High-resolution 3-dimensional images were acquired with a Zeiss LSM 710 confocal microscope using Zeiss ZEN 2.3 SP1 FP3 (black) (Version 14.0.18.201; Zeiss) software and a 20×/0.8 air or 63×/1.4 oil DIC M27 Plan-Apochromat objective, except for whole bowel E12.5 and E13.5 imaging, which employed a 5× objective and the ZEN Tile Scan function. Image stitching employed ZEN software. Z-stacks were created with ImageJ/FIJI. Increments between each slice were 1 μm, except for scanning the whole E10.5 embryo, where increments between slices were 3 μm. Videos of Z-stacks were created with the animation function of Imaris 9.02.
Zen 2.3 sp1 fp3 black
Manufactured by Zeiss
Sourced in Germany
The ZEN 2.3 SP1 FP3 (black) is a software package developed by Zeiss for microscopy imaging and analysis. It provides core functionalities for image processing and data management.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Lsm 880, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ethidium bromide, by Thermo Fisher Scientific (1 mentions)
Nvision40 sem, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Nir apo 60x wd 2, by Nikon (1 mentions)
Nova nanosem 230 microscope, by Thermo Fisher Scientific (1 mentions)
Eclipse ni u, by Nikon (1 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (1 mentions)
5 protocols using zen 2.3 sp1 fp3 black
1
High-Resolution 3D Imaging of Embryos
2
Live Cell Imaging with Fluorescent Dyes
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Live cell staining was performed using 10 mg/mL Hoechst 33,342 and 5 μg/mL ethidium bromide (both Life Technologies, New York, NY, USA) in a supplemented cell growth medium to stain body and nucleus of cells in green and red, respectively. Cells with Hoechst 33,342 were incubated for 15 min at 37 °C, and then with ethidium bromide for 5 min at 37 °C. The live imaging was conducted at day in vitro (DIV) 1 and DIV 2. The fluorescence imaging was performed via a laser scanning microscope (LSM880, Carl Zeiss, Berlin, Germany) using the Zen software (Zen 2.3 SP 1 FP3 Black, Carl Zeiss Microscopy GmbH, Jena, Germany) (see Figure S2 ). In each case, three images were acquired. The SEM images were obtained on a NVision 40 SEM (Zeiss, Oberkochen, Germany).
3
Confocal Microscopy Imaging Protocol
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The samples were observed with a Carl Zeiss LSM 780, utilizing the 100× objective with oil immersion, and the software ZEN 2.3 SP1 FP3 Black (Carl Zeiss), using an Airy Unit of 1 for all scans. Image acquisition was performed with as a z-stack at maximum scan speed, using a frame size of 512 × 512 pixels and an average of 2 with Line mode, Mean method, and 8 Bit depth, to obtain an image size of 85.0 μm × 85.0 μm.
Image processing of the CLSM images was done with the Imaris ver. 9.0.0 (Bitplane AG). In brief, the image was loaded into the “Surpass” function with “3D View” and “Smoothing” was performed with “Median Filter” on all channels with the filter size of 3 × 3 × 1, followed by “Background Subtraction” on all channels with the default “Filter Width”. “Volume” and “Ortho Slicer” was checked under “Scene,” with the “XY Plane” option was checked under “Slice Orientation,” the maximum thickness chosen for “Extended Section” and “Slice Position,” and the “Show Frame” option unchecked. The scale bar was adjusted accordingly before the image was exported as a TIFF image with the “Snapshot” function.
Image processing of the CLSM images was done with the Imaris ver. 9.0.0 (Bitplane AG). In brief, the image was loaded into the “Surpass” function with “3D View” and “Smoothing” was performed with “Median Filter” on all channels with the filter size of 3 × 3 × 1, followed by “Background Subtraction” on all channels with the default “Filter Width”. “Volume” and “Ortho Slicer” was checked under “Scene,” with the “XY Plane” option was checked under “Slice Orientation,” the maximum thickness chosen for “Extended Section” and “Slice Position,” and the “Show Frame” option unchecked. The scale bar was adjusted accordingly before the image was exported as a TIFF image with the “Snapshot” function.
4
Multimodal Imaging of Stretched Cells
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Fluorescence live cells images were acquired with Metamorph software using an upright microscope (Nikon eclipse Ni-U) with a 60× water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca Flash 4.0 camera (Hamamatsu). Fluorophore emission was collected every 3 s. Cells were imaged in a relaxed state and then for 3 min at 5% stretch, and for 3 min during the release of stretch. Fixed cells images were acquired either in the abovementioned acquisition system except for the experiments related to WAVE protein enrichment. In this case, images were acquired in a Zeiss Airyscan microscope (Zeiss LSM880 inverted confocal microscope objective, using Zeiss ZEN2.3 SP1 FP3 [black, version 14.0.24.201] software and a 63×1.46 NA oil immersion objective). Z-stack of single cells were acquired in full Airyscan mode to visualize the PM and the WAVE-C-GFP protein. SEM images were taken using the xTm Microscope Control software in a NOVA NanoSEM 230 microscope (FEI Company) under the high vacuum mode using ET and TL detectors to acquire high and ultra-high resolution images of the cell surface. TEM samples were observed in a Jeol 1010 microscope (Gatan, Japan) equipped with a tungsten cathode in the CCiTUB EM and Cryomicroscopy Units. Images were acquired at 80 kV with a CCD Megaview 1kx1k.
5
Confocal Microscopy Imaging Protocol
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Coverslips were imaged using a Zeiss LSM 710 laser scanning confocal microscope and Zeiss ZEN 2.3 SP1 FP3 (black) (version 14.0.18.201) software. Images were acquired with a ×20/0.8 air or ×63/1.4 oil DIC M27 Plan-Apochromat objective. The Zeiss LSM 710 condenser numerical aperture is 0.55. Confocal images were processed in ImageJ (NIH) to crop, scale, and uniformly color adjust. Confocal images are represented as “sum of slices” or “maximum intensity” projections after ImageJ processing.
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