Dataset Open Access

Dataset of confocal microscopy stacks from plant samples - ImageJ SurfCut: a user-friendly, high-throughput pipeline for extracting cell contours from 3D confocal stacks

Erguvan Özer; Verger Stéphane

This data set contains confocal stacks from Arabidopsis thaliana 35S::GFP-MBD light grown hypocotyl as well as propidium iodide stained cotyledon pavement cells and shoot apical meristem. This is the test dataset for the Fiji macro SurfCut (https://github.com/sverger/SurfCut; 10.5281/zenodo.2635737)

 

Material and methods:

Plant material and growth conditions

Arabidopsis thaliana wild type Col-0 and the microtubule reporter line GFP-MBD (WS-4, (Marc et al. 1998) were used. Seeds were cold treated for 48 hr to synchronize germination. Plants were then grown in a phytotron at 20°C, in a 16 hr light/8 hr dark cycle on solid Murashige and Skoog medium (MS medium, Duchefa, Haarlem, the Netherlands) with 0.8% agar, 1% sucrose, and no vitamin.

 

Confocal microscopy

Cell contour staining in the case of PC_PI_Col0_(1-8).tif and SAM_PI_Col-0.tif was performed by staining the cell wall with Propidium Iodide (PI). Plants were immersed in 0.2 mg/ml propidium iodide (PI, Sigma-Aldrich) for 10 min and washed with water prior to imaging. For imaging, samples were either placed on a solid agar medium and immersed in water, or placed between glass slide and coverslip separated by 400 μm spacers to prevent tissue crushing. Images were acquired using a Leica TCS SP8 confocal microscope, equipped with a water immersion objective (HCX IRAPO L 25x/0.95 W). PI excitation was performed using a 552 nm solid-state laser and fluorescence was detected at 600–650 nm. GFP excitation was performed using a 488 nm solid-state laser and fluorescence was detected at 495–535 nm. Stacks of 1024x1024 pixels (pixel size of 0.363 x 0.363 micron) optical section were generated with a Z interval of 0.5 μm.

 

File list:

Light grown hypocotyl, GFP-MBD reporter line:

- Hypocotyl_GFP-MBD.tif

Cotyledon’s pavement cells, PI staining:

- PC_PI_Col0_1.tif

- PC_PI_Col0_2.tif

- PC_PI_Col0_3.tif

- PC_PI_Col0_4.tif

- PC_PI_Col0_5.tif

- PC_PI_Col0_6.tif

- PC_PI_Col0_7.tif

- PC_PI_Col0_8.tif

Shoot apical meristem, PI staining:

- SAM_PI_Col-0.tif

 

Reference:

Marc, Jan, Cheryl L. Granger, Jennifer Brincat, Deborah D. Fisher, Teh-hui Kao, Andrew G. McCubbin, and Richard J. Cyr. 1998. “A GFP–MAP4 Reporter Gene for Visualizing Cortical Microtubule Rearrangements in Living Epidermal Cells.” The Plant Cell 10 (11): 1927–39. https://doi.org/10.1105/tpc.10.11.1927.

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