Chloral hydrate (as Hoyer's solution) has been used for the preservation of specimens since the late 19th century ( Hoyer, 1882), and has a high refractive index (1.428), which allows high penetration of light without scattering for a wide variety of plant tissues ( Villani et al., 2013).
Of these reagents, acidified chloral hydrate is most commonly used to clear plant tissues ( Lersten, 1967). Classically, a variety of chemical reagents have been used to improve the transparency of plant tissues.
#PLANT IMAGING SERIAL#
However, it is difficult to reconstruct a 3D representation of gene expression patterns from mechanical sections because of the laboriousness of serial sectioning and potential difficulty of obtaining sections for desired regions and orientations. In traditional observation methods, mechanical sectioning is required to obtain high-resolution images of deep plant tissues. These refractive index mismatches cause light scattering. In addition, plant tissues contain various components with different refractive indexes (e.g. However, 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. Recent advances in fluorescence imaging using fluorescent proteins (FPs), such as green fluorescent protein (GFP), reveal gene expression at the subcellular level. 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. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research. 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. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. 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. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. 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. Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms.