Airyscan confocal time-lapse data of cellulose synthase in developing protoxylem vessels of the Arabidopsis root.

Published: 20 January 2023| Version 1 | DOI: 10.17632/kxyfdgsbc3.1
Contributor:
Raymond Wightman

Description

These datasets are continuous time lapse of protoxylem vessels in roots of Arabidopsis thaliana. The aim is to study movement of cellulose synthase complexes (CSCs) during secondary wall formation. Timecourse 1 is a three channel movie showing CFP-Microtubules (CFP:MBD), YFP-CSCs (YFP:CESA7) and mCherry-Actin (mCherry:fABD2). Frame interval is 6 s. Timecourses 2, 3 and 4 are single channel movies showing YFP-CSCs. Frame interval is 0.57s (timecourse 2) or 0.69s (timecourses 3, 4). All data before Airyscan software processing, containing signal from each of the sub-airy detectors, is provided in the .zip archives ("Airyscan_raw"). Extracted files are Zeiss .czi format and can be opened in Zeiss Zen confocal software. Airyscan processed data is provided as .czi files ("Airyscan processed") and can be viewed in Zeiss ZEN software or ImageJ/Fiji BioFormats import tool. Multipage .tif files (see "steps to reproduce section") can be open natively by ImageJ/Fiji software. For viewing fast dynamic processes the airyscan datasets are viewed without further processing. For viewing slower process (e.g. CSCs moving below the emerging secondary cell wall thickenings), the fast time-lapse data (timecourses2-4) are subjected to a running average (ImageJ/Fiji plugins "walking average" or "running Z projector") of x6.

Files

Steps to reproduce

For timecourse1, imaging was carried out with the RS airyscan option on a Zeiss LSM880 with 63x 1.2 NA water immersion objective using line-sequential mode employing fast switching between 440nm, 514nm and 561nm lasers (out-of-objective powers of 24, 14, 38 uW, respectively) and a single band pass 420-480 + 495-620 emission filter and a long pass (LP) 460nm beamsplitter. A scan format of 1096x244, scan speed of 6, line average of 2 and zoom x2 was used. Images were taken continuously as part of a time course experiment at 6.02 s per frame. Airyscan processing used default values on Zen Black software. For timecourse 2, 3 and 4, Imaging was carried out as described for timecourse1 above except only the 514 nm laser was used (at an out-of-objective power of 7.8 uW) together with a BP 495-550 + LP 570 emission filter and a scan format of 888x140 pixels in airyscan SR mode. Timecourse2 exhibited a sudden lateral shift towards the end of the experiment and this part has been removed in the dataset "truncated_timecourse", For timecourse4, a small linear x-drift was observed and corrected manually using the CoordinateShift plugin for ImageJ/Fiji ("XDRIFT_CORRECTED").

Institutions

University of Cambridge Sainsbury Laboratory

Categories

Cellulose, Confocal Microscopy, Fluorescence Microscopy, Cell Wall, Super-Resolution Imaging, Root, Xylem

Funding

Gatsby Charitable Foundation

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