title: A Versatile Optical Clearing Protocol for Deep Tissue Imaging of Fluorescent Proteins in Arabidopsis thaliana.
abstract: Confocal microscopy is widely used to visualize gene expression patterns and developmental processes in plants. However, the imaging of plant tissue can be challenging due to its opacity, which often makes previous immersion in a clearing agent necessary. Many commonly-used chemicals suffer either from their incompatibility with fluorescent proteins or their complex and lengthy application. 2,2'-thiodiethanol (TDE) has recently been described as a clearing agent with an emphasis on high resolution microscopy due to its potential to adjust the refractive index. Here, we evaluate the use of TDE-based clearing for confocal as well as two-photon microscopy in various Arabidopsis thaliana tissue types. We demonstrate that tissue fixation is a mandatory prerequisite for the use of TDE, in order to preserve tissue integrity and fluorescent protein activity. TDE concentrations between 50-70% are a good compromise for imaging of technically challenging tissue to achieve good clearing without affecting fluorescent protein activity. TDE-based clearing is simple and rapid to use and allows for a flexible experimental setup while facilitating high quality imaging.
authors: Max Planck Institute for Developmental Biology, Department of Cell Biology, Spemannstrasse 35, 72076 Tuebingen, Germany.
authors: Thomas J
authors: TJ
authors: Musielak
authors: Max Planck Institute for Developmental Biology, Department of Cell Biology, Spemannstrasse 35, 72076 Tuebingen, Germany.
authors: Daniel
authors: D
authors: Slane
authors: Max Planck Institute for Developmental Biology, Light Microscopy Facility, Spemannstrasse 35, 72076 Tuebingen, Germany.
authors: Christian
authors: C
authors: Liebig
authors: Max Planck Institute for Developmental Biology, Department of Cell Biology, Spemannstrasse 35, 72076 Tuebingen, Germany.
authors: Martin
authors: http://orcid.org/0000-0001-5806-2253
authors: M
authors: Bayer
mesh_terms: D017360:Arabidopsis / Q000378:metabolism* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D017785:Photons; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D016707:Tissue Fixation
chemical_list: D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D049452:Green Fluorescent Proteins; C047519:2,2'-thiodiethanol