Confocal images were acquired using a camera (EM-CCD; Hamamatsu Photonics) and a spinning disk confocal microscope (CSU-10; Yokogawa) mounted on a microscope (Axiolmager; Carl Zeiss) with a Plan-APOCHROMAT 100×/1.4 oil differential interference contrast objective and controlled by by iVision software (Biovision Technologies) or microManager (Edelstein et al., 2010 ). All images within each experiment were acquired using identical settings. Acquired images were processed using ImageJ 1.46r and Photoshop (CS6 Extended; Adobe). 3D reconstructions were built from confocal z-stacks, analyzed, and exported as .mov files using IMARIS 9.0.2 (Bitplane, Inc.). Figures were constructed using Illustrator (CS6 Extended; Adobe), and graphs were exported from JMP Pro (SAS). Movies were annotated using Photoshop, compressed with Handbrake and combined to run alongside other movies using Pic Stitch. Quantitative analyses of AC-invadopodia, invasive protrusion, or BM breach formation was done using either ImageJ, Imaris, or both. For time-lapse microscopy, worms were anesthetized in 0.2% tricaine and 0.02% levamosile in M9 and then transferred to 5% noble agar pads, sealed with VALAP, and imaged at 23°C. See Kelley et. al. for detailed worm staging, microscopy and data handling protocols and video tutorials.
Imagej 1.46r
Manufactured by Adobe
ImageJ 1.46r is an open-source image processing software developed for scientific use. It provides a wide range of tools for image analysis, processing, and visualization. The software supports various file formats and can be used for a variety of applications, including cell biology, microscopy, and medical imaging.
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Lab products found in correlation
Szx16 microscope, by Olympus (2 mentions)
C dsd115 microscope, by Olympus (2 mentions)
Fv10 asw v4, by Olympus (2 mentions)
Prism 6, by Adobe (2 mentions)
Dp72 camera, by Olympus (2 mentions)
Jmp pro, by SAS Institute (2 mentions)
Em ccd, by Hamamatsu Photonics (1 mentions)
Axiolmager, by Zeiss (1 mentions)
Csu10, by Yokogawa (1 mentions)
Zen 2, by Zeiss (1 mentions)
Apotome 2 structured illumination slider, by Zeiss (1 mentions)
Orca flash 4.0 v2, by Zeiss (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Uplansapo 60x 1.20w, by Olympus (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
Fluoview fv1200 confocal microscope, by Olympus (1 mentions)
Axiovert 200 inverted microscope, by Olympus (1 mentions)
Uplansapo 10x, by Olympus (1 mentions)
Fv1200, by Olympus (1 mentions)
4 protocols using imagej 1.46r
1
Confocal Microscopy Imaging and Analysis
2
Quantifying Protein Co-localization in Fibroblasts
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Fixed NIH3T3 fibroblasts were imaged under epifluorescence illumination using an upright Axio Imager.M2 microscope equipped with a 63× 1.4NA objective and with an Apotome 2 structured illumination slider (all from Zeiss). Images were acquired with a black and white CMOS camera (Hamamatsu ORCA Flash 4.0 V2) and ZEN 2 software (Zeiss). For quantification of co‐localization of the EGFP‐tagged proteins with 594‐Phalloidin in fibroblasts, we analyzed the pixel intensities along a virtual line across the cell, excluding the nucleus (ImageJ, http://imagej.nih.gov/ij ). The placing of the line was done using the “actin channel”, and the experimenter was blinded to the transfections. We plotted the pixel intensities of both channels against each other and calculated the Pearson’s correlation coefficient (GraphPad Prism 7). Each individual experiment consists of 1–4 transfected wells. The person who analyzed the co‐localization and calculated Pearson’s coefficient values was blind to the transfection.
The dendritic spines of cultured hippocampal neurons were imaged using a Zeiss LSM 710 upright confocal microscope (63× 1.3NA objective) or an Axio Imager.M2 microscope (63× 1.4NA objective, Apotome 2 structured illumination slider). Image files were processed with ZEN 2012 (Carl Zeiss Microscopy GmbH), ImageJ 1.46r, and Photoshop CS4 (Adobe).
The dendritic spines of cultured hippocampal neurons were imaged using a Zeiss LSM 710 upright confocal microscope (63× 1.3NA objective) or an Axio Imager.M2 microscope (63× 1.4NA objective, Apotome 2 structured illumination slider). Image files were processed with ZEN 2012 (Carl Zeiss Microscopy GmbH), ImageJ 1.46r, and Photoshop CS4 (Adobe).
3
Confocal Imaging of Neural Crest Cells
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Confocal images were acquired using an Olympus Fluoview FV1200 confocal microscope and Olympus FV10-ASW v4.1 software. Olympus UPlanSApo 60X/1.20W and Olympus UPlanSApo 10X/0.45 objectives were used in this study. For all confocal imaging, embryos were embedded on cover slips in 1% low melt agarose. For analysis of filopodia dynamics, z-stacks of the leading edge of NC streams 1–2 (cranial, n = 6) or of trunk NC cells between somites 6–8 (trunk, n = 6) in 26 hpf Tg(sox10:rfpmb) and Tg(sox10:rfpmb); fscn1a MZ embryos were acquired every 4 minutes for one hour using the 60X water objective (S1 –S2 Movies). To monitor NC migration and individual cell behaviors, z-stacks were acquired every 25 minutes for 18 hours using the 10X objective (S3 –S4 Movies). Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CS5 and CS6, and Adobe Illustrator CS6 were used to generate figures.
4
Imaging Cranial Neural Crest Dynamics
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Confocal images were acquired using an Olympus Fluoview FV1000XY, FV10i or FV1200 confocal microscopes and Olympus FV10-ASW v4.1 software. All imaging was performed using Olympus UPlanSApo 60X water and Olympus UPlanSApo 10X objectives. Embryos were embedded in 1% low melt agarose on cover slips for all confocal imaging. For analysis of filopodia dynamics, z-stacks of the leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 of each) were acquired every 2 minutes for 1 hour using the 60X water objective. For analysis of NC stream depth, z-stacks were acquired of NC stream 3 in 26 hpf Tg(sox10:rfpmb; sox10:h2a-gfp) embryos injected with tp53MO or tp53MO plus fscn1aMO. Cranial NC migration was imaged in 22, 25, 28 and 36 hpf Tg(sox10:GFP) embryos using a Zeiss Axiovert 200 inverted microscope configured with an Olympus DP72 camera. Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CC 2014–2015, and Adobe Illustrator CC 2014–2015 were used to generate figures.
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