Csu x1 spinning disk head

Seedlings were scanned (HP Scanjet 8300) and germinated hypocotyl lengths were measured in ImageJ. For hypocotyl breakage experiments, three 6-day-old etiolated seedlings were arranged in parallel and clamped with small rubber-capped alligator clips at the tops and bottoms of the hypocotyls in a constant force extensometer (Durachko and Cosgrove, 2009 (link)). A 4 g counterweight (0.4 N force) was lowered gently by hand to allow the samples to become taut before releasing it. In most cases, breakage occurred almost immediately after the counterweight was released. To image breakage patterns using fluorescence microscopy, hypocotyls were stained with 10 µg/mL Propidium Iodide (PI; Life Technologies) and imaged on a Zeiss Axio Observer microscope with a Yokogawa CSU-X1 spinning disk head and a 20X 0.5 NA air objective using a 561 nm excitation laser and 617/73 nm emission filter. Different hypocotyl regions were defined by dividing the hypocotyls equally into thirds, and the breakage location was recorded for each hypocotyl. To image cell adhesion defects in seedlings overexpressing pectin-modifying enzymes, hypocotyls were stained with 10 µg/mL Propidium Iodide and imaged on a Zeiss Axio Observer microscope with a Yokogawa CSU-X1 spinning disk head and a 10X 0.3 NA air objective using a 561 nm excitation laser and 617/73 nm emission filter.

Primary intestinal epithelial organoids were grown as described before (Sato and Clevers, 2013 (link)). Briefly, the small intestine was flushed and cut into small pieces that were dissociated in PBS containing 2 mM EDTA for 30 min at 4°C. After extensive washing, the isolated crypts were pelleted and mixed with 50 μL of Matrigel (Corning) and put in a 24-well plate. After polymerization of the Matrigel, complete culture medium containing advanced DMEM/F12 (Gibco) supplemented with B27 supplement (0.02%, Invitrogen), N2 supplement (0.1%, Invitrogen), N-acetylcysteine (0.0025%, Sigma-Aldrich), mouse epidermal growth factor (mEGF; 0.001%, Invitrogen), and conditioned Rspondin and mNoggin medium. Organoids were seeded and imaged in an 8-well chamber (iBidi). Cell death was induced just before imaging and was monitored by an increase in PI positivity. Live-cell imaging was performed on an Axio Observer Z1 (Zeiss, Germany) equipped with a CSU-X1 spinning-disk head (Yokogawa) and AxioCam MRm (Zeiss), with a EC Plan-Neofluar 10× dry objective (numerical aperture [NA] 0.30). Images were acquired every 15 min for 16 hr. Data analysis and image reconstruction were performed with ImageJ (NIH).