A survey of clearing techniques for 3D imaging of tissues with special reference to connective tissue

Adriano Azaripour, Tonny Lagerweij, Christina Scharfbillig, Anna Elisabeth Jadczak, Brita Willershausen, Cornelis J. F. van Noorden

Research output: Contribution to journalReview articleAcademicpeer-review

121 Citations (Scopus)


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
Original languageEnglish
Pages (from-to)9-23
Number of pages15
JournalProgress in histochemistry and cytochemistry
Issue number2
Publication statusPublished - Aug 2016


  • Animals
  • Benzoates
  • Benzyl Alcohol
  • Connective Tissue
  • Fixatives
  • Fluorescent Dyes
  • Histocytochemistry
  • Humans
  • Imaging, Three-Dimensional
  • Journal Article
  • Microscopy, Confocal
  • Phenyl Ethers
  • Review
  • Specimen Handling
  • Staining and Labeling
  • Tissue Fixation

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