Samples were imaged using either DIC or epifluorescence on an Olympus IX81 inverted microscope. Fluorescence signal was acquired using a 485(20)-nm (fluorescein) or a 560(25)-nm (Alexa Fluor 594) excitation filter combined with a 525(30)-nm or a 607(36)-nm emission filter, respectively. Excitation light (Metal-Halide X-Cite 120) and emission wavelengths are split using a 4X4M-B quadriband dichroic mirror (Semrock). Oil immersion objectives 60× [Olympus PlanAPON60, 1.42 numerical aperture (NA)] or 100× (Olympus UPLFLN, 1.3 NA) were used for imaging, and a Hamamatsu Orca Flash4.0-V2 sCMOS (scientific Complementary Metal-Oxyde Semiconductor) 2048 × 2048 camera was used for detection. The acquisition and data analysis were performed with Volocity (Quorum Technologies) software.
Planapo n 60
Manufactured by Olympus
The PlanApo N 60 is a high-performance objective lens designed for use in advanced microscopy applications. It features a numerical aperture of 1.42 and a magnification of 60x, providing high-resolution imaging capabilities. The lens is plan-apochromatic, ensuring excellent flatness of field and chromatic correction.
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Lab products found in correlation
Fv1000, by Olympus (2 mentions)
Volocity, by Quorum Technologies (1 mentions)
Ix81 inverted microscope, by Olympus (1 mentions)
Orca flash4.0 v2 scmos, by Hamamatsu Photonics (1 mentions)
Uplfln, by Olympus (1 mentions)
Ultimate focus, by Olympus (1 mentions)
Deltavision elite, by Olympus (1 mentions)
Ix71 stand, by Olympus (1 mentions)
C9100 50 emccd camera, by Hamamatsu Photonics (1 mentions)
Tirf system, by Olympus (1 mentions)
Tcs sp5, by Leica (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Uapon 100, by Olympus (1 mentions)
Poly l lysine coated 35 mm glass bottom dishes, by Matsunami (1 mentions)
Deltavision microscope, by Cytiva (1 mentions)
Glass bottom dish, by MatTek (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
9 protocols using planapo n 60
1
Fluorescence Microscopy Imaging Protocol
2
Super-Resolution Imaging of GFP-NbMCTP7 in Leaves
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For 3D structured illumination microscopy (3D‐SIM), an epidermal peal was removed from a GFP‐NbMCTP7‐expressing leaf and mounted in perfluorocarbon PP11 106 under a high‐precision (170 μm ± 5 μm) coverslip (Marie Enfield). The sample chamber was sealed with non‐toxic Exaktosil N 21 (Bredent, Germany). 3D‐SIM images were obtained using a GE Healthcare/Applied Precision OMX v4 BLAZE with a 1.42NA Olympus PlanApo N 60× oil‐immersion objective. GFP was excited with a 488 nm laser and imaged with emission filter 504–552 nm (528/48 nm). SR images were captured using DeltaVision OMX software 3.70.9220.0. SR reconstruction, channel alignment and volume rendering were done using softWoRx V. 7.0.0.
3
High-Resolution Imaging System Protocol
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High-resolution imaging was performed using a General Electric (Boston, MA) DeltaVision Elite imaging system mounted on an Olympus (Japan) IX71 stand with a motorized XYZ stage, Ultimate Focus, cage incubator, ultrafast solid-state illumination with excitation/emission filter sets for DAPI, CFP, FITC, GFP, YFP, TRITC, mCherry, and Cy5, critical illumination, Olympus PlanApo N 60 ×/1.42 NA DIC (oil) and UPlanSApo 60 ×/1.3 NA DIC (silicone) objectives, Photometrics (Tucson, AZ) CoolSNAP HQ2 camera, SoftWoRx software with constrained iterative deconvolution, and vibration isolation table.
4
Confocal Microscopy Imaging and Uncaging
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Imaging was performed on an Olympus IX83 confocal laser-scanning microscope at 37 °C in a 5% CO2 high humidity atmosphere (EMBL incubation box). Imaging was performed using an Olympus Plan-APON × 60 (numerical aperture 1.4, oil) objective. The images were acquired utilizing a Hamamatsu C9100-50 EM CCD camera. Image acquisition was performed via FluoView imaging software, version 4.2. The green channel was imaged using the 488 nm laser line (120 mW cm−2) at 3% laser power and a 525/50 emission mirror. The red channel was imaged using the 559 nm laser (120 mW cm−2) at 2.0% laser power and a 643/50 emission filter. A pulsed 375 nm laser line (10 MHz) was applied for uncaging experiments. For uncaging experiments, circular regions of interest of 4–10 μm diameter were pre-defined. Pre-activation images were captured for five frames (5 s per frame), followed by 30 s of activation within the regions of interest. Recovery images were captured for 35 min at a frame rate of 5 s per frame.
5
Multi-modal Microscopy Techniques for Cellular Interactions
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A TIRF system (Olympus) was configured on a microscope (IX81). TIRF objective lenses (UAPON 100×, NA 1.49, oil; Olympus), dual excitation lasers, diode-pumped solid-state 488-nm and 561-nm laser systems (85BCD020 and 85YCA020; Melles Griot), and a charge-coupled device camera (ORCA flash4.0; Hamamatsu Photonics) were used. Dual-color videos were taken every 5 s for 10 min using Metamorph software (Molecular Devices). The images of the interaction between T cells and activated B cells that had been stimulated with LPS for 24 h were taken every 10 s using the IX81 microscope with an oil objective lens (Plan APO N 60×, NA 1.45; Olympus) under illumination with a Hg lamp. The intracellular calcium concentrations were also measured by using the IX81 microscope with Hg lamp illumination and an oil objective lens (UPlanAPO 40×, NA 1.00; Olympus). A microscope (TCS SP5; Leica Biosystems) with an oil immersion objective (HCX PL APO 100×, NA 1.44; Leica Biosystems) was used for fixed and stained samples. For detecting micro–adhesion rings formed in the cell–cell interaction, a hybrid detector (HyD; Leica Biosystems) was used to improve image quality.
6
Real-Time Live-Cell Imaging
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Cells were cultured in poly-L-lysine-coated 35-mm glass bottom dishes (Matsunami). Before observation, the DNA was counterstained with Hoechst 33342 for 15 min, followed by replacing the medium with phenol red-free DMEM containing 10% FBS, 2 mM L-glutamine, and 20 mM HEPES. Time-lapse observation was performed using a Delta Vision microscope (Applied Precision) equipped with a CO2 chamber set at 37°C. A 1.42 NA PlanApo N 60× oil immersion objective (Olympus) was used to observe cells at 3-min time intervals.
7
Immunofluorescence Staining of MEFs and T24 Cells
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MEFs and T24 cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min. The cells were blocked in PBST containing 0.4% bovine serum albumin (BSA) for 30 min and then incubated with primary antibodies in PBST. The coverslips were incubated with secondary antibodies in PBST for 1 h. Images were acquired with an FV1000 (Olympus) confocal microscope equipped with a PlanApo N 60× (numerical aperture [NA], 1.42; oil) or a UPlanApo 100× (NA, 1.40; oil) lens.
8
FRAP Analysis of GFP-SUN2 Dynamics
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For the FRAP experiments, HeLa cells were transfected with siRNAs or siNC and plated on a glass-bottom dish (Mat-Tek). After 24 h, cells were transfected with GFP-SUN2. Experiments were performed on a confocal microscope (FV-1000; Olympus) with a PlanApoN 60 (NA = 1.4) oil-immersion objective. After 2 images were obtained (0.2% 488 nm laser transmission), a rectangular region of the nuclear envelope was bleached (100% 488 nm laser transmission), and a further 60 images (10 s intervals) were collected using the original settings. Fluorescence intensity was measured using Metamorph (Molecular Devices, San Jose, CA, USA).
9
Poly-L-Lysine Microscopy Sample Preparation
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The samples were incubated for 30 min in plates treated with poly-L-Lysine (Sigma, St. Louis, MO, the United State). After treatment with PFA (4% in PBS), the samples were washed twice with PBS. Finally, the samples were observed with a confocal laser-scanning microscope Olympus FV1000 using a PlanApo N (60 × 1.42 NA) oil objective.
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