Abstract
To understand how the body of plants is made, it is essential to observe the morphology, structure, and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent (Kurihara et al. 2015), allowing direct observation of the inside of a plant body without inflicting damage on it, for example through physical cutting. However, because ClearSee is not effective in making some plant species and tissues transparent, in this study, we further improved its composition to prevent oxidation and have developed ClearSeeAlpha, which can be applied to a broader range of plant species and tissues. Sodium sulphite, one of the reductants, prevented brown pigmentation due to oxidation during clearing treatment. Using ClearSeeAlpha, we show that it is possible to obtain clear chrysanthemum leaves, tobacco and Torenia pistils, and fertilized Arabidopsis thaliana fruits—tissues which have hitherto been challenging to clear. Moreover, we show that the fluorescence intensity of purified fluorescent proteins emitting light of various colours was unaffected in the ClearSeeAlpha solution; only the fluorescence intensity of TagRFP was reduced by about half. ClearSeeAlpha should be useful in the discovery and analysis of biological phenomena occurring deep inside the plant tissues.
ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging