Improved clearing method contributes to deep imaging of plant organs

Abstract

Tissue clearing methods are increasingly essential for the microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls nor reduce the fluorescent signal of fluorescent proteins. Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. First, a caprylyl sulfobetaine was determined to efficiently remove chlorophylls from Arabidopsis thaliana seedlings without GFP quenching. Next, a weak alkaline solution restored GFP fluorescence, which was mainly lost during fixation, and an iohexol solution with a high refractive index increased sample transparency. These procedures were integrated to form iTOMEI. iTOMEI enables the detection of much brighter fluorescence than previous methods in tissues of A. thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h, and strong fluorescent signals were detected in the cleared brain.

3D-multiple immunoimaging using whole male organs in rice

“…iTOMEI clearing is a powerful method to reduce the autofluorescence of plant organs 13,18 . It was also applicable for the 3D-histochemical staining with PI in this study (Supplementary Fig.…”

“…The samples were then transferred and incubated in 50% iTOMEI solution (50% caprylyl sulfobetaine in 100 mM sodium phosphate buffer) for 10 minutes at 25 °C. Finally, the samples were transferred and incubated in 70.4% iTOMEI (70.4% iohexol in PBS) for 1 h at 25 °C 13 . The samples were mounted with 70.4% iTOMEI.…”

“…In order to investigate the localization of proteins and elucidate the molecular mechanism, imaging using fluorescent proteins is more established than immunostaining in plants 12 13 . However, this method requires an additional transformation step to generate plants with the target protein fused to a fluorescent protein.…”

Auxin Distribution in Lateral Root Primordium Development Affects the Size and Lateral Root Diameter of Rice

“…Crown root sections were sampled to observe DR5-NLS-3xVenus expression in the LRP of the WT and mutants. The samples were fixed in 4% paraformaldehyde (w/v) and stained with 5 μg/ml DAPI (4′,6-Diamidino-2-phenylindole Dihydrochloride; Wako, Japan) for 1 h, then cleared with iTOMEI (TCI, Japan) ( Sato et al, 2022 ; Sakamoto et al, 2022 ), or embedded in a 3% (w/v) agar medium then cross-sectioned into 50 μm thick slices with a linear slicer (DYK-1000N; Dosaka EM Co., Japan). The samples were viewed under a confocal laser scanning microscope (FV3000; Olympus, Japan).…”

3D multiple immunoimaging using whole male organs in rice

“…To investigate the localization of proteins and to elucidate the molecular mechanisms involved, imaging using fluorescent proteins is better established than immunostaining in plants 13 , 14 . Notably, Schnittger and colleagues have successfully developed live-cell imaging to observe meiotic progression using the buds of reporter lines in A. thaliana 15 , 16 .…”