achieved. [34,35]In addition to the CUBIC series of methods, a combination of detergents, PEG and urea are used to clear the tissue, and each variation has been optimized for specific tissue types or applications [36] (Table 3).

Hydrogel embedding tissue clearing
Although the aqueous-based method can clear small amounts of human tissue, the high concentration of detergents in the aqueous-based method would remove some proteins from the cleared tissue.Hydrogel-based methods attempt to address these issues by preserving tissuespecific proteins in the embedded hydrogel.The hydrogel is typically composed of a polymer matrix and can protect the biomolecules of samples under harsh treatment conditions while preserving endogenous fluorescence.The next step is to achieve a homogeneous RI by removing the lipids for weeks or quickly (days) in electrophoresis because the protein is fixed in the gel. [37]LARITY (cleared lipid-extracted acryl-hybridized rigid immunostaining/in situ hybridization-compatible tissue hydrogel), [38] the first tissue-clearing method for embedding hydrogels, is based on electrophoretic lipid extraction to achieve a homogenous RI, and it conveys the disadvantages of the heating process.Indeed, electrophoretic CLARITY is performed at 42 • C, which can damage the sample and expand it after clearing.Tissue-clearing methods developed to enhanced CLARITY method.Such as passive CLARITY technique (PACT), is an improved version of CLARITY that has addressed the sample expansion problem. [39]It improves tissue-clearing efficiency by diffusing sodium dodecyl sulfate (SDS) into the samples with moderate tissue expansion, while PACT-deCAL [40] and Bone CLARITY [41] are tailored for specific applications.By unifying and controlling the crosslinking reaction, SWITCH (system-wide control of interaction time and kinetics of chemicals), [42] and SHIELD (stabilization to harsh conditions via intramolecular epoxide linkages to prevent degradation) [43] methods enable the production of a chemical-and heat-resistant hybrid tissue/gel.While SWITCH labels specific cell types with a photoconvertible FP, allowing for selective removal of labeled cells.
CLARITY and its variants have played a crucial role in the development of tissue-clearing methods.ACT-PRESTO (active clarity technique-pressure-related efficient and stable transfer of macromolecules into organs) [44] and PRE-CLARITY [45] are among the variants that have modified the electrophoresis process and device to accelerate the clearing process and improve antibody penetration speed and depth.Choi et al. developed the FxClear (free-hydrogel electrophoretic tissue-clearing method) protocol, which removes acrylamide from the electrophoretic step of hydrogel, to improve image resolution and retention of fluorescence in cleared mouse brains. [46]MYOCLEAR (novel and highly reproducible muscle tissue-clearing protocol) is a hydrogel embeddingbased method that was developed to overcome the intolerance of using acrylamide-based techniques in muscle.It is suitable for various staining procedures and the quantification of mouse neuromuscular junctions and diaphragms. [47]nother method, clearing-enhanced 3D microscopy (Ce3D), uses acrylamide to form a hydrogel around the tissue, which is then cleared with detergents. [48]ocusClearTM [49] is a proprietary method that uses a water-soluble polymer to clear the tissue.In contrast, clearing for serial two-photon (STP) tomography [50] is a method optimized for electron microscopy that uses osmium tetroxide and thiocarbohydrazide to stain the tissue and increase its contrast.
Finally, scheme update on tissue transparency (SUT) [51] is a novel technique that combines electrophoretic tissue clearing, lipid extraction, and hydrogel embedding for efficient and versatile tissue clearing.Overall, the various hydrogel-based tissue-clearing methods have enabled researchers to visualize and study complex tissues in new ways, with different techniques suited for specific applications and sample types (Table 4).

APPLICATIONS OF OPTICAL TISSUE CLEARING METHODS FOR PARTICULAR HUMAN ORGANS
Tissue-clearing methods enable large-scale volumetric imaging of intact human tissues, allowing us to perceive complex spatial structures at the organ level.In recent years, various tissue-clearing methods have been widely used in human tissue research (Tables 5 and 6).

Eye
It is difficult to optically clear the entire human eye because using different protocols cannot completely get rid of pigment buildup in the retina.Hohberger et al. used the SeeDB protocol to transparent the sclera and adjacent optic nerve. [52]Studies on the optical clearing of the entire human eye were conducted using the SHANEL method, a solvent-based protocol for clearing human organ tissue. [24]In this method, the anatomical structures of the human eye cell nucleus were stained with TO-PRO-3 iodide (TO-PRO-3), and the autofluorescence signals showed the eye structure.

Skin
Although the human skin is a complex tissue with multiple layers of human skin that cause inhomogeneous light scattering, optical tissue-clearing methods can be used to examine the human skin biopsies.Fernandez and Marull-Tufeu used a confocal microscope to capture the entire epidermis using the solvent-based 3DISCO tissueclearing method and rhodamine B staining. [53]In clearing the human skin biopsies, Abadie et al. investigated different solvent-based protocols, such as 3DISCO, ethanol-BABB, and ethanol-dibenzyl ether (DBE).Although both 3DISCO and ethanol-DBE were equally fast in achiev-ing optical transparency of human skin, they were less effective than the ethanol-BABB method in terms of transparency. [20]

Bones
Many techniques have been used to clear bone tissue.Tissue-clearing methods based on aqueous reagents or hydrogel, [41] such as CLARITY, PACT, and CUBIC -R, [35] efficiently clear soft human tissues; however, they cannot clear hard human bone tissues as the RI of bone is approximately 1.60.Among the solvent-based tissueclearing methods, uDISCO had only moderate success in clearing mouse bones, but its tissue-clearing effect on human bone tissues has not been demonstrated. [17]any human tissue-clearing methods have considered direct RI-matching regents with bone tissue.In order to achieve greater optical transparency, the SHANEL method decalcified the human skull before incubating it in the RI-matching solution. [24]

Heart
Analyzing the anatomy of the human heart is extremely challenging because of its complex multicellular structures comprising rigid tissues. [54]The 3D structure of human heart tissue can be visualized using MRI, but it is not the best technique owing to its limited resolution. [55]The tissue-clearing technology provides access to a full 3D cellular resolution of human cardiac tissue that reflects the structure of human cardiac muscle under physiological or pathological conditions. [56]Recently, a variant of the SDS based and free of acrylamide hydrogel tissue-clearing method was used to get 3D imaging of myocardial slices with a thickness of 300 μm. [57]This method provides 3D information on the collagen content and its distribution in the cardiac tissue.The minimum distance required to study complex vascular networks with multiple regions  cannot be achieved due to the limited thickness of the myocardial slices used in this method.Therefore, the SHANEL tissue-clearing method exhibited a 3D vascular network of the entire human heart over long distances and found the adjusted changing of vasa vasorum following atherosclerosis in the optically transparent post-modern human heart. [24]

Pancreas
Knowledge of pancreatic anatomy helps to evaluate pancreatic pathology and develop interventions for pancreatic diseases. [58]In order to achieve this, many tissue-clearing methods have been developed and used on human pancreatic tissue. [59]PACT is a hydrogel-based method used for clearing the human pancreas while preserving the morphology of sections with a thickness of 1-2 mm. [60]nother organic solvent-based method was used to clear sections of the human pancreas with a thickness of 5 mm.This method labeled cytokeratin 19 to visualize the pancreatic ductal system and used the autofluorescent properties of collagen and elastin by visualizing blood vessels in normal pancreatic tissue. [61]Moreover, a tissue-clearing method was developed to adjust the RI-matching solution value of 1.52 to visualize better and identify the microenvironment of the human pancreas, allowing better light penetration and optical imaging of the microenvironment of islets and fat infiltration.In addition, it helps visualize neurovascular networks in a human pancreas tissue with a thickness of 350 μm from clinical patients. [62]Furthermore, pancreatic ductal adenocarcinoma is associated with marked lymph angiogenesis and remodeling of lymphatic vessels.In this context, a method was developed to clear human pancreatic cancer tissue with a thickness of 350 μm, [59] involving the 3D remodeling of lymphatic vessels by deep tissue staining.Another advanced tissue-clearing method is used for imaging thick human pancreatic tissue sections (thickness = 1 mm) using multifluorescent channels over two to three days and analyzing several hundred islets. [59]The entire human pancreas ductal system was visualized in 3D space, diminishing the significance of entire-organ imaging for assessing the pancreatic anatomy.

Urinary tract
Tissue-clearing protocols have been developed to resolve light scattering in the urinary system and improve antibody penetration into human kidney tissue. [63]However, many of these approaches are limited to human embryo size.For example, a tissue-clearing method used the passive CLARITY technique to examine an 11-week-old human fetal kidney and embed the tissue in an acrylamide hydrogel matrix to achieve optical transparency. [64]The human fetal kidney was then stained with anti-E-cadherin to detect branching morphogenesis of the ureteric bud epithelium using light sheet fluorescence microscopy (LSFM).Subsequently, another study applied the acrylamide-based hydrogel matrix technique to investigate morphological changes in the urogenital tract of the human fetus from the bisexual stage (<9 weeks of fetal age) to well-differentiate male and female organs (>13 weeks of fetal age).The development of the human genitourinary tract became more complex in 3D imaging after labeling the urethral plate and epidermis, urothelium, and dorsal aspect of the vestibular groove and imaging with the LSFM. [65]However, the major limitation of laboratory studies is that the tissue-clearing samples could not be applied to adult human kidneys. [64]An advanced LSFM with a large chamber and longer working distance of the objective has been developed for imaging larger human renal tissues.Also, the SHANEL method was used to label the entire kidney of vessels and cell nuclei using chemical dyes and extensively analyzing the human kidney structure. [24]

Brain
The existing tissue-clearing methods for 3D imaging of brain tissues can investigate the macroscopic organization of each brain region and visualize its neural network. [47,66,67]Many tissue-clearing methods, such as the CLARITY, SHIELD, and SWITCH methods, were originally developed for mouse brains before being improved to clear human brain slices. [67,68]The effective use of acridine orange, methylene blue, methyl green, and neutral red to label neurons on large samples of human tissues for visualization of neuron distribution in various brain regions. [69]However, many tissue-clearing methods have been introduced because the human brain differs from the rodent brain.A study described that using the hydrophilic reagent 1,2-hexanediol (HxD) resulted in fast delipidation and clearing of lipid-rich white matter in human brain tissue. [70]FASTClear was developed to overcome the limitations of acrylamides and provide 3D visualization of human brain tissue using SDS. [71]For 3D visualization of fresh and archived human brain tissues, OPTIClear enables the detection of neurons, glial cells, blood vessels, and pathological markers such as tau protein tangles. [72]ike the previous method that included a delipidation step and prevented labeling neuronal circuits, another method, hFRUIT, was developed as an immersion-based clearing method for the optical clearing of adult human brain samples with a thickness of 5 mm while preserving lipids and lipophilically labeled neuron circuits. [29]otably, 3D pathology patterns were found through the volume analysis of archived human brain tissues.CLAR-ITY allows for 3D visualization of Lewy bodies in cleared human brain tissues and observes greater diversity of amyloid plaques in 3D imaging, providing new insight into the mechanisms of neurodegeneration. [68,73]In studies on tissue-clearing cerebral vessels, new anatomical findings about the brain have been observed. [74]A comprehensive method for characterizing the vessels of the human cerebral cortex is required because vasculature plays a crucial role in many human brain diseases.The SHANEL method created 3D images of human cerebral cortex tissue blocks from aged patients immunized against Aβ42 and stained with fluorescent Gongo red.Vascular endothelium was labeled with Lycopersicon esculentum agglutinin (tomato lectin), and subsequently, all tissues were dehydrated and clarified using an adapted 3DISCO protocol.

MICROSCOPIES FOR CLEARED TISSUE IMAGING
Advances in developing fluorescence light microscopy have led to the application of tissue-clearing methods in various fields.The goal of fluorescence light microscopy aims to balance the image depth and resolution.Therefore, the best microscopy for optically transparent samples must be selected.In this context, cleared samples can be imaged in deep 3D using microscopies, such as confocal, [10,75] two-photon, [76] or LSFM.Two-photon microscopy (TPM) is an advanced version of confocal microscopy for deep imaging in living animals and thick fluorescent labeled tissues.However, even with cleared samples, TPM does not yield an appropriate image depth of cleared human tissues.Furthermore, TPM allows cellular 3D imaging of cleared samples with a depth of 5 mm, even with optimized objectives. [77]However, the slow scanning speed and unavoidable light bleaching make TPM unsuitable for large-volume cleared human tissue imaging.Nevertheless, LSFM enables volumetric imaging of considerably larger human tissues by selectively illuminating the entire layer of interest with a thin sheet of light while sparing the rest of the region from laser illumination, significantly reducing the damage from light bleaching and increasing scanning speed. [78]arge volumes of intact human tissues at cellular resolution can be investigated due to the advantages of LSFM and the development and optimization of technologies appropriate for human tissue clearing, [79] as listed in Table 7.For example, after tissue clearing using the organic solvent-based method to clear human organs, the size can be reduced by approximately 30%, allowing the cleared human organs to fit into the imaging chamber of LSFM. [17]he advantages of selective illumination are most apparent while observing complex and dynamic 3D fluorophore distributions, ranging from samples as large as entire embryos to objects as small as organelles. [80]Furthermore, LSFM enables fast acquisition of high-throughput cleared tissue sample images.Also, LSFM uses high-speed cameras for signal detection; thus, millions of voxels are acquired in parallel, and tens to thousands of images can be achieved in seconds. [17,81]ommercial and custom LSFMs are currently available in the market, and many companies offer different instruments with a wide range of lasers, objectives, and cameras, [79] as depicted in Figure 2. The single-view category includes a detection objective and a lattice light sheet through an illumination objective, such as the lattice light sheet microscopy. [82]In the two-opposing views category, the detection objective and Gaussian light sheet of the illumination objective are rotated by 90 • using simultaneous multi-view light sheet microscopy [83] and multiview microscopy with selective plane illumination. [84]bjectives are also used simultaneously for illumination and detection in the two-orthogonal and four-orthogonal view categories, such as the dual-view inverted selective plane illumination microscopy [85] and IsoView light sheet microscopy. [86]onsequently, by adapting LSFM with a tissue-clearing method, volumetric imaging could provide a powerful platform for imaging transparent human organs and enable ; Hohberger et al. [52] Skin 3DISCO, ethanol-BABB, ethanol-DBE Rhodamine B, H&E staining

Image compression
LSFM is a powerful imaging technique for visualizing transparent human tissues or organs with high spatial and temporal resolution. [87]However, this volume scanning of a human organ typically generates terabytes of complex image data containing information from 3D and multiple color channels.Thus, the next step of biological discovery may be hampered due to data management, particularly image processing, necessitating effective computing solutions for data management. [88]In order to prepare data for visualization, data management must involve fixing technical errors, reconstructing complex geometries, and compressing file size. [89]n this study, we compared various image compression formats.We focused on widely used formats that offer lossless compression because storing an unaltered version of data is required when using various analysis or visu-alization software packages.Joint photographic experts group (JPEG 2000) is a typical compressed image format.Although 3D compression is described in the JPEG 2000 standard, there are not many applications where this feature is included. [90]Furthermore, all JPEG 2000 encoding and decoding steps are difficult to parallelize, challenging for effective use of modern multicore computer hardware.Conversely, HDF5, another popular compression format for image files, [91] provides lossless data compression and storage data in blocks for fast access to arbitrary regions of interest.The hierarchical data format 5 (HDF5) interface does not parallelize writes and requires a high-performance computer.Since many visualization programs use Tiff files, layer-by-layer image compression using lempel-ziv-welch (LZW)-Tiff is recommended for most LSFM-generated image data.Additionally, it works well for clearing out large collections of multidimensional images of human tissues.

LSFM data visualization
Appropriate rendering tools are powerful in visualization after the stitching and volume fusion steps for cleared image sets of human organs.These rendering tools allow users to explore detailed characteristics of biological structures, complex geometries, and complex morphogenetic cascades of multiple human organs.Of course, finding Fiji's open-source plugin is the easiest step for rendering large volumes. [92]For instance, the Fiji Clear-Volume plugin can visualize large datasets during data acquisition. [93]Another open-source plugin from Fiji for rendering or creating animations using the 3Dscript plugin is the TeraFly extension [94] for Vaa3D. [95]Further, BigVolumeViewer is another Fiji plugin, an enhanced version of BigDataViewer that enables volume rendering regardless of dataset size. [96]Alternatively, UCSF Chimera is an open-source [97] that supports the rendering of 3D images and has been used to visualize the LSFM data.
In addition to open-source software packages, commercial software solutions with an easy-to-use graphical user interface and commercial microscope operating software integration are widely available.This commercial software would be preferable to render large human organs on a stable platform.Image stacks visualized using commercial software make comprehensive data processing solutions more accessible. [98]Examples of these software packages include Imaris (Bitplane), Amira ( ).Arivis Vision4D is the most suitable and widely used software for many human organs. [99]

THE PERSPECTIVE OF HUMAN TISSUE CLEARING
Even though we presented some successful applications of tissue-clearing methods for human tissues and organs, and their effectiveness has significantly improved in recent years, the existing human tissue-clearing methods require further improvement.According to the working distance and image depth of LSFM, one of the practical issues with cleared human tissue images is that they can be scanned with relatively low image resolution, making it difficult to analyze various cellular processes in 3D space.Therefore, large-area and fast-LSFM innovations are needed to allow new imaging applications.Furthermore, developing appropriate solutions for labeling, imaging, and data analysis requires a thorough understanding of the biophysical and biochemical principles of human tissues underlying tissue-clearing processes.In particular, faster and homogeneous antibodies, such as DNA and RNA labeling methods for large human tissues, are required as more accurate and comprehensible algorithms.Furthermore, a standard 3D atlas of human organs should be combined with single-cell RNA sequencing and proteomics to gain more spatial and omics about human organs.Finally, machine learningbased algorithms combined with tissue-clearing methods are required to achieve a faster and more accurate diagnosis with clarification in 3D human tissue histopathology.This is necessary because the combined system has the potential to significantly improve clinical applicants in the near future.
The technique of tissue clearing has the potential to revolutionize our understanding of fundamental biological questions and has significant implications for neuroscience research and regenerative medicine.The interdisciplinary technique of tissue clearing holds immense potential to revolutionize our understanding of fundamental biological questions, including the detailed mechanisms of disease development and complex processes such as neural circuitry and immune system interactions.The technique is already significant in neuroscience research, providing 3D visualization of the large scale human neural tissue at the cellular level, leading to insights into brain architecture and neural circuitry, with implications for brain function and treatment of neurological diseases.In addition, tissue clearing offers promising applications in regenerative medicine by enabling the study of the 3D cellular architecture of human tissues and organs, facilitating the identification of optimal strategies for next generation human tissue repair and regeneration.

A U T H O R C O N T R I B U T I O N S
Hongcheng Mai is responsible for studying concepts and design as well as writing and revising the manuscript.Dan Lu wrote and reviewed the manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

TA B L E 3
Quadrol

TA B L E 6
Abbreviation: LSFM, light sheet fluorescence microscopy.

F I G U R E 2
Summary of standard light sheet fluorescence microscopy (LSFM) configurations.(A) Single-view category LSFM with a single illumination and orthogonal imaging arm.(B) Two-opposite-view category LSFM with bidirectional illumination.(C) Two-opposite-view category LSFM with bidirectional illumination and imaging.(D) Two-orthogonal-view category LSFM with a reversed dual view achieved by selecting a single illumination and orthogonal imaging arm but in a reversed position.The red and blue arrows illustrate the illumination and detection paths, respectively, while the small dark arrow shows the direction in which the sample moves during imaging.
FEI), MetaMorph (Molecular Devices) (Zen [Zeiss], Leica application suite [Leica Microsystems], NIS Elements [Nikon Instruments], cellSense [Olympus], Huygens [SVI], and Arivis Vision4D [Arivis] This work was supported by grants received from the National Natural Science Foundation of China (82271304 and 81801150), the Science and Technology Planning Project of Guangdong Province, China (2019A050513005), the Natural Science Foundation of Guangdong Province (2018A0303130182), the Basic and Applied Basic Research Fund Project of Guangdong Province (2022A1515012311), the Science and Technology Planning Project of Guangzhou, China (202201010127), Science and Technology Program of Guangzhou: Key Lab of Guangzhou Basic and Translational Research of Pan-vascular Diseases (202201020042), Young Talent Support Project of Guangzhou Association for Science and Technology (QT-2023-024) and Science and Technology Projects in Guangzhou, China (SL2023A03J01214).The authors would like to thank TopEdit (www.topeditsci.com)for its linguistic assistance while preparing this manuscript.