Andor ixon ultra 897bv emccd camera

All seedlings for cotyledon experiments were floated on sterilized distilled water for 16–24 h before any treatments and/or imaging in order to minimize stimulation resulting from wounding. To observe actin filaments in cotyledons, an Olympus IX-83 inverted microscope equipped with a spinning disc confocal head (Yokogawa CSUX1-A1, Hamamatsu Photonics, Hamamatsu, Japan) and an Andor iXon Ultra 897BV EMCCD camera (Andor Technology, Concord, MA, USA) were utilized. All images were collected with an Olympus 100x oil objective (1.45 NA UPlanSApo; Olympus America, Inc., Waltham, MA, USA). To visualize GFP signal, cotyledons were excited with 488 nm light and 15 consecutive images of 12.5 μm total depth were captured with a 0.5 μm z-step interval and emission wavelength at 525–530 nm using MetaMorph version 7.8.8.0 software. A Gaussian blur filter was applied to the images for each z-stack, which was converted to a maximum intensity projection, followed by high-band pass filtration prior to analysis of percentage of occupancy or density [16 (link)]. All experiments were repeated at least three times independently to draw conclusions.

Epidermal cells from the apical region of 3-day-old dark-grown hypocotyls were imaged by spinning-disk confocal microscopy (SDCM). Image acquisition was performed using a Yokogawa scanning unit (CSU-X1-A1; Hamamatsu Photonics, Hamamatsu, Japan) mounted on an Olympus IX-83 microscope, equipped with a 100× 1.45 numerical aperture (NA) UPlanSApo oil objective (Olympus America Inc., Waltham, MA, USA) and an Andor iXon Ultra 897BV EMCCD camera (Andor Technology, Concord, MA, USA). YFP fluorescence was excited with a 514 nm laser line and emission collected through a 542/27 nm filter. For cortical and subcortical YFP-CESA6 imaging, z-series at 0.2 µm step sizes plus time lapse with 2 s intervals for 10 frames were collected.

Larvae at three dpf were settled on a glass-bottom dish, and imaging was performed at 28 °C. Time-lapse spinning disk confocal microscopy (SDCM) was performed with a Yokogawa scanning unit (CSU-X1-A1) mounted on an Olympus IX-83 microscope, equipped with a 20X 0.5–numerical aperture (NA) UPlanSApo oil objective (Olympus) and an Andor iXon Ultra 897BV EMCCD camera (Andor Technology). GFP and mCherry were excited with 488 nm and 561 nm and fluorescence emission collected through 525/30-nm and 607/36-nm filters, respectively, to determine the localization of stable actin in migrating neutrophils in vivo. Images were captured using MetaMorph version 7.8.8.0 software at 10 s intervals for a total of 5 min. Time-lapse fluorescence images in the head mesenchyme were acquired with a laser-scanning confocal microscope (LSM710, Zeiss) with a Plan-Apochromat 20×/0.8 M27 objective. The fluorescent stacks were flattened using the maximum intensity projection and overlaid with a single slice of the bright-field image. The velocity of neutrophils was quantified using ImageJ with the MTrackJ plugin.