Computational protocol: Simultaneous live-imaging of peroxisomes and the ER in plant cells suggests contiguity but no luminal continuity between the two organelles

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Protocol publication

[…] Plant tissue and seedlings were mounted in tap water on a glass depression slide and placed under a coverslip. For plants expressing the photoconvertible mEosFP-PTS1 protein the photoconversion time was varied according to the brightness of the respective organelles. In general, exposure times for peroxisomes were between 3 and 6 s and resulted in bright red organelles. The light source for photo-conversion was a HBO 100 W/2 Mercury Short Arc lamp and the Leica fluorescence filter set ā€œDā€ (Excitation filter: 355ā€“425 nm; Dichromatic mirror 455 nm; Suppression filter LP 470 nm). The epi-fluorescence setup consisted of a Leica DM-6000CS microscope. Photo-conversion was performed manually by controlling the diaphragm as described earlier (Mathur et al., ). Simultaneous imaging of peroxisomal and ER probes was carried out using a Leica TCS-SP5 confocal laser-scanning unit equipped with a 488 nm argon and a 543 nm helium-neon laser. All images were captured using at a color depth of 24bit RGB.All images and movies were cropped and processed for brightness/contrast as complete image or stacks using either Adobe Photoshop CS3 (http://www.adobe.com) or the ImageJ distribution Fiji (http://pacific.mpi-cbg.de/wiki/index.php/Fiji). Adobe Photoshop was used for annotation of movies. Imaris software (v. 6.4.0; Bitplane AG) was used to render iso-surface 3 D rendering of ER and peroxisomes from confocal image stacks and x-y-time series. All experiments were carried out at least five times. […]

Pipeline specifications

Software tools ImageJ, Imaris
Application Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae, Arabidopsis thaliana
Diseases Neoplasms by Site