title: A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue.
abstract: For 3-dimensional (3D) imaging of a tissue, 3 methodological steps are essential and their successful application depends on specific characteristics of the type of tissue. The steps are 1° clearing of the opaque tissue to render it transparent for microscopy, 2° fluorescence labeling of the tissues and 3° 3D imaging. In the past decades, new methodologies were introduced for the clearing steps with their specific advantages and disadvantages. Most clearing techniques have been applied to the central nervous system and other organs that contain relatively low amounts of connective tissue including extracellular matrix. However, tissues that contain large amounts of extracellular matrix such as dermis in skin or gingiva are difficult to clear. The present survey lists methodologies that are available for clearing of tissues for 3D imaging. We report here that the BABB method using a mixture of benzyl alcohol and benzyl benzoate and iDISCO using dibenzylether (DBE) are the most successful methods for clearing connective tissue-rich gingiva and dermis of skin for 3D histochemistry and imaging of fluorescence using light-sheet microscopy.
authors.affiliation: Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz 2, Mainz 55131, Germany; Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Electronic address: adrianoasso@hotmail.com.
authors.forename: Adriano
authors.initials: A
authors.lastname: Azaripour
authors.affiliation: Neuro-Oncology Research Group, VU University Medical Center, Cancer Center Amsterdam, Room 3.20, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
authors.forename: Tonny
authors.initials: T
authors.lastname: Lagerweij
authors.affiliation: Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz 2, Mainz 55131, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Scharfbillig
authors.affiliation: Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz 2, Mainz 55131, Germany.
authors.forename: Anna Elisabeth
authors.initials: AE
authors.lastname: Jadczak
authors.affiliation: Department of Operative Dentistry, University Medical Center of the Johannes Gutenberg University Mainz, Augustusplatz 2, Mainz 55131, Germany.
authors.forename: Brita
authors.initials: B
authors.lastname: Willershausen
authors.affiliation: Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
authors.forename: Cornelis J F
authors.initials: CJ
authors.lastname: Van Noorden
mesh_terms: D000818:Animals; D001565:Benzoates / Q000737:chemistry; D019905:Benzyl Alcohol / Q000737:chemistry; D003238:Connective Tissue / Q000648:ultrastructure*; D005404:Fixatives / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006651:Histocytochemistry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000295:instrumentation / Q000379:methods*; D018613:Microscopy, Confocal; D010647:Phenyl Ethers / Q000737:chemistry; D013048:Specimen Handling / Q000295:instrumentation / Q000379:methods; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D001565:Benzoates; D005404:Fixatives; D005456:Fluorescent Dyes; D010647:Phenyl Ethers; C076624:dibenzyl ether; D019905:Benzyl Alcohol; C006723:benzyl benzoate
keywords: 3D histochemistry; 3D imaging; Clearing; Connective tissue; Extracellular matrix; Gingiva; Light-sheet microscopy; Skin