Transgenic animals expressing Mito::GFP or mCherry were analyzed at the young adult stage (see table S1 ). To assess mitochondria density in GABAergic neurons after axotomy, selected axons in transgenic animals expressing Mito::GFP or mCherry were cut using a Micropoint laser from Photonic Instruments (10 pulses, 20 Hz). The axotomized animals were recovered to NGM plates and cultured at 20°C. 6, 12 or 24 hours later, images were acquired as 0.3~0.5 μm z-stacks at room temperature on an UltraVIEW Vex (PerkinElmer) spinning disc confocal microscope (Nikon Ti-E Eclipse inverted scope; Hamamatsu C9100-50 camera) with a 60× CFI Plan Apo numerical aperture (NA) 1.4 oil-immersion objective using Volocity software (Improvision). To score mitochondria density, Mito::mCherry puncta in individual GABAergic axons were counted and then the axon length was measured. The number of mitochondria was normalized by the axon length and converted to density per 100 μm axon length. To compare the volume of Mito::mCherry puncta, Volocity software (Improvision) was used to measure the volume of individual puncta. All animals within each experiment were imaged on the same day with identical conditions including camera gain, exposure settings, and fluorescence filters.
Volocity software
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Volocity software is a comprehensive image analysis and visualization platform designed for life science research. It provides tools for 3D and 4D image processing, analysis, and management. The software supports a wide range of microscopy techniques and file formats, enabling researchers to efficiently work with their imaging data.
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Lab products found in correlation
Ultraview vox, by PerkinElmer (31 mentions)
Axiovert 200m, by Zeiss (22 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (16 mentions)
Axiovert 200m microscope, by Zeiss (15 mentions)
Eclipse ti e, by Nikon (15 mentions)
Axio observer z1, by Zeiss (13 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (12 mentions)
Orca er, by Hamamatsu Photonics (11 mentions)
Orca er ccd camera, by Hamamatsu Photonics (11 mentions)
Ultraview, by PerkinElmer (11 mentions)
Lsm 510, by Zeiss (11 mentions)
Orca er camera, by Hamamatsu Photonics (10 mentions)
Eclipse ti, by Nikon (10 mentions)
Lsm 710, by Zeiss (10 mentions)
Em ccd camera, by Hamamatsu Photonics (9 mentions)
Vectashield, by Vector Laboratories (9 mentions)
Softworx, by Cytiva (9 mentions)
Axio imager m2 microscope, by Zeiss (9 mentions)
Imagej, by PerkinElmer (8 mentions)
Vectashield mounting medium, by Vector Laboratories (8 mentions)
827 protocols using volocity software
1
Quantifying Mitochondrial Density in GABAergic Neurons
2
Visualizing Pseudomonas Biofilm Polysaccharides
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PAO1-based strains were grown in LB or NB until Log phase and then diluted in NB 1.4% (v/v) or LB 1% to a final OD600 of 0.01. PA14 and PAO1 Δpsl were grown in LB to mid-log phase and then diluted in modified Jensen’s to a final OD600 of 0.05. Flow cell chambers were inoculated with the diluted cultures and incubated inverted for 1 h before initiation of flow. Biofilms, which were continuously supplied with fresh NB 1.4% or modified Jensen’s at 10 mL/h, were grown for 4 days at room temperature. When needed the media was supplemented with 0.05% arabinose. A Zeiss LSM 510 confocal laser scanning microscope (CLSM) was used to image the biofilms and Volocity software (Improvision) was used for image processing. The biomass was stained with 2.5 µM Syto 62 (Molecular Probes) to visualize the entire biomass before imaging. Fluorescent lectins were used to stain the different polysaccharides and were allowed to interact with the biofilm for 15 min. WFL lectin (100 μg/mL; Vector Laboratories) was used for Pel visualization, and HHA lectin (100 μg/mL; EY Laboratories) for Psl. LecB-FITC (Elicityl, Crolles, France) staining followed the same procedure used with HHA and WFL. To determine Psl localization, each condition had nine images from three different experiments analyzed using lines profile from Volocity software (Improvision)57 (link).
3
Chromosome Dynamics Analysis Pipeline
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The 3D images were first background-corrected with the background image in the Volocity software (PerkinElmer), and noise removal was achieved by convolving the raw images with the 3D Gaussian filter in Imaris (Bitplane). Chromosomes were tracked using the Imaris (Bitplane) 3D surface detection function, followed by manual corrections. The velocity and acceleration of chromosomes were analysed with Imaris using a centre projection function, and the images were processed with the Volocity software (PerkinElmer) and Image J (National Institutes of Health).
4
Measurement of Cellular Volumes in NB Lineages
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Clone volume of NB lineages was measured from 3D reconstructions of 1.5 μm spaced confocal Z stacks with Volocity software (PerkinElmer) or Imaris (Bitplane). Cellular volume, nuclear and nucleolar volumes were estimated with the formula 4/3πr3, with r measured from single confocal sections using the Leica LAS software to average orthogonal measurements of cell diameter (2r). NB diameter was measured from single confocal section using Volocity software as the average of orthogonal measurements. Eye disc clone volume was measured from 3D constructions of 1.5 μm spaced confocal Z stacks with Volocity software (PerkinElmer) or Imaris (Bitplane).
5
Visualizing Mitochondrial Dynamics in Cancer Cells
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Cells were plated in 35-mm glass-bottom dishes (MatTek, Ashland, MA, USA) coated with 10 μg/ml fibronectin (Sigma, Milwaukee, WI, USA) for MDA-MB-231 or 3.25 μg/cm2 Cell-Tak (BD Biosciences, San Diego, CA, USA) for MCF-10A cells. Cells were treated with CT20p and stained with 25 nM Mitotracker Green or 500 nM ER-Tracker Green (Life Technologies). Images were acquired with a PerkinElmer UltraView spinning disc confocal system, with AxioObserver.Z1 stand (Carl Zeiss, Thornwood, NY, USA), in a humidity and temperature-controlled chamber (LiveCell, Seoul, Korea). Time-lapse movies were acquired using a Plan-Apochromat 63 × Oil DIC objective. Images were processed with Volocity software (PerkinElmer, Shelton, CT, USA). Live cell imaging was also used to track mitochondrial movement and velocities calculated using the Volocity software (PerkinElmer).
6
Automated Microscopy Imaging Protocols
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Hematoxalin and eosin images were captured using a scanscope (Aperio CS model, Leica). Scanscope console imaging software was used for automated image capture and processing was performed using Volocity software (PerkinElmer). Immunofluorescence images were captured using an inverted microscope (DMI 6000b, Leica). Digital microscopy images were captured with a digital camera (ORCA-ER, Hamamatsu). Openlab imaging software was used for manual image capture, and processing was performed using Volocity software (PerkinElmer).
7
RSV Infection and Replication in HEp-2 Cells
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HEp-2 cells have been widely used in the study of respiratory viruses as they are readily infected; in this work, they were used as a read out of RSV infection and replication. HEp-2 cells (1 × 104/well) or ALMs (1 × 105/well) were plated in black, flat, clear-bottom 96-well plates and incubated overnight at 37 °C with 5% CO2. Afterwards, cells were washed with serum-free EMEM and incubated with RSV-GFP (MOI 1) for 90 min. Then, unbound virus was removed and fresh EMEM containing 10% (v/v) FBS was added to the cells. RSV-GFP-infected cells were imaged at 24 h intervals for 72 h on an inverted epifluorescence microscope (Nikon TE-2000), using a Hamamatsu C4742-12AG camera and Perkin Elmer Volocity software. The total fluorescence intensity was determined from 4 random fields per well (4× magnification) using Perkin Elmer Volocity software, as previously described [14 (link)].
8
Quantifying RSV Infection in PBMCs
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PBMCs were isolated from healthy volunteer donors who had signed an informed consent form. A total of 20 mL of whole blood was collected and subjected to a Histopaque-1077 (Sigma-Aldrich, cat 10771) gradient centrifugation for 30 min. PBMCs were seeded at 2 × 105/well in EMEM supplemented with 10% (v/v) heat-inactivated FBS overnight. Non-adherent cells were then removed by washing, and adherent cells were exposed to RSV-GFP (MOI 1) for 90 min. Then, unbound virus was removed and fresh EMEM containing 10% (v/v) FBS was added to the cells. RSV-GFP-infected cells were imaged at 24 h intervals for 72 h on an inverted epifluorescence microscope (Nikon TE-2000), using a Hamamatsu C4742-12AG camera and Perkin Elmer Volocity software. The total fluorescence intensity was determined from 4 random fields per well (4× magnification) using Perkin Elmer Volocity software, as previously described [14 (link)].
9
Quantifying HURP Binding on Microtubules
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Ten thousand cells were seeded on coverslips in 12-well plates and grown for 2 d. Cells were washed with PBS, pH 7.4 and then fixed with 4% paraformaldehyde in PBS at RT for 15 min. Cells were then permeabilized with 0.5% Triton X-100 for 10 min and then blocked with 10% horse serum for 30 min. The fixed cells were then stained by specific primary antibodies and AlexaFluor-conjugated secondary antibodies for 1 h at RT. The nuclei were visualized with Hoechst (Invitrogen). Images were acquired using an Olympus IX71 inverted microscope (Olympus America Incorporation), equipped with an Ultraview spinning disk confocal system (Ultraview ERS Rapid Confocal Imager; PerkinElmer) and controlled by Volocity software (PerkinElmer) utilizing an Olympus UPlanSApo 100x/1.4 oil objective. Analysis of images was performed by Volocity software (PerkinElmer). We measured the length of the spindle-associated HURP and the length of microtubules, stained with Tubulin. The length of HURP signal was then divided into that of Tubulin signal in each cell to obtain normalized spread of HURP signal on microtubules, and the data were then plotted and analyzed using Prism software. The significances in difference were analyzed by t test.
10
Confocal and Wide-field Live Cell Imaging Protocol
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For confocal live cell imaging, cells were seeded on glass-bottomed dishes (MatTek) coated with 10 mg/ml fibronectin (Sigma) and imaged in Leibovitz's L-15 Medium (Gibco) using an UltraView Vox (Perkin Elmer) spinning disc confocal microscope with a 60X (NA 1.4) or 100X (NA 1.4) oil objective equipped with a temperature-controlling environmental chamber. Images were acquired using a Hamamatsu ImagEM camera and Volocity software (Perkin Elmer). Wide-field live cell imaging was done using a Zeiss Axiovert 200 M microscope with a 20X objective (NA 0.4) equipped with a temperature-controlling environmental chamber, and images acquired using a Hamamatsu Orca AG camera and Volocity software (Perkin Elmer).
Nuclear envelope rupture experiments were done using an UltraView Photokinesis Device with 405 nm laser.
For the tubulin dilution calculations at NEP, cells were seeded in polydimethylsiloxane (PDMS) chambers incubated overnight with 0.1 mg/ml polyethylene glycol (PEG, Sigma) dissolved in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4.
Osmotic shock was induced either by diluting the imaging media by 50% with deionised water (hypo-osmotic shock) or by adding 4 mM sorbitol (Sigma) solution to an end concentration of 1.3 mM (hyper-osmotic shock). For the control experiments, imaging medium was added.
Nuclear envelope rupture experiments were done using an UltraView Photokinesis Device with 405 nm laser.
For the tubulin dilution calculations at NEP, cells were seeded in polydimethylsiloxane (PDMS) chambers incubated overnight with 0.1 mg/ml polyethylene glycol (PEG, Sigma) dissolved in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4.
Osmotic shock was induced either by diluting the imaging media by 50% with deionised water (hypo-osmotic shock) or by adding 4 mM sorbitol (Sigma) solution to an end concentration of 1.3 mM (hyper-osmotic shock). For the control experiments, imaging medium was added.
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