Abstract
The preparation of biological samples, especially for live-cell microscopy, remains a major experimental challenge in the lab despite technological advances. In addition, high-resolution microscopy techniques require higher sample quality and uniformity, which is difficult to ensure during manual preparation while maintaining “ideal” growth conditions. In this protocol, we provide a way out by growing Arabidopsis thaliana seedlings directly in an imaging chamber, which eliminates invasive sample preparation directly before imaging. This method hinges on the precise placement of seeds into imaging chambers, which can be grown in conventional climate chambers. We detail three methods to grow hypocotyls, cotyledons, leaves, and roots for high-resolution and long-term imaging of the plant cytoskeleton. Furthermore, we show that the growth and development of seedlings inside the chambers can be externally manipulated by the addition of chemicals.
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1 Electronic Supplementary Material(s)
Sample preparation for imaging hypocotyls. Transfer sterile seeds to the chambers by using sterile toothpicks to push individual seeds through the solid medium until they touch the glass coverslip (MP4 60916 kb)
Animation of large-scale 3D imaging of cotyledons (7-day-old seedling). Acquisition of the montage required 3 h of stable imaging (Leica Stellaris 8; HC PL APO 20×/0.75NA objective; 21 tiles, 125 slices; total imaging volume approximately 2 mm × 1 mm × 0.5 mm with 360 nm resolution in xy and 4 μm in z) (MP4 46775 kb)
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Ruhnow, F., Persson, S., Schneider, R. (2023). Noninvasive Long-Term Imaging of the Cytoskeleton in Arabidopsis Seedlings. In: Hussey, P.J., Wang, P. (eds) The Plant Cytoskeleton. Methods in Molecular Biology, vol 2604. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2867-6_24
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DOI: https://doi.org/10.1007/978-1-0716-2867-6_24
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