We imaged cells on a Nikon Eclipse Ti inverted microscope (Nikon Instruments) fitted with a CSU-X spinning disk confocal head (Yokogawa), four solid-state lasers (Nikon), IXon X3 EMCCD camera (Andor), and emission filter wheel (Sutter Instruments). The imaging area was kept at 37 °C with 5% CO2 (In Vivo Scientific, LCI). We used a 100 × 1.45 NA Plan Apo oil immersion objective (Nikon). Images were acquired using Nikon Elements. We generally imaged 3–7 z slices with 300 nm z spacing at 3 s time intervals. Cells were seeded on sterile 4-chambered or 8-chambered #1.5H (0.170 ± 0.005 mm) cover glasses (CellVis). We imaged the cells in media supplemented with HEPES and the antioxidant oxyrase (OxyFluor) with substrate lactate. For quantitative fluorescence experiments we limited the cells’ exposure to ambient light, brightfield light and 488 nm laser light prior to acquisition, and used the same laser power during acquisition to compare experiments (generally 10% acousto-optic tunable filter (AOTF) power). For most experiments, we used DMEM/F12 without phenol red (Thermo Fisher) supplemented with the protein supplement used in StemFlex media (Thermo Fisher), which gave similar fluorescence intensity results as cells imaged in StemFlex (which has phenol red).
Ixon x3 emccd camera
Manufactured by Oxford Instruments
Sourced in United States
The IXon X3 EMCCD camera is a high-performance imaging device designed for low-light applications. It features an electron-multiplying CCD sensor that enhances the signal-to-noise ratio, enabling the capture of faint signals. The camera offers fast frame rates and high quantum efficiency to provide sensitive, high-quality images.
Automatically generated - may contain errors
Lab products found in correlation
Metamorph software, by Molecular Devices (3 mentions)
Ff01 523 610 625 filter, by IDEX Corporation (2 mentions)
Ff01 523 35 filter, by IDEX Corporation (2 mentions)
Nis elements software, by Nikon (2 mentions)
Ti e inverted microscope, by Nikon (2 mentions)
Dmem f12 without phenol red, by Thermo Fisher Scientific (1 mentions)
Stemflex media, by Thermo Fisher Scientific (1 mentions)
Emission filter wheel, by Sutter Instruments (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Plan apo oil immersion objective, by Nikon (1 mentions)
Glass slide, by Matsunami (1 mentions)
W view gemini, by Hamamatsu Photonics (1 mentions)
Imagej, by Adobe (1 mentions)
Elements controlled eclipse 90i, by Nikon (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Clara cooled ccd camera, by Oxford Instruments (1 mentions)
Plan apo vc, by Oxford Instruments (1 mentions)
Nis elements 3, by Nikon (1 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
10 protocols using ixon x3 emccd camera
1
Live-cell Confocal Imaging of Cells
2
VIAFM Imaging of Root Epidermal Cells
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Root epidermal cells of 7-d-old seedlings expressing fluorescently tagged PICALM1 and VAMP721 or CLC were observed using an IX-71 (Olympus) equipped with a UAPON 100× O TIRF lens (Olympus) for VIAFM. The roots were placed on a glass slide (76 mm × 26 mm, Matsunami) and covered with a 0.12- to 0.17-mm-thick coverslip (24 mm × 60 mm, Matsunami). For VIAFM observations, GFP and mKO or TagRFP were simultaneously excited using 473- and 561-nm lasers, respectively. A FF01-523/610-625 filter (Semrock) was used to remove autofluorescence of samples. The fluorescence emission spectra were separated using a FF560-FDi01-25 × 36 LP dichroic mirror (Semrock) and filtered through a FF01-523/35 filter (Semrock) for GFP and a ET620/60M filter (Chroma) for mKO and TagRFP, using W-View GEMINI (Hamamatsu Photonics). VIAFM images were acquired using the iXon X3 EMCCD camera (Andor Technology) operated with Metamorph software (Molecular Devices). Each frame was exposed for 200 ms. The acquired images were analyzed using ImageJ and Photoshop CC (Adobe Systems).
3
Quantitative Live-Cell Microscopy Techniques
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Cells were imaged on a Ti-E inverted microscope (Nikon Instruments) with Nikon objectives Plan Apo λ 60× 1.42 NA and APO 100× 1.49 NA with a Spectra-X light engine (Lumencore) and environmental chamber at 37°C. A Clara cooled charge-coupled device (CCD) camera (Andor) and iXon X3 EMCCD camera (Andor) were used. The Ti-E microscope is driven by NIS Elements software (Nikon Instruments). Gliding filament assays were performed on a Nikon Elements controlled Eclipse 90i (Nikon) equipped with a 60× 1.4 NA (Nikon) objective and a Cool Snap HQ2 CCD camera (Roper). Spindle collapse assay cells were acquired using a 60× 1.4 NA objective (Olympus) on a DeltaVision Elite imaging system (GE Healthcare) equipped with a Cool SnapHQ2 CCD camera (Roper).
4
Microscopic imaging of molecular motors
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The cells were imaged on a Nikon Ti-E inverted microscope (Nikon Instruments) with Nikon objectives Plan Apo 40× DIC M N2 0.95 NA, Plan Apo λ 60× 1.42NA, and APO 100× 1.49 NA with a Spectra-X light engine (Lumencore) and environment chamber. The following cameras were used: Clara cooled-CCD camera (Andor) and iXon X3 EMCCD camera (Andor). The Nikon Ti-E microscope is driven by Nikon Instruments Software (NIS)-Elements software (Nikon Instruments). TIRF microscopy was performed at room temperature using an inverted Eclipse Ti-E microscope (Nikon) with a 100× Apo TIRF objective lens (1.49 N.A.) and dual iXon Ultra Electron Multiplying CCD cameras, running NIS-Elements version 4.51.01. Rhodamine-labeled microtubules were excited with a 561 and a 590/50 filter. GFP-WT KIF18A1–480 or GFP-sNL1 KIF18A1–480 motors were excited with a 488 laser and a 525/50 filter. Alexa 647–labeled tau was excited with a 640 laser and 655 filter. Alexa 532–labeled rigor kinesin was excited with a 561 laser and a 590/50 filter. All movies were recorded with an acquisition time of 100 ms.
5
Multimodal Fluorescence Imaging Protocol
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Except where noted otherwise, all images were acquired on Nikon Eclipse Ti inverted Yokogawa spinning disk confocal microscope fitted with Andor CSU-X spinning disc confocal equipment and controlled by Nikon Elements software. Imaging was performed using a 100× 1.45 NA Plan Apo λ oil immersion objective and an Andor IXon X3 EM-CCD camera. GFP, RFP, and Atto647 fluorescence was excited using 488-, 561- and 638-nm lasers, respectively. Images for figure panel S1C were collected using a Nikon Eclipse Ti microscope equipped with a 100× 1.4 NA Plan Apo VC oil objective and an Andor Neo sCMOS camera. The system was controlled using Metamorph software (Molecular Devices). Images for figure panels 4A (lower subpanel) and 4B (both subpanels) were acquired on a AxioObserver Zeiss LSM 710 Laser Scanning Confocal with 40× 1.4 NA Plan Apo oil immersion objective and PMT detector. GFP and Atto647 fluorescence was excited using 488- and 633-nm lasers, respectively. The system was controlled with Zen 2010 software. All imaging devices were kept in rooms maintained at 23 to 25°C.
6
STORM Imaging of FLAP and 5-LO
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After activation, cells were fixed and prepared for STORM as previously described [39 (link)]. The following day they were stained with antibodies to FLAP (Novus IMG 3160, 1:100) or to 5-LO (Santa Cruz H-120, sc-20785, RRID: AB_2226938, 1:20). Activator-reporter antibody was applied at 3 μg/mL for 1 h. Imaging buffer containing 147 mM βME and 1% (v/v) glucose oxidase with catalase (GLOX) was used to promote photoswitching and reduce photobleaching [39 (link)]. Cells were imaged in continuous mode on an inverted Nikon Ti-Eclipse STORM 3.0 system equipped with 100X/1.4 NA objective lens, iXon X3 EM CCD camera (Andor), and 647 nm (300 mW), 561 nm (150 mW) and 405 nm lasers (100 mW). 9,000 frames for each dye were collected at 30 ms exposure time. Localizations were identified with NIS Elements 3.0 (Nikon Instruments) and exported as tab-delimited text files.
7
Mechanically Induced Lethargus Disruption
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The 1-h deprivation protocol consisted of 3-min-long vibration pulses interspersed with 3-min-long pauses, starting during the first 30 min of L4 lethargus and lasting a total of 1 h. Animals expressing a fluorescent reporter were exposed to these mechanical stimuli in artificial dirt microfluidic devices placed inside a standard petri dish. They were imaged in the device immediately before and after the 1-h period of disruption. Imaging was performed at a magnification of 20× (0.5 numerical aperture, NA) using a Nikon Eclipse Ti microscope (Nikon Inc., Melville, NY, USA) and an Andor iXon X3 EMCCD camera (Andor, Belfast, UK). Fluorescence intensity was determined using custom Python scripts.
8
Single-Molecule FISH Imaging Protocol
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Bacterial cells were grown and treated as indicated above. Single-molecule fluorescence in situ hybridization (smFISH) experiments were performed following previously published procedures with the indicated modifications provided here and in the Supplementary Methods (27 (link)). Images were taken on a Nikon Eclipse –Ti-E microscope controlled by the NIS-Elements software using a 100 × N.A 1.45 oil immersion phase contrast objective lens (Nikon plan-apochromat 100 × 1.45 λ) and an Andor iXon X3 EMCCD camera. All the filters are from Chroma. The filters used were ET-534/30× for excitation, 560lp as dichroic mirror and ET-585/40M for the emission. A phase contrast image was acquired followed by a z-stack of 13 slices and 250 nm spacing of fluorescent images with 2 s integration time of each slice. Each sample was imaged at multiple locations to get a total of at least 500 cells.
9
Calcium Imaging in Microfluidic Devices
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To reduce background fluorescence, calcium imaging was performed in artificial dirt microfluidic devices [133 (link)]. Animals co-expressing GCaMP5 and mCherry in their vms were mounted in the presence of bacterial food on an epi-fluorescence Nikon Eclipse Ti inverted microscope. Each worm was imaged at a magnification of 20× (0.5 NA) and a frame rate of 6 frames per second. Images were captured with an Andor iXon X3 EMCCD camera. A Dual View (DV2) two-channel system was used for simultaneous imaging of the red and green channels (Photometrix, Tucson, AZ, USA). Each animal was tracked manually and continuously imaged for a total period of 30 min. Calcium transients were analyzed using custom Matlab scripts (The Mathworks Inc., Natick, MA, USA).
10
Live Imaging of Secretory Vesicles in Root Hairs
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Arabidopsis seedlings expressing GFP-SYP123 and TagRFP-VAMP727 were grown under continuous white light at 22 ± 2°C on 1/2 MS medium (1% sucrose, 1.2% agarose) in a 35-mm glass-base dish (Iwaki) for total internal reflection fluorescence microscopy. Growing root hairs were observed under an IX-71 (Olympus) equipped with a UAPON 100x O TIRF lens (Olympus), as described in Fujimoto et al. (2020), with some modifications. GFP and TagRFP were simultaneously excited using 473-and 561-nm lasers, respectively. An FF01-523/610-625 filter (Semrock) was used to remove autofluorescence. Fluorescence emission spectra were separated using a FF560-FDi01-25x36 LP dichroic mirror (Semrock) and filtered through a FF01-523/35 filter (Semrock) for GFP and an ET620/60M filter (Chroma) for TagRFP, using DV2 (Photometrics).
Images were acquired using an iXon X3 EMCCD camera (Andor Technology) operated with Metamorph software (Molecular Devices). Each frame was exposed for 150 ms. The images were analyzed using ImageJ.
Images were acquired using an iXon X3 EMCCD camera (Andor Technology) operated with Metamorph software (Molecular Devices). Each frame was exposed for 150 ms. The images were analyzed using ImageJ.
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