T-tubule power was determined by previously described methods. The WGA stained LV samples were imaged on an Olympus FV1000 confocal microscope (diode-pumped 559 nm laser, 60×/1.35 NA oil, FluoView 4.2) to a pixel size of 80 nm. 10–12 areas were imaged at 60 × per LV section. Images were analyzed in ImageJ (Fiji). All images underwent a 2 pixel median filter, and 0.3 pixel background subtract. FFT was performed along a selected 20 µm line (Extended Data 3). 2–4 individual myocytes were analyzed per image. T-tubule power was determined from a power spectrum obtained from the FFT.
Fluoview 4
Manufactured by Olympus
Sourced in Japan, Belgium
The FluoView 4.2 software is a versatile imaging software developed by Olympus for use with their FluoView confocal microscopy systems. It provides essential functionalities for image acquisition, processing, and analysis in a user-friendly interface.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000 confocal microscope, by Olympus (3 mentions)
Axiovision software, by Zeiss (1 mentions)
Fv10i, by Olympus (1 mentions)
Goat anti rabbit alexafluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield hard set antifade mounting media with dapi, by Vector Laboratories (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Fv1000d, by Olympus (1 mentions)
9 protocols using fluoview 4
1
Quantifying T-tubule Structure in LV
2
Ribosome Footprint Data Visualization and Analysis
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Ribosome footprint densities for individual transcripts in Figure 2 were visualized with Trips-Viz browser (Kiniry et al. 2019 (link)) using aggregated data listed in Supplemental Table S6 . All other plots were generated using Python Matplotlib library (Hunter 2007 (link)) and R ggplot2 package (Wickham 2009 ). Statistical analyses were performed with the Statistical Functions module from SciPy library and with R (R Core Team 2013 ). Analysis of microscopy and immunostaining data was performed using FluoView 4.2 software (Olympus) and Microsoft Excel.
3
Cardiomyocyte Cell Area Quantification
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Fluorescent images of labelled tissue sections were imaged on an Olympus FV1000 confocal microscope (diode-pumped 559 nm laser, 20×/0.8 NA oil, FluoView 4.2). 5–8 different regions from each stained LV slice were imaged at 20×. Analysis was performed using ImageJ (Fiji). A two-pixel median filter was applied to all images. Individual cardiomyocytes deemed to be lying parallel to the imaging, in full longitudinal view were traced around the circumference to measure cell area (µm2). For each region, 5–10 different cells were measured and an average cell size presented.
4
Neurite Outgrowth Visualization and Quantification
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Explants were fixed in 4% phosphate buffered PFA and neurite outgrowth was visualized by immunostaining for β-tubulin using the monoclonal mouse anti-β-tubulin III antibody (1:500, T8660 clone SDL.3D10, Sigma-Aldrich, MO, USA), as described[43 , 44 (link)]. β-tubulin III stained explants were imaged by the FV1000-D laser confocal scanning microscope controlled by Fluoview 4.0 software (Olympus Corporation, Japan) and analyzed using Axiovision software (Zeiss, Germany). Automated analysis of axonal outgrowth was performed by measuring the neurite outgrowth area, as previously described[43 , 44 (link)].
5
Visualizing Leaf Epidermal Cells
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At 3 dpi (days post infiltration), the epidermal strips of N. benthamiana leaves were mounted in water and added to a 22 × 22-mm cover slip for direct observation by fluorescence microscopy. Observations were made with a confocal microscope (OLYMPUS, FV10i) using an excitation wavelength of 405, 488, 556 nm, and the images were collected by Olympus Fluoview 4.0 software.
6
Immunofluorescent Localization of α-Chymase in Cardiomyocytes
7
NF-κB Activation in Hypoxic ARPE-19 Cells
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ARPE-19 cells were seeded at 3×104 cells/well and incubated for 12 h in poly-D-lysine-coated 24-well plates for cell attachment, followed by incubation at 1% O2 in a hypoxic incubator for 24 h. Cells were fixed with 4% paraformaldehyde for 5 min at room temperature and blocked with 5% NDS for 2 h in room temperature. Blocked cells were incubated with anti-p-NF-κB (1:1,600; cat. no. 3033) for overnight at 4°C. Subsequently, cells were incubated with donkey anti-rabbit immunoglobulin G secondary antibodies (1:200; cat. no. A-21206) for 1 h at room temperature and then mounted with ProLong Gold Antifade Mountant with DAPI overnight at room temperature (P36931). The images of coverslips were captured using fluorescence microscopy (magnification, ×200; Olympus Corporation). Data were analyzed using the Olympus Fluoview 4.2 software (Olympus Corporation).
8
Multimodal Imaging of ALS+FTD Pathology
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Wide‐field images for qualitative or quantitative analysis were acquired using a Zeiss Z2 Axioimager microscope with MetaSystems VSlide slide scanning software (20× dry magnification lens, 0.9 NA) with a Colibri 7 solid‐state fluorescent light source. Filters were described and validated previously [33 (link)]. Exposure times for each channel were set per case because of differences in immunoreactivity; UBQLN2‐linked ALS+FTD case MN17 showed poor immunoreactivity overall, likely because of long‐term fixation, therefore longer exposures were used. Confocal images were acquired using an Olympus FV1000 confocal microscope (100× magnification oil immersion lens, 1.4 NA, Z‐step 0.4–1 μm) with FluoView 4.2 software (Olympus). Maximum intensity Z‐projections were generated and processed using FIJI software (v1.53c, National Institutes of Health).
Final figures were compiled using FIJI and Adobe Illustrator (Adobe Systems Incorporated, v24.3). Extracted single‐channel images were imported into FIJI, merged and pseudocoloured. For each channel, image intensity was adjusted in FIJI to best view the marker of interest, reduce background autofluorescence and generate images representative of ubiquilin 2 and TDP‐43 aggregate burden in each region.
Final figures were compiled using FIJI and Adobe Illustrator (Adobe Systems Incorporated, v24.3). Extracted single‐channel images were imported into FIJI, merged and pseudocoloured. For each channel, image intensity was adjusted in FIJI to best view the marker of interest, reduce background autofluorescence and generate images representative of ubiquilin 2 and TDP‐43 aggregate burden in each region.
9
Retinal Whole-Mount Analysis with Confocal Microscopy
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Retinas were examined and photographed using a confocal laser confocal scanning microscope (FV1000-D, Olympus Corporation, Berchem, Antwerp, Belgium), controlled by Olympus Fluoview 4.2 software. Retinal whole-mount reconstructions were obtained by combining the different frames captured contiguously side-by-side in a raster scan pattern, with a 20× objective. A Z-stack of each frame of the entire RGC layer (20–30 planes, 3 μm interval) was created and a maximum intensity projection image was generated. All frames of a retina were combined automatically into a single image of the whole retina. Automated cell counting and isodensity maps were done using the in-house developed ImageJ script, as previously described [60 (link)].
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