Advertisement

Research Techniques Made Simple: Optical Clearing and Three-Dimensional Volumetric Imaging of Skin Biopsies

  • Yingrou Tan
    Affiliations
    Department of Research, National Skin Centre, Singapore

    Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
    Search for articles by this author
  • Carolyn Pei Lyn Chiam
    Affiliations
    School of Medicine, Dentistry & Nursing, University of Glasgow, Glasgow, United Kingdom
    Search for articles by this author
  • Yuning Zhang
    Affiliations
    Faculty of Science, National University of Singapore, Singapore
    Search for articles by this author
  • Author Footnotes
    7 These authors contributed equally to this work.
    Hong Liang Tey
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Department of Research, National Skin Centre, Singapore

    Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
    Search for articles by this author
  • Author Footnotes
    7 These authors contributed equally to this work.
    Lai Guan Ng
    Correspondence
    Correspondence: Lai Guan Ng, Functional Immune Imaging Group, Singapore Immunology Network, Singapore 138648.
    Footnotes
    7 These authors contributed equally to this work.
    Affiliations
    Singapore Immunology Network (SIgN), A*STAR (Agency for Science, Technology and Research), Biopolis, Singapore
    Search for articles by this author
  • Author Footnotes
    7 These authors contributed equally to this work.
      Skin histology is traditionally carried out using two-dimensional tissue sections, which allows for rapid staining, but these sections cannot accurately represent three-dimensional structures in skin such as nerves, vasculature, hair follicles, and sebaceous glands. Although it may be ideal to image skin in a three-dimensional manner, it is technically challenging to image deep into tissue because of light scattering from collagen fibrils in the dermis and refractive index mismatch owing to the presence of differing biological materials such as cytoplasm, and lipids in the skin. Different optical clearing methods have been developed recently, making it possible to render tissues transparent using different approaches. Here, we discuss the steps involved in tissue preparation for three-dimensional volumetric imaging and provide a brief overview of the different optical clearing methods as well as different imaging modalities for three-dimensional imaging.

      Abbreviations:

      2D (two-dimensional), 3D (three-dimensional), OTC (optical tissue clearing), RI (refractive index)

      Summary Points

      • Three-dimensional (3D) volumetric imaging of tissues provides a comprehensive view of 3D biological tissue architecture and has been used in ex vivo 3D skin pathology studies to investigate epidermal hyperplasia in psoriatic skin or neuronal innervation in pruritic skin as well as dermal blood flow in in vivo studies.
      • 3D volumetric imaging can potentially be used to carry out 3D spatial mapping of tissue-resident cells and structures within normal skin such as nerves, vasculature, hair follicles, and sebaceous glands.
      • Optical clearing renders tissue transparent by reducing the heterogeneity of light scattering within the tissue. Dissociation of collagen fibrils and refractive index matching are key to skin optical clearing.
      • Optical clearing methods can be broadly classified into solvent-based methods and aqueous-based methods. Aqueous-based methods can be further subdivided into simple immersion, hyperhydration, and hydrogel-embedding methods.
      • The tissue preparation workflow involves a series of steps from fixation, permeabilization, decolorization, and immunostaining to optical clearing or refractive index matching, which are crucial for acquiring high-quality images. The sequence of the steps may change depending on the optical clearing method being used.
      • The choice of microscope for 3D volumetric imaging depends on the resolution required. Although the light-sheet fluorescence microscope offers faster imaging speed and lower photobleaching, the confocal and two-photon microscopes generally provide higher imaging resolution. Serial two-photon tomography is one emerging 3D imaging technique, which uses a combination of imaging and sectioning to enable high-resolution and deep-tissue imaging with the two-photon microscope.

      Advantages

      • 3D volumetric imaging provides crucial spatial information of 3D structures such as nerves, vasculature, hair follicles, and sebaceous glands in the skin, which may not be accurately represented by traditional two-dimensional histology methods.

      Limitations

      • Relatively longer time required for tissue preparation, which can require up to 1 or 2 weeks if immunostaining is involved.
      • Depending on the optical clearing method chosen, the process can also be laborious, which makes it harder to have high sample throughput.
      • Existing approaches are currently unable to optically clear the melanin in pigmented skin. If the epidermis is being studied, this can be circumvented by imaging thinner sections of the skin with lower melanin content.

      Introduction

      Histology is traditionally carried out using tissue sections and viewed with the aid of a light microscope. Although such histology slides are useful for illustrating basic cellular anatomy through accurate and high-resolution images, the main flaw is that the images are two-dimensional (2D). This may pose a challenge for histopathological analysis because it may not be an accurate representation of three-dimensional (3D) biological structures. For example, in the excision of a skin tumor, knowledge of its full 3D structure is needed for the complete removal of the tumor. Mohs surgery is the existing gold standard for skin tumor removal, where the surgeon excises the visible tumor and then removes the skin layer by layer to check for tumor involvement in the margins (
      • Tolkachjov S.N.
      • Brodland D.G.
      • Coldiron B.M.
      • Fazio M.J.
      • Hruza G.J.
      • Roenigk R.K.
      • et al.
      Understanding mohs micrographic surgery: a review and practical guide for the nondermatologist.
      ). However, this procedure tends to be costly owing to the long time required for it. The alternative to Mohs surgery is serial transverse cross-sectioning or the bread loafing method. In this process, the specimen is cut into three or more pieces, embedded in a paraffin block, followed by sectioning only at a few locations for viewing under a microscope. However, this method has a low sensitivity and produces a substantial number of false negatives because only a small fraction of the entire tumor is sampled (
      • Jackson J.E.
      • Kelly B.
      • Petitt M.
      • Uchida T.
      • Wagner Jr., R.F.
      Predictive value of margins in diagnostic biopsies of nonmelanoma skin cancers.
      ).
      Alternatively, a 3D image of the tumor can be reconstructed from 2D serial sections. However, this approach is time-consuming and tedious because it involves volumetric reconstruction from hundreds of 2D sections, which may be further complicated by sectioning artifacts. Given the difficulties of imaging with 2D sections and the costs associated with Mohs surgery, 3D imaging provides a more attractive alternative, allowing for detection of tumor spatial heterogeneity (
      • Chen Y.
      • Shen Q.
      • White S.L.
      • Gokmen-Polar Y.
      • Badve S.
      • Goodman L.J.
      Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues.
      ).
      Although 3D imaging has been made possible with advances in microscopy such as the confocal microscope in recent years, it is still difficult to study most tissues of a certain thickness with 3D imaging techniques because tissues are opaque. This hinders the amount of light passing through and the sharpness of images collected owing to light scattering and absorption by the different components. (
      • Azaripour A.
      • Lagerweij T.
      • Scharfbillig C.
      • Jadczak A.E.
      • Willershausen B.
      • Van Noorden C.J.
      A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.
      ,
      • Tuchin V.V.
      Tissue optics and photonics: light-tissue interaction.
      )
      One method to solve this issue is optical tissue clearing (OTC). OTC renders tissues transparent by matching the different refractive index (RI) within the tissue with the aid of optical clearing agents (Figure 1a). With this method, it is possible to generate a 3D image without physically sectioning the tissue. OTC can be used in ex vivo 3D anatomical investigations to study skin pathology, for instance, epidermal hyperplasia in psoriatic skin (
      • Abadie S.
      • Jardet C.
      • Colombelli J.
      • Chaput B.
      • David A.
      • Grolleau J.L.
      • et al.
      3D imaging of cleared human skin biopsies using light-sheet microscopy: a new way to visualize in-depth skin structure.
      ) or neuronal innervation in pruritic skin (
      • Tan Y.
      • Ng W.J.
      • Lee S.Z.X.
      • Lee B.T.K.
      • Nattkemper L.A.
      • Yosipovitch G.
      • et al.
      3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
      ) as well as in in vivo skin imaging to measure dermal blood flow (
      • Wang J.
      • Shi R.
      • Zhu D.
      Switchable skin window induced by optical clearing method for dermal blood flow imaging.
      ) and alterations in skin structure in mice with diabetes (
      • Feng W.
      • Zhang C.
      • Yu T.
      • Zhu D.
      Quantitative evaluation of skin disorders in type 1 diabetic mice by in vivo optical imaging.
      ).
      Figure thumbnail gr1
      Figure 1Overview of different types of optical clearing protocols. Brief overview of tissue preparation steps for 3D volumetric imaging after fixation. (a) Solvent-based clearing. Tissues are first permeabilized before immunostaining, dehydration, and delipidation, followed by RI matching. (b) Simple immersion. Tissues are permeabilized, immunostained, followed by RI matching. (c) Hyperhydration. Tissue swelling is induced by hyperhydrating agents such as urea or formamide (
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      ). Some hyperhydrating protocols like CUBIC induce delipidation with high-detergent concentrations. This delipidation process also permeabilizes the tissue; the CUBIC protocol also induces tissue decolorization with amino alcohols, followed by immunostaining and RI matching. (d) Hydrogel embedding. An acrylamide hydrogel is formed by infusing the tissue with a buffer of acrylamide monomers and a temperature-sensitive crosslinker at low temperature, followed by transferring the tissue to 37 ⁰C to induce gel polymerization (
      • Chung K.
      • Wallace J.
      • Kim S.Y.
      • Kalyanasundaram S.
      • Andalman A.S.
      • Davidson T.J.
      • et al.
      Structural and molecular interrogation of intact biological systems.
      ). The hydrogel forms a scaffold to minimize protein loss during delipidation (using active or passive methods), and the tissue is permeabilized during delipidation. The tissue is then immunostained and RI matched. 3D, three-dimensional; RI, refractive index. Figure created using BioRender (https://biorender.com/).
      Optical clearing began in the early 1900s by
      • Spalteholz W.
      Über das Durchsichtigmachen von Menchlichen und Tierichen Präparaten und Seine Theoretischen Bedingungen.
      who started out using methyl salicylate, benzyl benzoate, and wintergreen oil. However, his technique caused necrosis of the outer layer of tissue, and it could not be applied to smaller tissue samples (
      • Azaripour A.
      • Lagerweij T.
      • Scharfbillig C.
      • Jadczak A.E.
      • Willershausen B.
      • Van Noorden C.J.
      A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.
      ). Numerous other optical clearing methods have since been developed to better visualize whole organs in 3D, which will be discussed later.

      Tissue preparation for 3D volumetric imaging

      Before understanding tissue clearing, we need to understand why tissues are opaque. Tissues lack transparency because different cellular organelles and biomolecules have differing RIs. For instance, the RI of cell membranes is 1.45, whereas that of water is 1.33 (
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      ). Although light travels in straight lines, light scattering occurs whenever light interacts with molecules of differing RIs, resulting in the bending of light (
      • Tuchin V.V.
      Tissue optics and photonics: light-tissue interaction.
      ). In the dermis of the skin, collagen fibrils form a major component within skin dermis and are major light scatterers within the tissue; dealing with collagen in the skin is thus important for skin optical clearing (
      • Zhu D.
      • Larin K.V.
      • Luo Q.
      • Tuchin V.V.
      Recent progress in tissue optical clearing.
      ). Light scattering combined with light absorption, caused by the presence of pigments like melanin in skin epidermis, causes blurry and dim images with progressively poorer image quality deeper within a tissue.
      Hence, the goal of OTC is to decrease the heterogeneity of light scattering and absorption by substances in the tissue so that light can travel in a straight line through the tissue, making it transparent (
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      ). Preparation of the tissue before imaging involves multiple steps, and each step is crucial for getting good images: fixation, permeabilization, decolorization, immunostaining, and optical clearing or RI matching (Figure 1).
      After harvest, skin can first be fixed in paraformaldehyde or glutaraldehyde or embedded together with hydrogel to preserve structures of interest (
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      ). Use of typical chemical fixatives can cause loss of fluorescence or reduce antigen–antibody binding; hence, alternative cross-linking strategies such as the stabilization to harsh conditions through intramolecular epoxide linkages to prevent degradation have been developed to improve the preservation of protein fluorescence and antigenicity (
      • Park Y.G.
      • Sohn C.H.
      • Chen R.
      • McCue M.
      • Yun D.H.
      • Drummond G.T.
      • et al.
      Protection of tissue physicochemical properties using polyfunctional crosslinkers.
      ).
      Permeabilization is then required to promote the substitution of water with high RI cell-permeable molecules to allow for RI matching and for immunostaining to allow antibodies to diffuse deep into the tissue. Permeabilization reagents can be classified into three groups: water-miscible polar solvents such as methanol, detergents that remove lipids that have a high RI, or hyperhydration reagents without delipidation (
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      ).
      Water-miscible polar solvents aid in replacing water within the tissue for RI matching. In contrast, permeabilization by delipidation decreases interactions between dyes and tissue molecules either by using an electric field to remove lipids in tissue in CLARITY-related protocols (
      • Tomer R.
      • Ye L.
      • Hsueh B.
      • Deisseroth K.
      Advanced CLARITY for rapid and high-resolution imaging of intact tissues.
      ) or high detergent concentrations with the reagent ScaleCUBIC-1 in CUBIC (
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Yukinaga H.
      • Kuno A.
      • Ueda H.R.
      Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.
      ,
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Kishino F.
      • Tawara T.
      • Watanabe T.M.
      • et al.
      Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.
      ,
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      ). Hyperhydration using urea to increase osmotic pressure or relax protein fibers can also increase the permeation of fluorescent molecules in ScaleS (
      • Hama H.
      • Hioki H.
      • Namiki K.
      • Hoshida T.
      • Kurokawa H.
      • Ishidate F.
      • et al.
      ScaleS: an optical clearing palette for biological imaging.
      ) and ScaleCUBIC-1 (
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Yukinaga H.
      • Kuno A.
      • Ueda H.R.
      Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.
      ,
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Kishino F.
      • Tawara T.
      • Watanabe T.M.
      • et al.
      Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.
      ,
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      ). Treatment with small molecules to extract cholesterol from the plasma membrane with cyclodextrins and loosen collagen fiber structure using N-acetyl-L-hydroxyproline has also been found to enhance tissue permeability (
      • Hama H.
      • Hioki H.
      • Namiki K.
      • Hoshida T.
      • Kurokawa H.
      • Ishidate F.
      • et al.
      ScaleS: an optical clearing palette for biological imaging.
      ). Dehydration‒rehydration using methanol and PBS in iDISCO also aids in permeabilization (
      • Renier N.
      • Wu Z.
      • Simon D.J.
      • Yang J.
      • Ariel P.
      • Tessier-Lavigne M.
      iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.
      ), although the exact biochemical mechanism for this phenomenon is not fully understood.
      Furthermore, decolorization removes endogenous molecules in the tissue that absorb light, especially heme and melanin (
      • Tuchin V.V.
      Tissue optics and photonics: light-tissue interaction.
      ), which increases transparency by increasing the amount of light passing through the tissues. The presence of blood is particularly tricky to deal with in clinical samples such as skin biopsies where it may not always be possible to ensure a sample with lower blood content. For preclinical mouse specimens, heme can be removed by removing red blood cells in circulation by perfusion of buffer through the circulatory system during tissue harvest; alternatively, when perfusion is not possible, the skin specimen can be treated with hydrogen peroxide, although this could potentially cause oxidative damage to the tissues (
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      ). The amino-alcohol component in ScaleCUBIC-1 was serendipitously discovered to be capable of decolorizing blood by eluting heme, which aids in decolorizing blood-rich organs like the liver, spleen, and kidney (
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      ). Melanin pigmentation is much harder to remove. Besides genetic modification to block melanin production, which is only applicable to transgenic animal models, the only other method is to image tissues in the near-infrared region of the electromagnetic spectrum where the tissue is unable to absorb light (
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      ).
      Fluorescent proteins are typically used for visualizing cells or structures in reporter mouse models or for tracking cells in in vivo–transfer experiments. However, fluorescent proteins are not an option for labeling clinical specimens; some solvent-based OTC methods such as the DISCO methods also tend to cause quenching of fluorescent proteins. To circumvent this, modifications of the solvent-based OTC methods have been developed to preserve endogenous fluorescence, such as uDISCO (
      • Pan C.
      • Cai R.
      • Quacquarelli F.P.
      • Ghasemigharagoz A.
      • Lourbopoulos A.
      • Matryba P.
      • et al.
      Shrinkage-mediated imaging of entire organs and organisms using uDISCO.
      ), FDISCO (
      • Qi Y.
      • Yu T.
      • Xu J.
      • Wan P.
      • Ma Y.
      • Zhu J.
      • et al.
      FDISCO: advanced solvent-based clearing method for imaging whole organs.
      ), and FluoClearBABB (
      • Schwarz M.K.
      • Scherbarth A.
      • Sprengel R.
      • Engelhardt J.
      • Theer P.
      • Giese G.
      Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
      ,
      • Stefaniuk M.
      • Gualda E.J.
      • Pawlowska M.
      • Legutko D.
      • Matryba P.
      • Koza P.
      • et al.
      Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.
      ), which uses either the adjustment of pH and temperature or specific chemicals for fluorescence preservation, such as α-tocopherol, tert-butanol (uDISCO, FluoClearBABB), and polyethylene glycol (PEGASOS) (
      • Jing D.
      • Zhang S.
      • Luo W.
      • Gao X.
      • Men Y.
      • Ma C.
      • et al.
      Tissue clearing of both hard and soft tissue organs with the PEGASOS method.
      ).
      If it is not possible to use a fluorescent reporter animal model, immunolabeling is a crucial step for visualizing cells or structures in tissue preparation protocols. Passive diffusion is typically used for immunolabeling, but this tends to be a slow process and can be problematic for tissues that are millimeters thick. To circumvent this, a rotational electrical field can be used to disperse antibodies within a sample more rapidly in a method termed stochastic electrotransport (
      • Kim S.Y.
      • Cho J.H.
      • Murray E.
      • Bakh N.
      • Choi H.
      • Ohn K.
      • et al.
      Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.
      ); alternatively, centrifugal force or convectional flow using a syringe to apply pressure could be a more rapid manner to carry out immunolabeling of tissues (
      • Lee E.
      • Choi J.
      • Jo Y.
      • Kim J.Y.
      • Jang Y.J.
      • Lee H.M.
      • et al.
      ACT:PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.
      ). A miniaturized version of antibodies—nanobodies could be used in combination with high-pressure transcardial perfusion for whole-body labeling as shown in the vDISCO protocol (
      • Cai R.
      • Pan C.
      • Ghasemigharagoz A.
      • Todorov M.I.
      • Förstera B.
      • Zhao S.
      • et al.
      Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.
      ).
      Finally, RI matching needs to be carried out for tissues to become optically clear. Tissues appear opaque because of light scattering caused by RI mismatch within the tissue, and when light scattering is reduced, the tissue is made more transparent. Water has a lower RI (1.33), and lipid and protein have a higher RIs (1.4–1.6) (
      • Johnsen S.
      • Widder E.A.
      The physical basis of transparency in biological tissue: ultrastructure and the minimization of light scattering.
      ,
      • Tuchin V.V.
      Tissue optics and photonics: light-tissue interaction.
      ); thus, removing water and lipid and replacing them with a substance of similar RI in the remaining components minimizes light scattering and makes tissues more transparent (
      • Susaki E.A.
      • Ueda H.R.
      Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.
      ). Alternatively, lipids can be removed and the tissue hyperhydrated for RI matching to occur (
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      ).
      A variety of optical clearing methods have been developed in recent years. These methods can be broadly split into two categories: organic solvent–based clearing or aqueous-based technique (Table 1, Figure 1). Organic solvent–based clearing such as the DISCO protocols (
      • Cai R.
      • Pan C.
      • Ghasemigharagoz A.
      • Todorov M.I.
      • Förstera B.
      • Zhao S.
      • et al.
      Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.
      ,
      • Ertürk A.
      • Becker K.
      • Jährling N.
      • Mauch C.P.
      • Hojer C.D.
      • Egen J.G.
      • et al.
      Three-dimensional imaging of solvent-cleared organs using 3DISCO.
      ,
      • Pan C.
      • Cai R.
      • Quacquarelli F.P.
      • Ghasemigharagoz A.
      • Lourbopoulos A.
      • Matryba P.
      • et al.
      Shrinkage-mediated imaging of entire organs and organisms using uDISCO.
      ,
      • Renier N.
      • Wu Z.
      • Simon D.J.
      • Yang J.
      • Ariel P.
      • Tessier-Lavigne M.
      iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.
      ) usually consists of dehydration using lipid solvation followed by RI matching to the dehydrated tissue and typically provides better transparency with shorter incubation time (Figure 1a). Owing to the inability of many of these methods at that point in time to preserve fluorescent protein emission and tissue architecture, aqueous-based methods were developed. These consist of either simple immersion in an optical clearing agent to carry out RI matching (Figure 1b); hyperhydration methods such as the CUBIC (
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Yukinaga H.
      • Kuno A.
      • Ueda H.R.
      Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.
      ,
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      ) and Scale (
      • Hama H.
      • Hioki H.
      • Namiki K.
      • Hoshida T.
      • Kurokawa H.
      • Ishidate F.
      • et al.
      ScaleS: an optical clearing palette for biological imaging.
      ,
      • Hama H.
      • Kurokawa H.
      • Kawano H.
      • Ando R.
      • Shimogori T.
      • Noda H.
      • et al.
      Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.
      ) protocols, where high concentrations of detergent are used for delipidation together with urea or formamide to induce hyperhydration to lower the RI of the tissue components followed by RI matching (Figure 1c); hydrogel-embedding methods such as the CLARITY (
      • Chung K.
      • Wallace J.
      • Kim S.Y.
      • Kalyanasundaram S.
      • Andalman A.S.
      • Davidson T.J.
      • et al.
      Structural and molecular interrogation of intact biological systems.
      ,
      • Tomer R.
      • Ye L.
      • Hsueh B.
      • Deisseroth K.
      Advanced CLARITY for rapid and high-resolution imaging of intact tissues.
      ) protocols, which utilize chemical cross-linking of proteins with a hydrogel to retain proteins during the delipidation process (Figure 1d). Expansion microscopy combines hydrogel embedding with hyperhydration to expand the protein-gel complex for super-resolution imaging of structures such as the cytoskeleton (
      • Chen F.
      • Tillberg P.W.
      • Boyden E.S.
      Optical imaging. Expansion microscopy.
      ,
      • Chozinski T.J.
      • Halpern A.R.
      • Okawa H.
      • Kim H.J.
      • Tremel G.J.
      • Wong R.O.
      • et al.
      Expansion microscopy with conventional antibodies and fluorescent proteins.
      ).
      Table 1Overview of Different Optical Clearing Methods (Method Classification Adapted from
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      )
      Organic Solvent–Based TechniquesAqueous-Based Techniques
      High RIs (>1.50)Lower RIs (<1.49)
      Generally, better transparencyGenerally, less transparency (
      • Renier N.
      • Wu Z.
      • Simon D.J.
      • Yang J.
      • Ariel P.
      • Tessier-Lavigne M.
      iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.
      )
      Significant endogenous fluorescence loss (Circumvented with the development of newer fluorescence-preserving protocols)Less endogenous fluorescence loss
      General solvent-based protocolsSimple immersion
       Spalteholz (
      • Spalteholz W.
      Über das Durchsichtigmachen von Menchlichen und Tierichen Präparaten und Seine Theoretischen Bedingungen.
      )
       Sucrose (
      • Feng W.
      • Shi R.
      • Ma N.
      • Tuchina D.K.
      • Tuchin V.V.
      • Zhu D.
      Skin optical clearing potential of disaccharides.
      )
       BABB (
      • Dodt H.U.
      • Leischner U.
      • Schierloh A.
      • Jährling N.
      • Mauch C.P.
      • Deininger K.
      • et al.
      Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.
      ),
       FocusClear (proprietary formula)
       3DISCO (
      • Ertürk A.
      • Mauch C.P.
      • Hellal F.
      • Förstner F.
      • Keck T.
      • Becker K.
      • et al.
      Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.
      ,
      • Ertürk A.
      • Becker K.
      • Jährling N.
      • Mauch C.P.
      • Hojer C.D.
      • Egen J.G.
      • et al.
      Three-dimensional imaging of solvent-cleared organs using 3DISCO.
      ), iDISCO (
      • Renier N.
      • Wu Z.
      • Simon D.J.
      • Yang J.
      • Ariel P.
      • Tessier-Lavigne M.
      iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.
      ), vDISCO (
      • Cai R.
      • Pan C.
      • Ghasemigharagoz A.
      • Todorov M.I.
      • Förstera B.
      • Zhao S.
      • et al.
      Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.
      )
       RapiClear (proprietary formula)
       Ethanol/Ethyl cinnamate (
      • Klingberg A.
      • Hasenberg A.
      • Ludwig-Portugall I.
      • Medyukhina A.
      • Männ L.
      • Brenzel A.
      • et al.
      Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using Lightsheet microscopy.
      )
       ClearT and ClearT2 (
      • Kuwajima T.
      • Sitko A.A.
      • Bhansali P.
      • Jurgens C.
      • Guido W.
      • Mason C.
      ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.
      )
      Fluorescence preservation protocols SeeDB (
      • Ke M.T.
      • Fujimoto S.
      • Imai T.
      SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.
      ); SeeDB2 (
      • Ke M.T.
      • Nakai Y.
      • Fujimoto S.
      • Takayama R.
      • Yoshida S.
      • Kitajima T.S.
      • et al.
      Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent.
      )
       FluoClearBABB (
      • Schwarz M.K.
      • Scherbarth A.
      • Sprengel R.
      • Engelhardt J.
      • Theer P.
      • Giese G.
      Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
      )
       FRUIT (
      • Hou B.
      • Zhang D.
      • Zhao S.
      • Wei M.
      • Yang Z.
      • Wang S.
      • et al.
      Scalable and DiI-compatible optical clearance of the mammalian brain.
      )
       uDISCO (
      • Pan C.
      • Cai R.
      • Quacquarelli F.P.
      • Ghasemigharagoz A.
      • Lourbopoulos A.
      • Matryba P.
      • et al.
      Shrinkage-mediated imaging of entire organs and organisms using uDISCO.
      ), FDISCO (
      • Qi Y.
      • Yu T.
      • Xu J.
      • Wan P.
      • Ma Y.
      • Zhu J.
      • et al.
      FDISCO: advanced solvent-based clearing method for imaging whole organs.
      )
       TDE (
      • Aoyagi Y.
      • Kawakami R.
      • Osanai H.
      • Hibi T.
      • Nemoto T.
      A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.
      ,
      • Costantini I.
      • Ghobril J.P.
      • Di Giovanna A.P.
      • Allegra Mascaro A.L.
      • Silvestri L.
      • Müllenbroich M.C.
      • et al.
      A versatile clearing agent for multi-modal brain imaging.
      )
       PEGASOS (
      • Jing D.
      • Zhang S.
      • Luo W.
      • Gao X.
      • Men Y.
      • Ma C.
      • et al.
      Tissue clearing of both hard and soft tissue organs with the PEGASOS method.
      )
       Ce3D (
      • Li W.
      • Germain R.N.
      • Gerner M.Y.
      Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D).
      ,
      • Li W.
      • Germain R.N.
      • Gerner M.Y.
      High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging.
      )
      Tissue-specific protocols RTF (
      • Yu T.
      • Zhu J.
      • Li Y.
      • Ma Y.
      • Wang J.
      • Cheng X.
      • et al.
      RTF: a rapid and versatile tissue optical clearing method.
      )
       Adipo-clear (adapted from iDISCO) (
      • Chi J.
      • Wu Z.
      • Choi C.H.J.
      • Nguyen L.
      • Tegegne S.
      • Ackerman S.E.
      • et al.
      Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density.
      )
       FOCM (
      • Zhu X.
      • Huang L.
      • Zheng Y.
      • Song Y.
      • Xu Q.
      • Wang J.
      • et al.
      Ultrafast optical clearing method for three-dimensional imaging with cellular resolution.
      )
      Hyperhydration
       Scale and ScaleS (
      • Hama H.
      • Hioki H.
      • Namiki K.
      • Hoshida T.
      • Kurokawa H.
      • Ishidate F.
      • et al.
      ScaleS: an optical clearing palette for biological imaging.
      ,
      • Hama H.
      • Kurokawa H.
      • Kawano H.
      • Ando R.
      • Shimogori T.
      • Noda H.
      • et al.
      Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.
      )
       CUBIC (
      • Susaki E.A.
      • Tainaka K.
      • Perrin D.
      • Kishino F.
      • Tawara T.
      • Watanabe T.M.
      • et al.
      Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.
      ,
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      ); CUBIC-L and CUBIC-R (
      • Tainaka K.
      • Murakami T.C.
      • Susaki E.A.
      • Shimizu C.
      • Saito R.
      • Takahashi K.
      • et al.
      Chemical landscape for tissue clearing based on hydrophilic reagents.
      )
       UbasM (
      • Chen L.
      • Li G.
      • Li Y.
      • Li Y.
      • Zhu H.
      • Tang L.
      • et al.
      UbasM: an effective balanced optical clearing method for intact biomedical imaging.
      )
       FUnGI (
      • Rios A.C.
      • Capaldo B.D.
      • Vaillant F.
      • Pal B.
      • van Ineveld R.
      • Dawson C.A.
      • et al.
      Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging.
      )
      Hydrogel embedding
       CLARITY (
      • Chung K.
      • Wallace J.
      • Kim S.Y.
      • Kalyanasundaram S.
      • Andalman A.S.
      • Davidson T.J.
      • et al.
      Structural and molecular interrogation of intact biological systems.
      ,
      • Tomer R.
      • Ye L.
      • Hsueh B.
      • Deisseroth K.
      Advanced CLARITY for rapid and high-resolution imaging of intact tissues.
      )
       PACT-PARS (
      • Treweek J.B.
      • Chan K.Y.
      • Flytzanis N.C.
      • Yang B.
      • Deverman B.E.
      • Greenbaum A.
      • et al.
      Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.
      ,
      • Yang B.
      • Treweek J.B.
      • Kulkarni R.P.
      • Deverman B.E.
      • Chen C.K.
      • Lubeck E.
      • et al.
      Single-cell phenotyping within transparent intact tissue through whole-body clearing.
      )
       ACT- PRESTO (
      • Lee E.
      • Choi J.
      • Jo Y.
      • Kim J.Y.
      • Jang Y.J.
      • Lee H.M.
      • et al.
      ACT:PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.
      )
       SWITCH (
      • Murray E.
      • Cho J.H.
      • Goodwin D.
      • Ku T.
      • Swaney J.
      • Kim S.Y.
      • et al.
      Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.
      )
       MAP (
      • Ku T.
      • Swaney J.
      • Park J.Y.
      • Albanese A.
      • Murray E.
      • Cho J.H.
      • et al.
      Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.
      )
       Expansion Microscopy (
      • Chen F.
      • Tillberg P.W.
      • Boyden E.S.
      Optical imaging. Expansion microscopy.
      ,
      • Chozinski T.J.
      • Halpern A.R.
      • Okawa H.
      • Kim H.J.
      • Tremel G.J.
      • Wong R.O.
      • et al.
      Expansion microscopy with conventional antibodies and fluorescent proteins.
      )
      Protein-crosslinking
       SHIELD (
      • Park Y.G.
      • Sohn C.H.
      • Chen R.
      • McCue M.
      • Yun D.H.
      • Drummond G.T.
      • et al.
      Protection of tissue physicochemical properties using polyfunctional crosslinkers.
      )
      Tissue-specific protocols
       Bone CLARITY (adapted from CLARITY) (
      • Greenbaum A.
      • Chan K.Y.
      • Dobreva T.
      • Brown D.
      • Balani D.H.
      • Boyce R.
      • et al.
      Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow.
      )
       PEA-CLARITY (adapted from CLARITY for clearing plants) (
      • Palmer W.M.
      • Martin A.P.
      • Flynn J.R.
      • Reed S.L.
      • White R.G.
      • Furbank R.T.
      • et al.
      PEA-CLARITY: 3D molecular imaging of whole plant organs.
      )
       Organoid specific fructose-glycerol clearing (
      • Dekkers J.F.
      • Alieva M.
      • Wellens L.M.
      • Ariese H.C.R.
      • Jamieson P.R.
      • Vonk A.M.
      • et al.
      High-resolution 3D imaging of fixed and cleared organoids.
      )
      Abbreviations: 3DISCO, 3D imaging of solvent-cleared organs; ACT-PRESTO, Active Clarity Technique-Pressure Related Efficient and Stable Transfer of macromolecules into Organs; BABB, Benzyl Alcohol and Benzyl Benzoate; Ce3D, Clearing-Enhanced 3D; CLARITY, Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel; CUBIC, Clear, Unobstructed Brain or body Imaging Cocktails and computational analysis; FOCM, ultraFast Optical Clearing Method; FUnGI, Fructose, Urea, and Glycerol for Imaging; MAP, Magnified Analysis of the Proteome; PACT-PARS, PAssive CLARITY Technique-Perfusion-assisted Agent Release in Situ; PEGASOS, polyethylene glycol (PEG)-Associated Solvent System; RI, refractive index; RTF, Rapid clearing method based on Triethanolamine and Formamide; SeeDB, See Deep Brain; SHIELD, stabilization to harsh conditions through intramolecular epoxide linkages to prevent degradation; SWITCH, System-Wide control of Interaction Time and kinetics of CHemicals; TDE, 2,2′-thiodiethanol; UbasM, Urea-based amino-sugar Mixture.
      Disruption of collagen fibril structure is key to optically clearing skin because a large part of the skin extracellular matrix is composed of collagen (
      • Yeh A.T.
      • Choi B.
      • Nelson J.S.
      • Tromberg B.J.
      Reversible dissociation of collagen in tissues.
      ) (Figure 2a–c). Alcohols with hydroxyl groups such as glycerol are able to dissociate collagen and successfully clear skin; molecular simulations have revealed that alcohols with hydroxyl groups further apart on the carbon backbone are more effective for dissociating collagen fiber structure than those with hydroxyl groups next to each other (
      • Hirshburg J.M.
      • Ravikumar K.M.
      • Hwang W.
      • Yeh A.T.
      Molecular basis for optical clearing of collagenous tissues.
      ,
      • Zhu D.
      • Larin K.V.
      • Luo Q.
      • Tuchin V.V.
      Recent progress in tissue optical clearing.
      ). However, in formaldehyde-fixed tissues, some optical clearing occurs in the presence of glycerol despite the lack of collagen fiber dissociation (
      • Yeh A.T.
      • Choi B.
      • Nelson J.S.
      • Tromberg B.J.
      Reversible dissociation of collagen in tissues.
      ). This is likely to be because of RI matching caused by the hyperosmotic nature of glycerol, causing tissue dehydration and the influx of glycerol into the tissue (
      • Vargas G.
      • Chan E.K.
      • Barton J.K.
      • Rylander 3rd, H.G.
      • Welch A.J.
      Use of an agent to reduce scattering in skin.
      ). This optical clearing ability is enhanced at higher incubation temperatures probably owing to more rapid permeation of glycerol into the skin (
      • Deng Z.
      • Liu C.
      • Tao W.
      • Zhu D.
      Improvement of skin optical clearing efficacy by topical treatment of glycerol at different temperatures.
      ). RI matching for skin using solutions closer to the collagen RI (1.43, fully hydrated; 1.53, dry), such as FocusClear (
      • Song E.
      • Ahn Y.
      • Ahn J.
      • Ahn S.
      • Kim C.
      • Choi S.
      • et al.
      Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin.
      ), benzyl alcohol benzoate (
      • Abadie S.
      • Jardet C.
      • Colombelli J.
      • Chaput B.
      • David A.
      • Grolleau J.L.
      • et al.
      3D imaging of cleared human skin biopsies using light-sheet microscopy: a new way to visualize in-depth skin structure.
      ), 3DISCO, or uDISCO, also worked better for skin clearing (
      • Xu J.
      • Ma Y.
      • Yu T.
      • Zhu D.
      Quantitative assessment of optical clearing methods in various intact mouse organs.
      ).
      Figure thumbnail gr2
      Figure 2Ex vivo and in vivo examples of skin optical clearing and imaging. (a) Photomicrograph of uncleared and optically cleared human skin (reprinted from
      • Tan Y.
      • Ng W.J.
      • Lee S.Z.X.
      • Lee B.T.K.
      • Nattkemper L.A.
      • Yosipovitch G.
      • et al.
      3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
      ) (b) Bright-field images of skin from BALB/c-nu/nu mice treated with CUBIC reagents and PBS using the 10-day clearing protocol (reprinted from
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      with permission from Elsevier). (c) Reversible effect of glycerol on collagen fibrils in the rodent dermis by imaging with the multiphoton microscope. Bar = 8 μm. Typical collagen fibril organization within the dermis before glycerol application (left). Collagen fibril dissociation after glycerol application (middle). Reorganization of collagen fibrils in the dermis after rehydration with PBS (right) (reprinted from
      • Yeh A.T.
      • Choi B.
      • Nelson J.S.
      • Tromberg B.J.
      Reversible dissociation of collagen in tissues.
      ). (d) In vivo imaging of dermal microvessels in rat skin using laser speckle temporal contrast microscopy. Typical visual photos (top row) and corresponding laser speckle temporal contrast images (bottom row) of in vivo rat skin before and after clearing in comparison with application of saline. Arrows 1–4 indicate typical blood vessels (bottom row) (reprinted from
      • Wang J.
      • Shi R.
      • Zhu D.
      Switchable skin window induced by optical clearing method for dermal blood flow imaging.
      with permission from SPIE). (e) 3D image of immunostained healthy skin. Epidermal nerves, green; dermal nerves, blue; traced nerves, yellow. Bar = 20 μm. Immunostained epidermal nerve signal was used for manual nerve tracing for analysis of the epidermal nerves (reprinted from
      • Tan Y.
      • Ng W.J.
      • Lee S.Z.X.
      • Lee B.T.K.
      • Nattkemper L.A.
      • Yosipovitch G.
      • et al.
      3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
      ]). (f) 3D images of a reporter mouse skin expressing green fluorescence protein (green) in all tissues except erythrocytes and hair with nuclear stain (red). Bar = 2 mm. Enlarged images indicated by a white box on z plane images are shown. Bar = 1 mm (reprinted from
      • Tainaka K.
      • Kubota S.I.
      • Suyama T.Q.
      • Susaki E.A.
      • Perrin D.
      • Ukai-Tadenuma M.
      • et al.
      Whole-body imaging with single-cell resolution by tissue decolorization.
      with permission from Elsevier). 3D, three-dimensional.
      Part of the challenge in optically clearing skin is in clearing the pigmented epidermis. If the researcher’s focus is on studying structures present within the epidermis, quality data can still be obtained by imaging thin cross-sections (100–300 μm) of skin samples. Where possible, skin samples of lower melanin content can also be selected to improve the image quality. If the focus is on structures such as sebaceous glands or hair follicles present within the dermis, thick sections of the dermis can be relatively easily cleared and imaged (
      • Foster D.S.
      • Nguyen A.T.
      • Chinta M.
      • Salhotra A.
      • Jones R.E.
      • Mascharak S.
      • et al.
      A clearing technique to enhance endogenous fluorophores in skin and soft tissue.
      ).
      As the skin is an easily accessible organ, in vivo clearing of the skin is an attractive prospect to make noninvasive observations. However, topical applications of optical clearing agents are rendered ineffective owing to the stratum corneum functioning as a barrier, preventing optical clearing agents from reaching the dermis (
      • Zhu D.
      • Larin K.V.
      • Luo Q.
      • Tuchin V.V.
      Recent progress in tissue optical clearing.
      ). To overcome this, physical methods such as tape stripping to remove the stratum corneum or chemical penetration enhancers such as the application of thiazone (
      • Zhu D.
      • Larin K.V.
      • Luo Q.
      • Tuchin V.V.
      Recent progress in tissue optical clearing.
      ) or hyaluronic acid (
      • Liopo A.
      • Su R.
      • Tsyboulski D.A.
      • Oraevsky A.A.
      Optical clearing of skin enhanced with hyaluronic acid for increased contrast of optoacoustic imaging.
      ) could potentially improve optical clearing agent permeation into the dermis. Alternatively, a dermal injection can be used, but the concentration of optical clearing agents is still an issue; too low and skin will not be sufficiently cleared, and too high and problems such as necrosis (

      Mao Z, Han Z, Wen X, Luo Q, Zhu D. Influence of glycerol with different concentrations on skin optical clearing and morphological changes in vivo. Paper presented at: Proc. SPIE 7278, Photonics and Optoelectronics Meetings (POEM) 2008: Fiber Optic Communication and Sensors, 72781T. 24-27 November 2008; Wuhan, China.

      ) or blood vessel occlusions can occur (
      • Vargas G.
      • Readinger A.
      • Dozier S.S.
      • Welch A.J.
      Morphological changes in blood vessels produced by hyperosmotic agents and measured by optical coherence tomography.
      ). Further development of safe methods of delivering biosafe optical clearing agents into the dermis in vivo would enable visualizing dynamic processes such as dermal blood flow with greater clarity (Figure 2d).
      For a more comprehensive overview of the different clearing methods, we would like to refer the reader to Table 2, which lists several excellent reviews on this topic.
      Table 2List of Optical Clearing Reviews and How Each Review Would Be Helpful for the Reader
      Review ReferenceHow the Review Is Helpful
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      Discusses physical principles of optical clearing; one of the first reviews providing a useful framework for classifying different optical clearing methods on the basis of the mechanism of tissue treatment.
      • Ariel P.
      A beginner’s guide to tissue clearing.
      Easy to read and an excellent overview for a beginner in tissue clearing.
      • Silvestri L.
      • Costantini I.
      • Sacconi L.
      • Pavone F.S.
      Clearing of fixed tissue: a review from a microscopist's perspective.
      Useful classification of optical clearing methods based on the application and microscope used; helpful checklist for the user to assess newly published clearing methods.
      • Susaki E.A.
      • Ueda H.R.
      Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.
      Excellent discussion of tissue-specific light scatterers and absorbers and how specific chemicals improve clearing; good overview of cell labeling strategies and discussion of microscopy setups and image informatics pipeline for dealing with big data.
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      Excellent mechanistic discussion of the chemical reactions involved in the 3D volumetric imaging workflow (fixation, permeabilization, and clearing); helpful summary tables of different optical clearing protocols.
      • Yu T.
      • Qi Y.
      • Gong H.
      • Luo Q.
      • Zhu D.
      Optical clearing for multiscale biological tissues.
      Classifies optical clearing methods based on different tissue types—tissue blocks; embryos and/or neonatal samples; intact-adult samples; whole-body clearing.
      • Tuchin V.V.
      Tissue optics and photonics: biological tissue structures.
      ,
      • Tuchin V.V.
      Tissue optics and photonics: light-tissue interaction.
      Overview of the composition of different biological tissues and detailed mathematical description of phenomena of light reflection, refraction, absorption, and scattering.
      3D, three-dimensional.

      3D volumetric imaging

      After clearing, various microscope modalities can be used for ex vivo volumetric imaging depending on the resolution required, including confocal microscopy, two-photon microscopy, or light-sheet fluorescence microscopy. In the case of OTC at cellular to subcellular imaging, the objective lens chosen should have a significant optical resolution and a long working distance to image deep into tissues (
      • Susaki E.A.
      • Ueda H.R.
      Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.
      ). Furthermore, in some cases, a specialized objective lens may be used, for instance, water-immersion or oil-immersion objective lens increases the resolution of the microscope compared with air owing to decreased refraction by the spherical lens. Alternatively, an index-optimized objective lens would be more suitable for higher resolution or deeper imaging (
      • Marx V.
      Microscopy: seeing through tissue.
      ).
      Confocal and two-photon microscopes are point-scanning imaging techniques where only one point in the sample is illuminated at a time and an image is constructed point-by-point. Confocal microscopy uses a pinhole to filter out-of-focus light, whereas two-photon microscopy uses the fact that fluorescence excitation only occurs when two photons are absorbed simultaneously, which most likely occurs at the point of highest light intensity. As both methods are point-scanning microscopes, it takes a significant amount of time to image a 3D stack of the field of interest (Table 3). In addition, a significant amount of photobleaching occurs when imaging with the confocal microscope because the entire 3D volume is illuminated when scanning through different depths. Despite these drawbacks, both confocal and two-photon microscopes can give images of relatively high-resolution if the right objectives are used.
      Table 3Comparison between Confocal and Light-Sheet Microscope Imaging Speeds for a Field of View of a Similar Size
      ConfocalLight Sheet
      Field of view (μm)1,272 × 1,2721,663 × 1,404
      Aspect ratio (pixel)800 × 8002,560 × 2,160
      Duration/frame (s)2.560.2
      Duration/stack (s)1,280100
      Duration/stack (min)21.331.67
      Abbreviation: sCMOS, scientific complementary metal-oxide-semiconductor.
      Calculations are based on realistic imaging parameters with the following assumptions: Olympus FV1000 Confocal microscope:
      Imaging with a ×10 objective with a pixel dwell time of 4 μs/pixel for a stack of 500 Z slices. The time taken for each frame is calculated by the total number of pixels per frame × pixel dwell time.
      La Vision Biotec Ultramicroscope I (Light-sheet microscope): Imaging with an sCMOS camera chip size of 2,560 × 2,160 pixels for a stack of 500 Z slices; ×10 magnification using the Olympus Zoombody with a pixel size of 0.65 μm. Exposure time for each frame is 200 ms.
      In light-sheet fluorescence microscopy, a thin slice of the sample is illuminated from the side, perpendicular to the angle of observation, producing a high-resolution image of the entire plane at each exposure. The thin light sheet can be generated either by a cylindrical lens (selective plane illumination microscopy) or by laser scanning (digital scanned laser light-sheet fluorescence microscopy) (
      • Keller P.J.
      • Ahrens M.B.
      Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy.
      ). Optically sectioned images are obtained in succession, which are put together and visualized with imaging software to form a 3D image. This is a more rapid manner of data acquisition than point-scanning systems because the entire focal plane is illuminated for a fixed exposure time. Light-sheet fluorescence microscopy is advantageous for imaging whole tissues like the brain, with faster imaging speed (hours to days rather than days to weeks) and minimized photobleaching and phototoxicity compared with confocal and two-photon microscopy (Table 2). For skin biopsies, which are generally much smaller in size, light-sheet fluorescence microscopy provides the ability to complete imaging multiple samples within a day. Unfortunately, there is still a trade-off between resolution and the volume of the tissue that can be imaged. The thinnest light sheet (5 to <1 μm) provides high-resolution images but only allows imaging of tissue a few hundreds of μm thick, whereas thicker light sheets allow for deeper imaging with a corresponding loss in resolution (
      • Richardson D.S.
      • Lichtman J.W.
      Clarifying tissue clearing.
      ).
      Another emerging imaging technique that is useful for obtaining high-resolution images from thicker tissues is serial two-photon tomography. This involves scanning a 3D volume of the cleared tissue using a two-photon microscope, followed by mechanical sectioning with an automated microtome to expose the next section of the tissue for imaging (
      • Costantini I.
      • Ghobril J.P.
      • Di Giovanna A.P.
      • Allegra Mascaro A.L.
      • Silvestri L.
      • Müllenbroich M.C.
      • et al.
      A versatile clearing agent for multi-modal brain imaging.
      ,
      • Ragan T.
      • Kadiri L.R.
      • Venkataraju K.U.
      • Bahlmann K.
      • Sutin J.
      • Taranda J.
      • et al.
      Serial two-photon tomography for automated ex vivo mouse brain imaging.
      ). This combinational approach of imaging and sectioning enables high-resolution and deep-tissue imaging with the two-photon microscope and would be particularly useful when imaging nerves or blood vessels at high-resolution in the skin. The downside of this technique is the requirement for a specialized microscope setup, which may not be readily available in most microscope core facilities.

      Applications of optical clearing and volumetric imaging

      The combination of tissue optical clearing together with volumetric imaging makes it possible to obtain anatomically representative 3D views of tissues and organs, which was previously not possible with 2D sections. This makes it possible to visualize structures that span the whole body such as nerves and blood vessels at the macro level or at cellular resolution, thereby creating whole organ atlases of healthy and diseased tissues.
      The initial driving force for developing optical clearing protocols was to image the whole brain to understand neuronal connections, leading to the development of optical clearing protocols such as the suite of DISCO, CLARITY, CUBIC, and Scale protocols, which are customized for brain clearing and imaging. The general interest for clearing other tissues has expanded the application of these protocols to other organ types such as spleen, intestine, lung, liver, muscle, lymph node, and skin to name a few, as well as to the development of specialized tissue clearing protocols such as Bone CLARITY (
      • Greenbaum A.
      • Chan K.Y.
      • Dobreva T.
      • Brown D.
      • Balani D.H.
      • Boyce R.
      • et al.
      Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow.
      ) and Adipo-Clear (
      • Chi J.
      • Wu Z.
      • Choi C.H.J.
      • Nguyen L.
      • Tegegne S.
      • Ackerman S.E.
      • et al.
      Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density.
      ). In recent years, optical clearing has been broadly applied to a variety of species and organs, ranging from in vitro cultured organoids (
      • Dekkers J.F.
      • Alieva M.
      • Wellens L.M.
      • Ariese H.C.R.
      • Jamieson P.R.
      • Vonk A.M.
      • et al.
      High-resolution 3D imaging of fixed and cleared organoids.
      ) to human and marmoset brains; mouse embryos (
      • Tainaka K.
      • Kuno A.
      • Kubota S.I.
      • Murakami T.
      • Ueda H.R.
      Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
      ) to human embryos (
      • Belle M.
      • Godefroy D.
      • Couly G.
      • Malone S.A.
      • Collier F.
      • Giacobini P.
      • et al.
      Tridimensional visualization and analysis of early human development.
      ); pill bugs (
      • Konno A.
      • Okazaki S.
      Aqueous-based tissue clearing in crustaceans.
      ) to mosquitoes (
      • Mori T.
      • Hirai M.
      • Mita T.
      See-through observation of malaria parasite behaviors in the mosquito vector.
      ).
      In the skin, ex vivo OTC has been applied for studying hair follicles of the mouse pinna (
      • Song E.
      • Ahn Y.
      • Ahn J.
      • Ahn S.
      • Kim C.
      • Choi S.
      • et al.
      Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin.
      )—dermal architecture of vasculature and adipocytes and the expansion of keratinocytes during wound healing of mouse dorsal skin. Cleared human skin was imaged to reveal auto-fluorescent sweat glands, sebaceous glands, hair follicles, and the cutaneous plexus (
      • Foster D.S.
      • Nguyen A.T.
      • Chinta M.
      • Salhotra A.
      • Jones R.E.
      • Mascharak S.
      • et al.
      A clearing technique to enhance endogenous fluorophores in skin and soft tissue.
      ); epidermal hyperplasia in psoriatic skin (
      • Abadie S.
      • Jardet C.
      • Colombelli J.
      • Chaput B.
      • David A.
      • Grolleau J.L.
      • et al.
      3D imaging of cleared human skin biopsies using light-sheet microscopy: a new way to visualize in-depth skin structure.
      ); and epidermal nerve atrophy in pruritic skin such as atopic dermatitis and psoriasis (
      • Tan Y.
      • Ng W.J.
      • Lee S.Z.X.
      • Lee B.T.K.
      • Nattkemper L.A.
      • Yosipovitch G.
      • et al.
      3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
      ) (Figure 2e).
      In vivo, OTC has been applied to the skin for visualizing blood flow in the skin (
      • Vargas G.
      • Readinger A.
      • Dozier S.S.
      • Welch A.J.
      Morphological changes in blood vessels produced by hyperosmotic agents and measured by optical coherence tomography.
      ,
      • Wang J.
      • Shi R.
      • Zhang Y.
      • Zhu D.
      Ear skin optical clearing for improving blood flow imaging/Optisches Clearing der Ohrhaut zur verbesserten Bildgebung des Blutflusses.
      ,
      • Wang J.
      • Shi R.
      • Zhu D.
      Switchable skin window induced by optical clearing method for dermal blood flow imaging.
      ,
      • Zhu D.
      • Wang J.
      • Zhi Z.
      • Wen X.
      • Luo Q.
      Imaging dermal blood flow through the intact rat skin with an optical clearing method.
      ) and cutaneous vascular permeability in mice suffering from diabetes (
      • Feng W.
      • Zhang C.
      • Yu T.
      • Zhu D.
      Quantitative evaluation of skin disorders in type 1 diabetic mice by in vivo optical imaging.
      ) and for improving the imaging of diseased skin in a variety of skin diseases such as hemangioma and epidermoid cyst (
      • Shan H.
      • Liang Y.
      • Wang J.
      • Li Y.
      Study on application of optical clearing technique in skin diseases.
      ) as well as increasing laser penetration into the dermis during tattoo removal (
      • Liu C.
      • Shi R.
      • Chen M.
      • Zhu D.
      Quantitative evaluation of enhanced laser tattoo removal by skin optical clearing.
      ).
      The 3D volumetric imaging provides crucial spatial information in both ex vivo and in vivo studies, especially of 3D structures in the skin, which may not be accurately represented by traditional 2D histology methods (Figure 2f; Supplementary Movie S1). One key limitation of 3D volumetric imaging is the length of time required for tissue preparation, which often requires up to 1 or 2 weeks if immunostaining is involved. Depending on the optical clearing method chosen, the process can also be laborious, which makes it harder to achieve high sample throughput.
      As further technological developments are made in 3D volumetric imaging, we envision that this tool will enable 3D spatial mapping of tissue-resident cells and structures within normal skin such as nerves, vasculature, hair follicles, and sebaceous glands. Generation of such a 3D healthy skin atlas would provide a useful point of reference for the study of skin diseases, especially to delineate the spatial localization of infiltrating immune cells in inflammatory skin diseases such as atopic dermatitis, psoriasis, or pressure ulcers (
      • Goh C.C.
      • Evrard M.
      • Chong S.Z.
      • Tan Y.
      • Tan L.L.
      • Teng K.W.W.
      • et al.
      The impact of ischemia-reperfusion injuries on skin resident murine dendritic cells.
      ). The generation of 3D skin atlases would also serve as a useful reference in the application of deep learning techniques to detect pathological changes within whole-skin biopsies for the next generation of derma(histo)pathological research.

      Conflict of Interest

      The authors state no conflicts of interest.

      Multiple Choice Questions

      • 1.
        What are the benefits of three-dimensional (3D) imaging?
        • A.
          Accurate representation of 3D biological structures
        • B.
          Less time-consuming and tedious than two-dimensional imaging with 3D reconstruction as it does not involve volumetric reconstruction
        • C.
          Avoids sectioning artifacts
        • D.
          All of the above
      • 2.
        In general, the tissue is first harvested before going through optical tissue clearing and 3D imaging of the tissue. What is the sequence of steps of optical tissue clearing for solvent-based methods?
        • A.
          Permeabilization, Fixation, Immunostaining, Clearing
        • B.
          Fixation, Immunostaining, Permeabilization, Clearing
        • C.
          Fixation, Permeabilization, Immunostaining, Clearing
        • D.
          Fixation, Permeabilization, Clearing, Immunostaining
      • 3.
        Which statement below is FALSE?
        • A.
          There are multiple organic and aqueous optical tissue clearing protocols
        • B.
          Aqueous-based methods cannot preserve fluorescent protein emission
        • C.
          Organic-based methods confer better transparency than aqueous-based methods
        • D.
          Aqueous-based methods work better for softer tissues.
      • 4.
        Which statement below is FALSE about light-sheet fluorescent microscopy (LSFM) as compared with confocal and two-photon microscopy?
        • A.
          LSFM has a faster imaging speed
        • B.
          LSFM minimizes photobleaching and phototoxicity
        • C.
          LSFM uses a pinhole to eliminate out-of-focus light
        • D.
          There is a trade-off between resolution and the volume of the tissue that can be imaged using LSFM
      • 5.
        Which of the following CANNOT be studied using optical tissue clearing and 3D volumetric imaging?
        • A.
          3D reconstruction of tissue microstructures and vasculature
        • B.
          Pathological conditions in the tissue environment
        • C.
          Skin as the stratum corneum that prevents optical clearing agents from reaching the dermis
        • D.
          None of the above

      Acknowledgments

      We apologize to those in the field whose work could not be included because of space constraints. This work is supported by the A∗STAR-NHG-NTU skin research grant (SRG/14019) and the National Medical Research Council grant (NMRC/CSA-INV/0023/2017). This research was funded by Singapore Immunology Network core funding, Agency for Science, Technology and Research , Singapore. LGN is supported by Singapore Immunology Network core funding. Clinically related correspondence and queries can be directed to HLT ( [email protected] ).

      Author Contributions

      Conceptualization; YT, HLT, LGN; Funding Acquisition: HLT, LGN; Resources, HLT, LGN; Supervision; HLT, LGN; Visualization; YT, YZ; Writing - Original Draft Preparation; YT, CPLC; Writing - Review and Editing; YT, YZ, HLT, LGN

      Detailed Answers

      • 1.
        What are the benefits of three-dimensional (3D) imaging?
      • CORRECT ANSWER: D. All of the above.
      • 3D imaging better represents 3D biological structures than two-dimensional sections. It does not require intensive volumetric reconstruction of 3D volumes from two-dimensional sections and avoids sectioning artifacts associated with two-dimensional sections.
      • 2.
        In general, the tissue is first harvested before going through optical tissue clearing and 3D imaging of the tissue. What is the sequence of steps of optical tissue clearing for solvent-based methods?
      • CORRECT ANSWER: C. Fixation, Permeabilization, Immunostaining, Clearing
      • After tissue harvest, chemical fixation is required for tissue preservation; then is needed permeabilization to allow antibodies and optical clearing agents to diffuse in, followed by immunostaining of structures of interest and optical clearing.
      • 3.
        Which statement below is FALSE?
      • CORRECT ANSWER: B. Aqueous-based methods cannot preserve fluorescent protein emission
      • One of the motivations for developing aqueous-based clearing methods is to preserve fluorescent protein emission, which is quenched by organic solvent–based methods.
      • 4.
        Which statement below is FALSE about light-sheet fluorescent microscopy as compared with confocal and two-photon microscopy?
      • CORRECT ANSWER: C. Confocal microscopy uses a pinhole to eliminate out-of-focus light
      • Confocal microscopy uses a pinhole to eliminate out-of-focus light to reduce blurred background, whereas light-sheet fluorescent microscopy uses a light sheet to illuminate a thin slice of
      • the sample from the side perpendicular to the angle of observation to produce an
      • image of the entire plane at each exposure.
      • 5.
        Which of the following CANNOT be studied using optical tissue clearing and 3D volumetric imaging?
      • CORRECT ANSWER: D. None of the above
      • Skin has also been successfully cleared and imaged in recent years, although it is more technically challenging. This has allowed for 3D imaging of blood vessels and nerves of the skin.

      Supplementary Material

      References

        • Abadie S.
        • Jardet C.
        • Colombelli J.
        • Chaput B.
        • David A.
        • Grolleau J.L.
        • et al.
        3D imaging of cleared human skin biopsies using light-sheet microscopy: a new way to visualize in-depth skin structure.
        Skin Res Technol. 2018; 24: 294-303
        • Aoyagi Y.
        • Kawakami R.
        • Osanai H.
        • Hibi T.
        • Nemoto T.
        A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.
        PLoS One. 2015; 10e0116280
        • Ariel P.
        A beginner’s guide to tissue clearing.
        Int J Biochem Cell Biol. 2017; 84: 35-39
        • Azaripour A.
        • Lagerweij T.
        • Scharfbillig C.
        • Jadczak A.E.
        • Willershausen B.
        • Van Noorden C.J.
        A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.
        Prog Histochem Cytochem. 2016; 51: 9-23
        • Belle M.
        • Godefroy D.
        • Couly G.
        • Malone S.A.
        • Collier F.
        • Giacobini P.
        • et al.
        Tridimensional visualization and analysis of early human development.
        Cell. 2017; 169: 161-173.e12
        • Cai R.
        • Pan C.
        • Ghasemigharagoz A.
        • Todorov M.I.
        • Förstera B.
        • Zhao S.
        • et al.
        Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections.
        Nat Neurosci. 2019; 22: 317-327
        • Chen F.
        • Tillberg P.W.
        • Boyden E.S.
        Optical imaging. Expansion microscopy.
        Science. 2015; 347: 543-548
        • Chen L.
        • Li G.
        • Li Y.
        • Li Y.
        • Zhu H.
        • Tang L.
        • et al.
        UbasM: an effective balanced optical clearing method for intact biomedical imaging.
        Sci Rep. 2017; 7: 12218
        • Chen Y.
        • Shen Q.
        • White S.L.
        • Gokmen-Polar Y.
        • Badve S.
        • Goodman L.J.
        Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues.
        Sci Rep. 2019; 9: 5624
        • Chi J.
        • Wu Z.
        • Choi C.H.J.
        • Nguyen L.
        • Tegegne S.
        • Ackerman S.E.
        • et al.
        Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density.
        Cell Metab. 2018; 27: 226-236.e3
        • Chozinski T.J.
        • Halpern A.R.
        • Okawa H.
        • Kim H.J.
        • Tremel G.J.
        • Wong R.O.
        • et al.
        Expansion microscopy with conventional antibodies and fluorescent proteins.
        Nat Methods. 2016; 13: 485-488
        • Chung K.
        • Wallace J.
        • Kim S.Y.
        • Kalyanasundaram S.
        • Andalman A.S.
        • Davidson T.J.
        • et al.
        Structural and molecular interrogation of intact biological systems.
        Nature. 2013; 497: 332-337
        • Costantini I.
        • Ghobril J.P.
        • Di Giovanna A.P.
        • Allegra Mascaro A.L.
        • Silvestri L.
        • Müllenbroich M.C.
        • et al.
        A versatile clearing agent for multi-modal brain imaging.
        Sci Rep. 2015; 5: 9808
        • Dekkers J.F.
        • Alieva M.
        • Wellens L.M.
        • Ariese H.C.R.
        • Jamieson P.R.
        • Vonk A.M.
        • et al.
        High-resolution 3D imaging of fixed and cleared organoids.
        Nat Protoc. 2019; 14: 1756-1771
        • Deng Z.
        • Liu C.
        • Tao W.
        • Zhu D.
        Improvement of skin optical clearing efficacy by topical treatment of glycerol at different temperatures.
        J Phys Conf Ser. 2011; 277012007
        • Dodt H.U.
        • Leischner U.
        • Schierloh A.
        • Jährling N.
        • Mauch C.P.
        • Deininger K.
        • et al.
        Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.
        Nat Methods. 2007; 4: 331-336
        • Ertürk A.
        • Becker K.
        • Jährling N.
        • Mauch C.P.
        • Hojer C.D.
        • Egen J.G.
        • et al.
        Three-dimensional imaging of solvent-cleared organs using 3DISCO.
        Nat Protoc. 2012; 7: 1983-1995
        • Ertürk A.
        • Mauch C.P.
        • Hellal F.
        • Förstner F.
        • Keck T.
        • Becker K.
        • et al.
        Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury.
        Nat Med. 2011; 18: 166-171
        • Feng W.
        • Shi R.
        • Ma N.
        • Tuchina D.K.
        • Tuchin V.V.
        • Zhu D.
        Skin optical clearing potential of disaccharides.
        J Biomed Opt. 2016; 21081207
        • Feng W.
        • Zhang C.
        • Yu T.
        • Zhu D.
        Quantitative evaluation of skin disorders in type 1 diabetic mice by in vivo optical imaging.
        Biomed Opt Express. 2019; 10: 2996-3008
        • Foster D.S.
        • Nguyen A.T.
        • Chinta M.
        • Salhotra A.
        • Jones R.E.
        • Mascharak S.
        • et al.
        A clearing technique to enhance endogenous fluorophores in skin and soft tissue.
        Sci Rep. 2019; 9: 15791
        • Goh C.C.
        • Evrard M.
        • Chong S.Z.
        • Tan Y.
        • Tan L.L.
        • Teng K.W.W.
        • et al.
        The impact of ischemia-reperfusion injuries on skin resident murine dendritic cells.
        Eur J Immunol. 2018; 48: 1014-1019
        • Greenbaum A.
        • Chan K.Y.
        • Dobreva T.
        • Brown D.
        • Balani D.H.
        • Boyce R.
        • et al.
        Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow.
        Sci Transl Med. 2017; 9: eaah6518
        • Hama H.
        • Hioki H.
        • Namiki K.
        • Hoshida T.
        • Kurokawa H.
        • Ishidate F.
        • et al.
        ScaleS: an optical clearing palette for biological imaging.
        Nat Neurosci. 2015; 18: 1518-1529
        • Hama H.
        • Kurokawa H.
        • Kawano H.
        • Ando R.
        • Shimogori T.
        • Noda H.
        • et al.
        Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.
        Nat Neurosci. 2011; 14: 1481-1488
        • Hirshburg J.M.
        • Ravikumar K.M.
        • Hwang W.
        • Yeh A.T.
        Molecular basis for optical clearing of collagenous tissues.
        J Biomed Opt. 2010; 15055002
        • Hou B.
        • Zhang D.
        • Zhao S.
        • Wei M.
        • Yang Z.
        • Wang S.
        • et al.
        Scalable and DiI-compatible optical clearance of the mammalian brain.
        Front Neuroanat. 2015; 9: 19
        • Jackson J.E.
        • Kelly B.
        • Petitt M.
        • Uchida T.
        • Wagner Jr., R.F.
        Predictive value of margins in diagnostic biopsies of nonmelanoma skin cancers.
        J Am Acad Dermatol. 2012; 67: 122-127
        • Jing D.
        • Zhang S.
        • Luo W.
        • Gao X.
        • Men Y.
        • Ma C.
        • et al.
        Tissue clearing of both hard and soft tissue organs with the PEGASOS method.
        Cell Res. 2018; 28: 803-818
        • Johnsen S.
        • Widder E.A.
        The physical basis of transparency in biological tissue: ultrastructure and the minimization of light scattering.
        J Theor Biol. 1999; 199: 181-198
        • Ke M.T.
        • Fujimoto S.
        • Imai T.
        SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction.
        Nat Neurosci. 2013; 16: 1154-1161
        • Ke M.T.
        • Nakai Y.
        • Fujimoto S.
        • Takayama R.
        • Yoshida S.
        • Kitajima T.S.
        • et al.
        Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent.
        Cell Rep. 2016; 14: 2718-2732
        • Keller P.J.
        • Ahrens M.B.
        Visualizing whole-brain activity and development at the single-cell level using light-sheet microscopy.
        Neuron. 2015; 85: 462-483
        • Kim S.Y.
        • Cho J.H.
        • Murray E.
        • Bakh N.
        • Choi H.
        • Ohn K.
        • et al.
        Stochastic electrotransport selectively enhances the transport of highly electromobile molecules.
        Proc Natl Acad Sci USA. 2015; 112: E6274-E6283
        • Klingberg A.
        • Hasenberg A.
        • Ludwig-Portugall I.
        • Medyukhina A.
        • Männ L.
        • Brenzel A.
        • et al.
        Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using Lightsheet microscopy.
        J Am Soc Nephrol. 2017; 28: 452-459
        • Konno A.
        • Okazaki S.
        Aqueous-based tissue clearing in crustaceans.
        Zool Lett. 2018; 4: 13
        • Ku T.
        • Swaney J.
        • Park J.Y.
        • Albanese A.
        • Murray E.
        • Cho J.H.
        • et al.
        Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.
        Nat Biotechnol. 2016; 34: 973-981
        • Kuwajima T.
        • Sitko A.A.
        • Bhansali P.
        • Jurgens C.
        • Guido W.
        • Mason C.
        ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue.
        Development. 2013; 140: 1364-1368
        • Lee E.
        • Choi J.
        • Jo Y.
        • Kim J.Y.
        • Jang Y.J.
        • Lee H.M.
        • et al.
        ACT:PRESTO: rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging.
        Sci Rep. 2016; 6: 18631
        • Li W.
        • Germain R.N.
        • Gerner M.Y.
        Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D).
        Proc Natl Acad Sci USA. 2017; 114: E7321-E7330
        • Li W.
        • Germain R.N.
        • Gerner M.Y.
        High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging.
        Nat Protoc. 2019; 14: 1708-1733
        • Liopo A.
        • Su R.
        • Tsyboulski D.A.
        • Oraevsky A.A.
        Optical clearing of skin enhanced with hyaluronic acid for increased contrast of optoacoustic imaging.
        J Biomed Opt. 2016; 21081208
        • Liu C.
        • Shi R.
        • Chen M.
        • Zhu D.
        Quantitative evaluation of enhanced laser tattoo removal by skin optical clearing.
        J Innov Opt Health Sci. 2015; 08: 1541007
      1. Mao Z, Han Z, Wen X, Luo Q, Zhu D. Influence of glycerol with different concentrations on skin optical clearing and morphological changes in vivo. Paper presented at: Proc. SPIE 7278, Photonics and Optoelectronics Meetings (POEM) 2008: Fiber Optic Communication and Sensors, 72781T. 24-27 November 2008; Wuhan, China.

        • Marx V.
        Microscopy: seeing through tissue.
        Nat Methods. 2014; 11: 1209-1214
        • Mori T.
        • Hirai M.
        • Mita T.
        See-through observation of malaria parasite behaviors in the mosquito vector.
        Sci Rep. 2019; 9: 1768
        • Murray E.
        • Cho J.H.
        • Goodwin D.
        • Ku T.
        • Swaney J.
        • Kim S.Y.
        • et al.
        Simple, scalable proteomic imaging for high-dimensional profiling of intact systems.
        Cell. 2015; 163: 1500-1514
        • Palmer W.M.
        • Martin A.P.
        • Flynn J.R.
        • Reed S.L.
        • White R.G.
        • Furbank R.T.
        • et al.
        PEA-CLARITY: 3D molecular imaging of whole plant organs.
        Sci Rep. 2015; 5: 13492
        • Pan C.
        • Cai R.
        • Quacquarelli F.P.
        • Ghasemigharagoz A.
        • Lourbopoulos A.
        • Matryba P.
        • et al.
        Shrinkage-mediated imaging of entire organs and organisms using uDISCO.
        Nat Methods. 2016; 13: 859-867
        • Park Y.G.
        • Sohn C.H.
        • Chen R.
        • McCue M.
        • Yun D.H.
        • Drummond G.T.
        • et al.
        Protection of tissue physicochemical properties using polyfunctional crosslinkers.
        Nat Biotechnol. 2018; 37: 73-83
        • Qi Y.
        • Yu T.
        • Xu J.
        • Wan P.
        • Ma Y.
        • Zhu J.
        • et al.
        FDISCO: advanced solvent-based clearing method for imaging whole organs.
        Sci Adv. 2019; 5: eaau8355
        • Ragan T.
        • Kadiri L.R.
        • Venkataraju K.U.
        • Bahlmann K.
        • Sutin J.
        • Taranda J.
        • et al.
        Serial two-photon tomography for automated ex vivo mouse brain imaging.
        Nat Methods. 2012; 9: 255-258
        • Renier N.
        • Wu Z.
        • Simon D.J.
        • Yang J.
        • Ariel P.
        • Tessier-Lavigne M.
        iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging.
        Cell. 2014; 159: 896-910
        • Richardson D.S.
        • Lichtman J.W.
        Clarifying tissue clearing.
        Cell. 2015; 162: 246-257
        • Rios A.C.
        • Capaldo B.D.
        • Vaillant F.
        • Pal B.
        • van Ineveld R.
        • Dawson C.A.
        • et al.
        Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging.
        Cancer Cell. 2019; 35: 953
        • Schwarz M.K.
        • Scherbarth A.
        • Sprengel R.
        • Engelhardt J.
        • Theer P.
        • Giese G.
        Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
        PLoS One. 2015; 10e0124650
        • Shan H.
        • Liang Y.
        • Wang J.
        • Li Y.
        Study on application of optical clearing technique in skin diseases.
        J Biomed Opt. 2012; 17: 115003
        • Silvestri L.
        • Costantini I.
        • Sacconi L.
        • Pavone F.S.
        Clearing of fixed tissue: a review from a microscopist's perspective.
        J Biomed Opt. 2016; 21081205
        • Song E.
        • Ahn Y.
        • Ahn J.
        • Ahn S.
        • Kim C.
        • Choi S.
        • et al.
        Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin.
        Opt Laser Technol. 2015; 73: 69-76
        • Spalteholz W.
        Über das Durchsichtigmachen von Menchlichen und Tierichen Präparaten und Seine Theoretischen Bedingungen.
        S Hirzel Verlag, Leipzig1914 ([in German])
        • Stefaniuk M.
        • Gualda E.J.
        • Pawlowska M.
        • Legutko D.
        • Matryba P.
        • Koza P.
        • et al.
        Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.
        Sci Rep. 2016; 6: 28209
        • Susaki E.A.
        • Tainaka K.
        • Perrin D.
        • Kishino F.
        • Tawara T.
        • Watanabe T.M.
        • et al.
        Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.
        Cell. 2014; 157: 726-739
        • Susaki E.A.
        • Tainaka K.
        • Perrin D.
        • Yukinaga H.
        • Kuno A.
        • Ueda H.R.
        Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.
        Nat Protoc. 2015; 10: 1709-1727
        • Susaki E.A.
        • Ueda H.R.
        Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals.
        Cell Chem Biol. 2016; 23: 137-157
        • Tainaka K.
        • Kubota S.I.
        • Suyama T.Q.
        • Susaki E.A.
        • Perrin D.
        • Ukai-Tadenuma M.
        • et al.
        Whole-body imaging with single-cell resolution by tissue decolorization.
        Cell. 2014; 159: 911-924
        • Tainaka K.
        • Kuno A.
        • Kubota S.I.
        • Murakami T.
        • Ueda H.R.
        Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.
        Annu Rev Cell Dev Biol. 2016; 32: 713-741
        • Tainaka K.
        • Murakami T.C.
        • Susaki E.A.
        • Shimizu C.
        • Saito R.
        • Takahashi K.
        • et al.
        Chemical landscape for tissue clearing based on hydrophilic reagents.
        Cell Rep. 2018; 24: 2196-2210.e9
        • Tan Y.
        • Ng W.J.
        • Lee S.Z.X.
        • Lee B.T.K.
        • Nattkemper L.A.
        • Yosipovitch G.
        • et al.
        3-Dimensional optical clearing and imaging of pruritic atopic dermatitis and psoriasis skin reveals downregulation of epidermal innervation.
        J Invest Dermatol. 2019; 139: 1201-1204
        • Tolkachjov S.N.
        • Brodland D.G.
        • Coldiron B.M.
        • Fazio M.J.
        • Hruza G.J.
        • Roenigk R.K.
        • et al.
        Understanding mohs micrographic surgery: a review and practical guide for the nondermatologist.
        Mayo Clin Proc. 2017; 92: 1261-1271
        • Tomer R.
        • Ye L.
        • Hsueh B.
        • Deisseroth K.
        Advanced CLARITY for rapid and high-resolution imaging of intact tissues.
        Nat Protoc. 2014; 9: 1682-1697
        • Treweek J.B.
        • Chan K.Y.
        • Flytzanis N.C.
        • Yang B.
        • Deverman B.E.
        • Greenbaum A.
        • et al.
        Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.
        Nat Protoc. 2015; 10: 1860-1896
        • Tuchin V.V.
        Tissue optics and photonics: biological tissue structures.
        J Biomed Photonics Amp Eng. 2015; 1: 3-21
        • Tuchin V.V.
        Tissue optics and photonics: light-tissue interaction.
        J Biomed Photonics Amp Eng. 2015; 1: 98-134
        • Vargas G.
        • Chan E.K.
        • Barton J.K.
        • Rylander 3rd, H.G.
        • Welch A.J.
        Use of an agent to reduce scattering in skin.
        Lasers Surg Med. 1999; 24: 133-141
        • Vargas G.
        • Readinger A.
        • Dozier S.S.
        • Welch A.J.
        Morphological changes in blood vessels produced by hyperosmotic agents and measured by optical coherence tomography.
        Photochem Photobiol. 2003; 77: 541-549
        • Wang J.
        • Shi R.
        • Zhang Y.
        • Zhu D.
        Ear skin optical clearing for improving blood flow imaging/Optisches Clearing der Ohrhaut zur verbesserten Bildgebung des Blutflusses.
        Photonics Lasers Med. 2013; 2: 37-44
        • Wang J.
        • Shi R.
        • Zhu D.
        Switchable skin window induced by optical clearing method for dermal blood flow imaging.
        J Biomed Opt. 2013; 18061209
        • Xu J.
        • Ma Y.
        • Yu T.
        • Zhu D.
        Quantitative assessment of optical clearing methods in various intact mouse organs.
        J Biophotonics. 2019; 12e201800134
        • Yang B.
        • Treweek J.B.
        • Kulkarni R.P.
        • Deverman B.E.
        • Chen C.K.
        • Lubeck E.
        • et al.
        Single-cell phenotyping within transparent intact tissue through whole-body clearing.
        Cell. 2014; 158: 945-958
        • Yeh A.T.
        • Choi B.
        • Nelson J.S.
        • Tromberg B.J.
        Reversible dissociation of collagen in tissues.
        J Invest Dermatol. 2003; 121: 1332-1335
        • Yu T.
        • Qi Y.
        • Gong H.
        • Luo Q.
        • Zhu D.
        Optical clearing for multiscale biological tissues.
        J Biophotonics. 2018; 11e201700187
        • Yu T.
        • Zhu J.
        • Li Y.
        • Ma Y.
        • Wang J.
        • Cheng X.
        • et al.
        RTF: a rapid and versatile tissue optical clearing method.
        Sci Rep. 2018; 8: 1964
        • Zhu D.
        • Larin K.V.
        • Luo Q.
        • Tuchin V.V.
        Recent progress in tissue optical clearing.
        Laser Photonics Rev. 2013; 7: 732-757
        • Zhu D.
        • Wang J.
        • Zhi Z.
        • Wen X.
        • Luo Q.
        Imaging dermal blood flow through the intact rat skin with an optical clearing method.
        J Biomed Opt. 2010; 15026008
        • Zhu X.
        • Huang L.
        • Zheng Y.
        • Song Y.
        • Xu Q.
        • Wang J.
        • et al.
        Ultrafast optical clearing method for three-dimensional imaging with cellular resolution.
        Proc Natl Acad Sci USA. 2019; 116: 11480-11489