Images were obtained with the Leica TCS SP5 and the Zeiss LSM880 and LSM780 confocal microscopes. The same imaging acquisition parameters were used for all the comparative analyses. Images were processed with the Imaris Software (Oxford Instruments), Fiji, or Photoshop CS4 (Adobe). For DNA quantification and nuclear size of salivary glands, DNA staining intensity in the salivary glands and tracheal cells was obtained from z stacked images every 0.25 μm of DAPI-stained L3 larvae. Image analysis was performed using Fiji. For the volumetric calculation of the wing pouch region in Imaris Software (Oxford Instruments), the regions of interest were selected based on the RFP fluorescence (induced by nub-Gal4) in confocal stacks that included the whole disc. The surface function of Imaris was used to segment the wing pouch and the surface volume was calculated by the software. Adult flies, nymphal parts, and adult cockroach images were acquired using AxioImager.Z1 (ApoTome 213 System, Zeiss) microscope, and images were subsequently processed using Photoshop CS4 (Adobe).
Imaris software
Manufactured by Oxford Instruments
Sourced in Switzerland, United Kingdom, United States, Germany
Imaris is a powerful 3D/4D visualization and analysis software developed by Oxford Instruments. It allows users to render, analyze, and quantify complex biological and material science data from a variety of microscopy techniques, including confocal, multiphoton, and light sheet imaging.
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Lab products found in correlation
Lsm 880, by Zeiss (14 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
Lsm 710, by Zeiss (7 mentions)
Lsm 780, by Zeiss (7 mentions)
Sp8 confocal microscope, by Leica (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Lsm 700, by Zeiss (6 mentions)
Lsm 510, by Zeiss (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Fluoview fv1000, by Olympus (6 mentions)
Axio observer z1, by Zeiss (6 mentions)
Sp8x confocal microscope, by Leica (6 mentions)
Lsm 880 confocal microscope, by Zeiss (6 mentions)
Sp5 confocal microscope, by Leica (5 mentions)
Zen black software, by Zeiss (5 mentions)
Axio observer, by Zeiss (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Tcs sp5 confocal microscope, by Leica (4 mentions)
Matlab, by MathWorks (4 mentions)
319 protocols using imaris software
1
Confocal Imaging and Image Analysis Protocol
2
Quantifying Infected Satellite Cells in Myofiber Cultures
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Analysis was carried out using a Leica TCS SPE confocal microscope. Images were processed with either Adobe Photoshop CS5 software (Adobe Systems) or ImageJ (version 1.47v; National Institutes of Health, USA, ImageJ.nih.gov/ij">http://ImageJ.nih.gov/ij ).
Infected satellite cells in myofiber cultures were directly counted under a Leica fluorescent microscope at 40x and 100x magnification.
Mean ± standard error (SEM) was given. The single (*), double (**) and triple (***) asterisks represent p-values p < 0.05, p < 0.01, and p < 0.001 respectively by Student's unpaired t-test or Mann–Whitney U-test. All experiments have been performed on at least three independent experiments for each condition.
Supplementary Movies were performed using a DSD2 Workstation with Imaris software (ANDOR).
Infected satellite cells in myofiber cultures were directly counted under a Leica fluorescent microscope at 40x and 100x magnification.
Mean ± standard error (SEM) was given. The single (*), double (**) and triple (***) asterisks represent p-values p < 0.05, p < 0.01, and p < 0.001 respectively by Student's unpaired t-test or Mann–Whitney U-test. All experiments have been performed on at least three independent experiments for each condition.
Supplementary Movies were performed using a DSD2 Workstation with Imaris software (ANDOR).
3
Quantifying Apical Area of Salivary Gland Cells
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SGs in early (pre-invagination; invagination depth, 0 µm) and late (during invagination; invagination depth, 5–10 µm) stage 11 of embryos were used. Confocal images of the SG were obtained from embryos immunostained for the Ecad (adherens junctions) and CrebA (SG cell nuclei) and used for cell segmentation. A maximum intensity projection was generated using the LasX program from three apical z-sections of confocal images of SG cells encompassing the adherens junction. Using the maximum intensity projection, cells were segmented using Ecad signals as a marker for each cell boundary using Imaris software (Andor). The apical area was calculated using Imaris and individual cells were color-coded based on the size of their apical areas. The frequency distribution of the apical area of the cells was analyzed using GraphPad Prism with the data exported from Imaris. Since the output data from Imaris for the apical area quantification is a sum of the area of both sides of the cell layer, we further divided the values by 2 to obtain the apical area of SG cells.
Scatter plots were generated using the X and Y coordinates for each SG cell, which were determined with respect to the A/P and D/V axes of the tissue using Imaris. The cell at the center of the SG placode was used as the origin point (X=0, Y=0).
Scatter plots were generated using the X and Y coordinates for each SG cell, which were determined with respect to the A/P and D/V axes of the tissue using Imaris. The cell at the center of the SG placode was used as the origin point (X=0, Y=0).
4
Visualizing Autophagy with mCherry-GFP-LC3B
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Cells transfected with pDEST-CMV mCherry-GFP-LC3B WT (a gift from Robin Ketteler, Addgene plasmid # 123230, Watertown, MA, USA) were exposed to different treatments. Cells were washed with PBS and then the coverslips were carefully placed and slight pressure was applied to remove the remaining bubbles. Confocal images were obtained with an Andor BC43 Benchtop confocal microscope (Andor, Belfast, UK) at the single-line excitation (529 nm for GFP, or 600 nm for mCherry). Images were acquired with the Fusion software (Andor) and processed with the Imaris software (Andor).
5
Multiphoton Microscopy for 3D Tumor Vasculature
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Bright field images were obtained on a MVX10 Research MacroView Microscope (Olympus) with a DP73 camera at 16-bits (Olympus). Multiphoton fluorescence images were acquired in frame scanning mode with 12 bits per pixel, x = 256, y = 256. The z stack depth was variable between 200 and 300 samples depending on the experiment (each z step = 2 μm) and thickness of the tumor. Image stacks were sampled at 5 minute intervals due to the time required to acquire multiple planes in the z-axis. Images stacks were segmented and rendered to 3D volumes. 3D volumes and fluorescence intensity were automatically quantified by Imaris software (Bitplane AG, Switzerland). Vessel diameters were measured manually using the «Measuring Distances» tool in Imaris, by choosing delimiting points on each side of the vessel walls. Images were background corrected and median filtered equally across treated samples and controls. Figures were prepared using Adobe Photoshop CS6 (version 13.0) after conversion of RGB or grayscale images.
6
Differential Phase Contrast Imaging of Carotid Plaques
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The presented data sets were obtained by synchrotronbased X-ray tomography using the differential phase contrast technique [4, (link)6] at a photon energy of 25 keV. Carotid artery segments were placed in a falcon tube filled with PBS. To measure the phase shift, a grating interferometer was used and series of projection images were taken while the carotid plaque specimens rotated. A 300 lm thick LAG:Ce scintillator converted the X-rays to visible light, which were captured by a CMOS camera (PCO.Edge, PCO AG, Kelheim, Germany). The total scan time for a segment with a field of view (FOV) of 12 mm 9 3.5 mm was about 90 min. More details about components and parameters are provided in Table S1.
To obtain 3-dimensional images of the DPC data sets, individual Tiff files were converted to a multilayer Tiff file and processed by Imaris software (Bitplane AG, Zurich, Switzerland).
To obtain 3-dimensional images of the DPC data sets, individual Tiff files were converted to a multilayer Tiff file and processed by Imaris software (Bitplane AG, Zurich, Switzerland).
7
Collagen Quantification in 3D Multiphoton Imaging
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As previously described [26] , 3D stacks of 425x425x60 μm were acquired using a 7MP multiphoton laser scanning microscope (Carl Zeiss, Jena, Germany), equipped with a 20x objective and coupled to a Tisapphire femtosecond laser, Chameleon Ultra 2 (Coherent Inc) tuned to 800 nm. Organized fibrillar collagens were detected by second harmonic generation at a wavelength of 400 nm.
The acquired 3D images and collagens quantification were analyzed off-line with Imaris software (Bitplane AG). Briefly the volume occupied by the collagen was calculated by measuring the volume occupied by the isosurface of the SHG in a cube of defined volume (80,1 x 80,1 x 80 µm). Three independent samples were used for each condition.
The acquired 3D images and collagens quantification were analyzed off-line with Imaris software (Bitplane AG). Briefly the volume occupied by the collagen was calculated by measuring the volume occupied by the isosurface of the SHG in a cube of defined volume (80,1 x 80,1 x 80 µm). Three independent samples were used for each condition.
8
Quantifying Matrigel Viscoelasticity and Cell Migration
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Each experiment was repeated at least three times. The error bars represent the mean ± s.e.m. For viscoelasticity measurements and western blotting analysis, open source Image J (National Institutes of Health) with some plugins was used (Figs 1b,d and 5d ). Displacement of latex beads embedded in the matrigel was analyzed by using Imaris software (Bitplane AG) (Fig. 3a and Supplementary Figure 2a ), and was described by box-plot using R software (R Development Core Team, Vienna, Austria) (Fig. 3b and Supplementary Figure 2b ). We performed all statistical analyses using student’s t-test, and considered a P-value of <0.05 as significant.
9
Liver Targeting Ability of Nanoparticles
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To detect the liver targeting ability of the nanoparticles, immunofluorescent staining was performed on the liver sections. In brief, the rats were fasted overnight before the study and then administered orally with FITC-labeled nanoparticles. After treatment for 4 h, the rats were sacrificed, and the livers were isolated. Then, the frozen sections of the liver were obtained using a cryostat and stained using anti-ASGPR rabbit pAb as the primary antibody, and Alexa 647 labeled goat anti-rabbit IgG as the secondary antibody. Afterward, the liver sections were stained with DAPI, and the colocalization signals of nanoparticles with ASGPRs were observed by CLSM. The colocalization coefficient (R) was quantified using Imaris software (Bitplane AG, Switzerland).
10
Two-Photon Imaging of Secretory Organs
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WGA-stained or immuno-stained secretory organs were observed with an upright two-photon microscope (FV1200MPE: Olympus, Tokyo, Japan) equipped with a water immersion objective lens (XLPlaN25 × 1.05WMP, Olympus) which was connected to a femtosecond laser source, Ti:sapphire laser (MaiTaiHP: Spectra Physics, Santa Clare, Ca, USA). The excitation wavelength for YC3.60 probe, WGA, and Alexa-555 was 830 nm, and the emission was simultaneously detected through a band-pass filter for YC3.60 (510−550 nm) and for WGA and Alexa 555 (575−630 nm). Fluorescence images at a depth of approximately 200 µm from the surface of each secretory organ were reconstructed from 200 images acquired at z-step sizes of 1 µm using Imaris software (Bitplane AG, Zurich, Switzerland).
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