Three-Dimensional Multiphoton Imaging of Transcription Factor by ClearSee
In plants, transcription factors often act as cell-to-cell trafficking mobile proteins and specify cell fate. Thus, to visualize spatiotemporal expression pattern and localization of transcription factors are essential to understand their functions during development. Several protocols have been developed to observe fluorescent protein. However, plant-specific autofluorescent compounds and various tissue components with different refractive indexes interfere with detection of fluorescent signals of your interest. Furthermore, cell fate specification often occurs in a limited number of cells covered by lateral/layers of organs. To overcome those issues, the plant clearing method, ClearSee, was recently developed for high-resolution imaging inside tissues by making background transparent. In this chapter, we provide three-dimensional imaging of fluorescent-protein-fused transcription factors by two-photon excitation microscopy in Arabidopsis and rice. Complex cell patterning with gene expression could be observed from any direction three-dimensionally. This method could be applicable to visualize any protein of your interest or it can readily be adapted in various other plants.
Key wordsDeep imaging ClearSee Two-photon excitation microscopy Fluorescent proteins Pistil Shoot meristem Arabidopsis thaliana Oryza sativa
This work was supported by grants from the Japan Science and Technology Agency (JST; PRESTO) Grant number JPMJPR15QC to Y.M. and by JSPS KAKENHI Grant JP16K18637 to K.T.
3D image of ClearSee-treated Arabidopsis ovary. Related to Fig. 2. The movie was created by cropping the 3D models displayed in “3D Volume View.” Cropping plane is shown as a green line (MP4 2023 kb)
3D image of ClearSee-treated rice inflorescence meristems. Related to Fig. 4a. The movie with a of 360° turnaround y-axis was created by the 3D model displayed in “3D Volume View,” as shown in Fig. 4a. The 3D model was generated from 101 z-stack images with 3 μm intervals. Color is associated with z depth (MP4 2410 kb)
A series of optical sections of ClearSee-treated rice shoot apex. Related to Fig. 4b, c. A series of xy images of rice inflorescence meristems after clearing by ClearSee for 1 month. The inflorescence was at the stage In3-4 . Optical xy stack was generated from 101 z-stack images with 3-μm intervals. Chitin in the cell wall is shown as cyan and magenta. Scale bars = 100 μm (MP4 21266 kb)
3D image of ClearSee-treated young rice flower. Related to Fig. 5a. The movie with a 360° turnaround y-axis was created by the 3D model displayed in “3D Volume View,” as shown in Fig. 5a. The 3D model was generated from 101 z-stack images with 3-μm intervals. Chitin in the cell wall is shown as cyan and magenta. (MP4 4871 kb)
A series of optical sections of ClearSee-treated young rice flower. Related to Fig. 5b, c. A series of xy images of young rice flower after clearing by ClearSee for 1 month. The florals stage was at Sp7 . Optical xy stack was generated from 101 z-stack images with 3-μm intervals. Chitin in the cell wall is shown as cyan and magenta. Scale bars = 100 μm (MP4 28080 kb)
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