Volumetric imaging of Arabidopsis thaliana root cells

Arabidopsis thaliana seeds were surface sterilized, germinated and grown in Murashigue and Skoog medium at pH 5.7 and supplemented with vitamins (0.1 mg l-1 pyridoxine, 0.1 mg l-1 nicotinic acid), 0.8% agar, and 1% sucrose. Plants were grown at 21°C, 16/8-hour light/dark periods at 105 µmol/m²s² light intensity.

Using a Zeiss Axiovert 200M microscope and a C-APO 63X, 1.2NA objective (Oberkochen, Germany), confocal volumetric imaging of the plant material was performed, with a pixel size of 404 nm and Z step size of 500 nm. Excitation of the sample was provided by a 488 nm laser, and a filter cube with 525/45 nm and 630/92 nm bandpass filters was used for yellow and red emission light collection, respectively.

An inverted Olympus FV1000-IX81 confocal microscope equipped with a LUMFLN×60, 1.3NA S objective was used for root cell nuclear imaging. Sample excitation was achieved with a 543 nm laser and a BA560-660 filter was used for emission light collection. Pixel size was 41 nm, with a Z step size of 100 nm.

A custom-built selective plane illumination microscopy (SPIM) system was used for imaging of primary root cells expressing p35s:H2B-R-RF. Sample excitation was achieved with a 561 nm laser using stroboscopic illumination. Emission was filtered via a multi-bandpass emission filter (Semrock, FF01-446/523/600/677-25 BrightLine). Volumetric imaging was done by mounting the sample on a four dimensional (XYZ, and Y rotation) motorized stage (Picard Industries). A sCMOS sensor (Hamamatsu, ORCA-Flash4.0 V2) was used for signal recording. The OpenSPIM plugin of µmanager (v.1.4 for windows) was used for control of acquisition parameters, sample translation and stroboscopic illumination. Collected images had a pixel size of 0.325µm, Z step size of 100 nm and a Y rotation step of 1.8°.

Experimental procedures were approved by the Bioethics Committee of the Biotechnology Institute of the National Autonomous University of Mexico.