研究目的
To develop a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages, enabling the investigation of the morphology of internal tissues of the ovule with cellular resolution.
研究成果
The protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages with cellular resolution, allowing the generation of digital three-dimensional models of ovules. Such models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species.
研究不足
The method is incompatible with live imaging and requires fixed tissue. Cells of the mature gametophyte cannot be stained well and need to be marked by other means.
1:Experimental Design and Method Selection:
The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide.
2:Sample Selection and Data Sources:
Fixed Arabidopsis ovules of all stages were used.
3:List of Experimental Equipment and Materials:
Confocal laser scanning microscopy was performed on an upright Leica TCS SP8 X WLL2 HyVolution 2 equipped with GaAsP (HyD) detectors and a 63× glycerol objective.
4:Experimental Procedures and Operational Workflow:
Ovules were dissected, fixed, cleared in ClearSee, stained with SR2200 and TO-PRO-3 iodide, and mounted in Vectashield for imaging.
5:Data Analysis Methods:
Z-stacks of ovules were imported into MorphographX for processing using watershed-based segmentation followed by surface mesh formation.
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