Abstract
Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.
Highlights
To understand how cell patterning changes with gene expression, an important challenge in developmental biology is visualization of three-dimensional (3D) morphology with gene expression in intact tissues at the cellular level
Confocal and two-photon imaging of ClearSee-treated tissues Recently, we showed that 2PEM is valuable for in vivo deep imaging while avoiding autofluorescence in plant tissues (Mizuta et al, 2015)
The H2B-mRFP signal was clearly observed even in the apical region of the gametophore, as well as in the gametophore leaf cells, following ClearSee treatment (Fig. 8B, ClearSee, H2B-mRFP). These results suggest that the ClearSee clearing method is not limited to angiosperm tissues but is suitable for non-vascular plant tissues while maintaining the stability of fluorescent proteins (FPs)
Summary
To understand how cell patterning changes with gene expression, an important challenge in developmental biology is visualization of three-dimensional (3D) morphology with gene expression in intact tissues at the cellular level. Recent advances in fluorescence imaging using fluorescent proteins (FPs), such as green fluorescent protein (GFP), reveal gene expression at the subcellular level. It is difficult to observe such FPs in intact plant tissues because plant tissues contain a variety of autofluorescent compounds (Müller et al, 2013), which results in non-specific background fluorescence. Plant tissues contain various components with different refractive indexes (e.g. air, 1.00; cell wall, 1.42; cytoplasm; 1.36) (Kumar and Silva, 1973; Vogelmann et al, 1996). These refractive index mismatches cause light scattering. Mechanical sectioning is required to obtain high-resolution images of deep
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