Youtor
Dissecting Arabidopsis Flowers in Water Needle Technique for Precise Floral Removal
>> YOUR LINK HERE: ___ http://youtube.com/watch?v=3rMRjrU_JTo
More post: https://www.scenseme.com/post/dissect... • Arabidopsis sepals, with their small size, are excellent models for analyzing growth at a cellular resolution under a confocal microscope. However, imaging deep cell layers of sepals poses challenges due to the low signal-to-noise ratio in the deeper tissues, especially in live imaging datasets. To study how differential growth in connected cell layers generates distinct organ morphologies, it is critical to live-image deep into the tissue, despite these difficulties. Here, we provide an optimized methodology for live imaging Arabidopsis sepals and subsequent image processing to overcome some of these challenges. • For optimal dissection, we prefer six- to seven-week-old Arabidopsis plants grown in 21°C long-day conditions, which prolongs vegetative growth phase relative to 24 hours light, resulting in larger meristem sizes and thicker inflorescence axes. Although plants can be grown under short day conditions as well, which leads to larger meristem size compared to long-day/24 hour light conditions (Hamant et al., 2019), plants exhibit significant delay in flowering under short day conditions (Mouradov et al., 2002), which is not necessary for our dissection purposes. Dissection can either be performed while the inflorescence is on the plant or on a clipped inflorescence to allow for easier maneuvering during dissection. However, clipping the inflorescence bears the risk of faster dehydration, and thus requires quicker dissection. The choice of whether to dissect in planta or on a clipped inflorescence depends on the researcher. • Although the dissection techniques vary among researchers, we describe the procedure that works best for us, resulting in minimal tissue damage. For dissection, we grip the inflorescence in one hand, and use the tweezers to press the base of the pedicel and pull the flower backwards in a gentle yet swift manner. We adopt a stage-wise dissection approach by rotating the inflorescence, starting with the oldest flowers first and gradually progressing to younger ones, all the way down to stages 7 to 8 (Watch video 1: • Precision Dissection of Arabidopsis F... ) [Figures 1A–C, staging performed according to (Smyth et al., 1990)]. We then submerge the inflorescence in water for 3 to 4 hours to allow for rehydration at room temperature, a critical step in ensuring sample viability (Figure 1D). Following rehydration, dissection of flowers below stages 7 to 8 is performed under a stereo microscope (Zeiss Stemi 2000-C), while the inflorescence remains submerged in water. This retains sample hydration during dissection of younger flowers, as it requires increased caution and is therefore more time consuming. While using tweezers is an option, we prefer using a needle in favor of its sharper blade and increased precision in dissection, to avoid damaging younger adjacent flowers. Dissection in water involves rotating the plate to position the flower to be dissected at a convenient angle, pulling the flower bud backwards using the needle and nipping it from the pedicel base. These steps are repeated until we dissect down to flowers of stages 3 to 4 (Figure 1E). Post dissection, we drain the water and transfer the sample to a fresh plate. The sample is then allowed to recover for 24 hours under initial growth conditions. Assuming no flowers were accidentally damaged during dissection, all flowers should transition through their respective developmental stages (Figures 1E–G). • For live imaging, we choose a mid-stage 4 (Smyth et al., 1990) flower (Figures 1F, G). The inflorescence is repositioned at an angle in which the flower to be imaged directly faces the confocal objective (Figure 1H). The sample is then submerged in 0.01% SILWET-77 (surfactant) in water solution to reduce surface tension, which helps displace the air bubbles that can obstruct imaging. Any persisting air bubbles can be removed by gently pipetting liquid solution towards air bubble. We then allow the sample to stabilize for five to ten minutes, following which the flower is imaged under a confocal microscope using a 20X water dipping objective (Figure 1I). • Keywords: growth, 2.5D segmentation, live imaging, image processing, deep tissue imaging, Arabidopsis, MorphoGraphX, sepals • Citation: Singh Yadav A and Roeder AHK (2024) An optimized live imaging and multiple cell layer growth analysis approach using Arabidopsis sepals. Front. Plant Sci. 15:1449195. doi: https://doi.org/10.3389/fpls.2024.144... • Received: 14 June 2024; Accepted: 29 July 2024; • Published: 03 September 2024. • Attribution 4.0 International — CC BY 4.0 - Creative Commons
#############################
