Improved clearing method contributes to deep imaging of plant organs

Abstract

Tissue clearing methods are increasingly essential for microscopic observation of internal tissues of thick biological organs. We previously developed TOMEI, a clearing method for plant tissues; however, it could not entirely remove chlorophylls and reduced the fluorescent signal of fluorescent proteins (FPs). Here, we developed an improved TOMEI method (iTOMEI) to overcome these limitations. We show that iTOMEI efficiently removes chlorophylls using caprylyl sulfobetaine solution and restores fluorescence of FPs, mainly lost by fixation, using a weak alkaline solution. iTOMEI enables detection of much brighter FP fluorescence than previous methods within 26 h in tissues of Arabidopsis thaliana, Oryza sativa, and Marchantia polymorpha. Moreover, a mouse brain was also efficiently cleared by the iTOMEI-Brain method within 48 h and strong fluorescent signals were detected in the cleared brain.

Three-dimensional morphological analysis revealed the cell patterning bases for the sexual dimorphism development in the liverwortMarchantia polymorpha

“…Germline specification and gametangiophore development initiate simultaneously in M. polymorpha To elucidate the cell-level patterning processes underlying the sexual dimorphism development in M. polymorpha, we performed 3D cell segmentation analyses of early-stage gametangiophore primordia. Briefly, segments containing gametangiophore primordia were excised from the apical notch region of thalli, subjected to tissue clearing by iTOMEI (Sakamoto et al 2022), and stained with the fluorescence dye Renaissance 2200 to visualize cell walls. 3D cell wall patterns were reconstructed from Z-stack confocal images and used for cell segmentation analysis by the MorphoGraphX program (Figure 2A-2D) (Strauss et al 2022).…”

Comparing the efficiency of six clearing methods in developing seeds of Arabidopsis thaliana

“…5). Recently, Sakamoto et al (2022) used iohexol, a mounting medium with a high refractive index, to improve tissue transparency. We combined FAST9 clearing with 60% iohexol as a mounting medium to test whether it would better preserve seed morphology (Fig.…”

“…PBS-T best preserved the seed morphology, but limited the laser penetration resulting in a reduced sharpness of images. Recently, iohexol was tested as a mounting medium in the TOMEI clearing method (Sakamoto et al 2022). Iohexol has a high refractive index and appears to be a good mounting solution to combine with FAST9 to efficiently clear Arabidopsis seeds while mostly preserving the seed shape and fluorescence of FPs.…”

“…Cleared samples with FAST9, as described above, were mounted in iohexol with modifications on the final iohexol concentration (Sakamoto et al 2022). In brief, after overnight washing with PBS-T, the cleared samples were incubated at room temperature in a concentration series of 20%, 50%, and 60% (w/w) iohexol (TCI) in PBS for 10 minutes each to avoid osmotic stress and seed collapse.…”

Whole-Tissue Three-Dimensional Imaging of Rice at Single-Cell Resolution

“…The samples were transferred to a decolorization solution (100 mM sodium phosphate buffer at pH 8.0 with 20% ( w / v ) caprylyl sulfobetaine (TCI, Tokyo, Japan)) and incubated for 24 h for SAMs and florets or 3 days for roots at room temperature to decolorize the tissues [ 29 ]. The samples were transferred to a clearing solution (56.2% ( w / w ) Histodenz (Merck, Darmstadt, Germany) in PBS buffer) and incubated for 1 h at room temperature for SAMs, florets, and roots [ 29 ]. For anther, fixed anther was transferred to ClearSee and incubated for 1 week at room temperature.…”