A ClearSee-Based Clearing Protocol for 3D Visualization of Arabidopsis thaliana Embryos

Abstract

Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here, we describe a ClearSee-based clearing protocol which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining and enables high-quality 3D imaging of embryo morphology and expression of fluorescent reporters with the cellular resolution. Our protocol provides a reliable method that is applicable to the analysis of morphogenesis and gene expression patterns in Arabidopsis thaliana embryos.

Keywords: 3D imaging; Arabidopsis thaliana; GFP; cell wall staining; clearing; confocal microscopy; embryo; fluorescent reporter.

Figure 1

Dissection of Arabidopsis thaliana embryos: (A) a seed processed with the original ClearSee protocol [11], not transparent and exhibiting a brown color; (B) seeds excised from a fruit in 7% glucose solution (left) and schematic diagram of its internal structure (right); (C) the procedure of embryo isolation, where half of the seed is excised along the red line (left) and the region around the micropyle marked with a red circle (right) is pushed several times until the embryo pops out; and (D–F) the effects of illumination of a stereo microscope. In ClearSee solution, embryos are clearly visible with oblique transmitted illumination (D) whereas they are almost invisible with bright-field (E) or dark-field (F) illumination. The arrowheads indicate the positions of embryos. c, chalaza; e, embryo; and m, micropyle. Bars = 100 μm. Diagrams in B and C are modified from Hughes, 2009 [17].

Figure 2

Confocal microscopic images of a ClearSee-processed embryo with a Green Fluorescent Protein (GFP) reporter: (A) frontal optical section of a heart stage embryo carrying DR5rev::GFP [18] and (B,C) 3D reconstruction of 157 serial optical sections obtained from the same embryo as in A in frontal (B) and top (C) views. The signals of Calcofluor White and endoplasmic reticulum (ER)-localized GFP are represented in magenta and green, respectively. Bars = 20 μm.

Figure 3

Confocal microscopic images of ClearSee-processed embryos with various fluorescent reporters: (A–C) frontal optical sections of heart (A), torpedo (B), and bent-cotyledon (C) stage embryos of pARF5-n3GFP and (D) a frontal optical section of the gWUS-GFP3 pCLV3:H2B-mScarlet embryo, displaying signals of the two fluorescent proteins (left), GFP alone (middle), and mScarlet alone (right) together with Calcofluor White signals. In (A–C) and the left panel of D, the signals of Calcofluor White, GFP, and mScarlet are represented in grey scale, green, and magenta, respectively. In the middle and right panels of D, the signals of Calcofluor white, GFP, and mScarlet are all represented in grey scale. Bars = 20 μm.