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Improved clearing method contributes to deep imaging of plant organs
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Improved clearing method contributes to deep imaging of plant organs
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Journal Article
Improved clearing method contributes to deep imaging of plant organs
2022
Overview
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.
Sakamoto et al. demonstrate an improved optical clearing method, iTOMEI, for plant imaging. The new method can achieve fast clearing and effective removal of autofluorescence signals, and at the same time preserve signals from desired fluorescence proteins.
