Imagem em ccd

Embryo preparation and mounting has been described elsewhere (Pohl and Bao, 2010 (link); Dutta et al., 2015 (link)). Mounting was modified by using differently sized polystyrene (15 µm, 20 µm, 25 µm; Polysciences, Hirschberg, Germany) and polymethylmethacrylate spheres (12 µm and 13.5 µm, PolyAn, Berlin, Germany). Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. All acquisitions were performed at 21–23°C using a Leica HC PL APO 63×/1.4-0.6 oil objective. Cell cortex ablations were performed using a pulsed 355 nm UV laser mounted on the same microscope. One ablation cycle was performed per acquisition with a residence time per pixel of 3.5 ms. Acquisitions pre and post ablation were performed at 200-ms intervals.

Live embryos/explants expressing fluorescent proteins were mounted in 0.75% low-melt agarose, and fixed embryos/explants subjected to immunofluorescent staining were mounted in 2% methylcellulose in glass- bottomed 35 mm Petri dishes for imaging using a modified Olympus IX81 inverted spinning disc confocal microscope equipped with Voltran and Cobolt steady-state lasers and a Hamamatsu ImagEM EM CCD digital camera. For live time-lapse series, 60–100 μm z-stacks with a 1–2 μm step were collected every 3–5 minutes (depending on the experiment) for 3 hours using a 20x or 40x dry objective lens for intact embryos and a 20x objective for explants. Temperature was maintained at 28.5°C during imaging using a Live Cell Instrument stage heater. For immuno-stained embryos and explants, 100 μm z-stacks with a 1 or 2 μm step were collected using a 10x or 20x dry objective lens, depending on the experiment. Bright-field and transmitted light images of live embryos and in situ hybridizations were collected using a Nikon AZ100 macroscope.

Sporulated haploid strains were grown to log phase in SC-Trp medium and were adhered on a 25 mm coverslip that was first incubated for 10 min at room temperature with 40 µl of Concanavalin A (100 µg/ml) and then washed with SC-Trp medium. Cells were then imaged on the coverslip in 40 µl of SC-Trp at room temperature. The imaging was done with an Olympus IX81 inverted microscope equipped with an Olympus 100 x/1.45 NA TIRF objective, a GFP-3035C-OMF single-band filter set (Semrock) and a Hamamatsu ImagEM EMCCD set at full gain. Cells were excited for 80 ms (Rvs167-GFP) or 100 ms (Sla1-GFP) with a 488 nm laser.

Bone marrow-derived macrophages were cultured with invasion-blocked or untreated mCherry-expressing cpsII parasites (MOI = 1) and LysoTracker Green DND-26 (Life Technologies) was added prior to imaging, following the manufacturer's instructions. Images were collected using a Leica DMI4000 microscope equipped with a Yokogawa CSU10 spinning disk confocal unit and a Hamamatsu ImagEM EMCCD camera. Images were analyzed using ImageJ software.

Laterally mounted embryos were imaged on 2.5% agarose pads, ventrally mounted embryos were imaged using clay feet as spacers between the slide and coverslip. Embryos were imaged using a spinning disk confocal microscope with a Nikon TiE stand and a 60X 1.4NA Plan Apo immersion oil objective (Nikon), CSUXI spinning disk head (Yokogawa), and an ImagEM EMCCD (Hamamatsu). For analysis of coupling, images were collected in sets where the membrane was imaged on the 1st and 7th frames, and myosin was imaged in every frame. Optical sections of 0.5 μm were collected to a depth of 2 μm from the surface of the embryo. In doing this, a membrane volume was collected every ~34.3 seconds while myosin volumes were collected every ~5.7 seconds. Z-projections were analyzed using ImageJ and our automated analysis pipeline (see Methods below).

Myofiber images were captured on a Leica DMRXA upright spinning disc confocal microscope. Objectives were 10×/0.3 NA HC Plan Fluotar, 20×/0.7 NA HC Plan Apo, or 40×/0.85 NA HC X Plan Apo (correction collar). Leica was equipped with a Yokagawa CSU10B spinning disk, and images were taken with a Hamamatsu ImagEM EM-CCD. Muscle sections were imaged on either the Leica or Zeiss 510 LSM. Objectives used on the Zeiss were 10×/0.3 NA EC Plan Neofluar, 20×/0.8 NA Plan Apo Chromat, or 63×/1.4 NA oil differential interference contrast Plan Apo Chromat M27 lens (Carl Zeiss). Images were processed using MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices) or the FIJI ImageJ version 1.47 package (NIH) with the additional MacMaster BioPhotonics Facility plugin set. Confocal stacks were projected as maximum intensity images for each channel, background subtracted, and merged into a single image in ImageJ. Brightness and contrast were adjusted for the entire image as necessary. Images were either cropped or merged as necessary, and individual color channels were extracted without color correction or ɣ-adjustment. Images were adjusted and counted manually.

Imaging was executed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany). The system consists of a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. Z-sectioning was performed with a Piezo-driven motorized stage (Applied Scientific Instrumentation, Eugene, OR, United States) using a Leica HC PL APO 63X/1,4-0,6 oil objective. All acquisitions were performed at 20–23°C.
For most experiments, we collected z-stacks with 45 steps at 1.0 μm distance each with 2, 3, 4, or 5 min intervals, respectively, for a total duration of 1–3 h for acquiring early lima bean to 1.5-fold stage embryos. For imaging of the head-on-view, we used 3–4 min intervals to avoid tipping of embryos. For lineaging, we performed long-term imaging for 250 time points at 3 min intervals and with z sampling of 1 μm over a distance of 30 μm. For acquiring of embryos for one time point before and a time-lapse series after UV laser ablation, we used 2 min intervals and z sampling at 1 μm over a distance of 40–45 μm.