The chambers were mounted on a fluorescence microscope (Nikon, Eclipse TE-2000U) with a 60× objective (Nikon, Plan Apo VC 60× (NA=1.40)). Images were recorded in two ways depending on the temporal and spatial requirements. The first method used an EM-CCD TV camera (Hitachi Kokusai Electric, KP-E500) attached to the side-port of the microscope while the second method used a conventional digital camera (Canon, EOS Kiss X7i) attached to the front port of the microscope. TV images were recorded on a PC by an image grabber board (The Imaging Source, DFG/SVI PCI grabber) and controller (The Imaging Source, IC Capture) in an uncompressed AVI format (UYVY, 640×480 pixels, 30 fps). Still images were recorded in TIFF format (0.5 sec exposure, ISO=12800, 3456×3456 pixels). A wide-angle image was obtained by an All-in-one Fluorescence Microscope (Keyence, BZ-X700). Images were analyzed on a PC by Image-J software.
Plan apo vc 60
Manufactured by Nikon
Sourced in Japan
The Plan Apo VC 60x is a high-magnification objective lens designed for microscopy applications. It features a numerical aperture of 1.40 and a working distance of 0.21 mm, providing excellent optical performance and resolution. The lens is part of Nikon's Plan Apo series, which is known for its flat field, high-contrast imaging capabilities.
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Lab products found in correlation
Ti e inverted microscope, by Nikon (2 mentions)
Eos kiss x7i, by Canon (1 mentions)
Bz x700, by Keyence (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Alexa488 conjugated anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Hm 500 om, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Fujifilm (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
A1 confocal laser microscope, by Nikon (1 mentions)
35 mm glass bottom microwell dish, by MatTek (1 mentions)
Fcs2 live cell chamber, by Bioptechs (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
A1 laser scanning confocal microscope, by Nikon (1 mentions)
L α phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
L α phosphatidylserine, by Avanti Polar Lipids (1 mentions)
Mowiol, by Merck Group (1 mentions)
Coolled pe 300, by Nikon (1 mentions)
Dynabeads m 270 streptavidin, by Thermo Fisher Scientific (1 mentions)
Nis elements c software, by Nikon (1 mentions)
Uplanfl 100, by Olympus (1 mentions)
9 protocols using plan apo vc 60
1
Fluorescence Microscope Imaging Protocol
2
Immunostaining of Mouse Brain Slices
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To immunostain brain slices, adult mouse brains were infused with Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen and sectioned at 10 µm using a cryostat (HM500-OM, Carl Zeiss, Oberkochen, Germany). The sections were mounted on silane-coated coverslips and dried for ~30 min at room temperature. The samples were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl, pH 7.4) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 10% FCS in PBS, the samples were incubated with the anti-RNG105 antibody over night at 4°C. After washing with PBS, the samples were incubated with Alexa488-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries) for 1 hr at room temperature to label RNG105 and nuclei, respectively. To immunostain cultured neurons, neurons at 12 DIV were fixed and stained in the same way. Fluorescence images were acquired using an A1 confocal laser microscope equipped with a Ti-E inverted microscope (Nikon, Tokyo, Japan) with a 10 × objective lens or a PlanApo VC60 × oil objective lens.
3
Live Cell Imaging of GFP Dynamics
4
Visualizing His-GFP-JRAB/MICAL-L2 Droplets
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To observe the droplets, 25% of PEG (final concentration: 5%) was added to a mixture of purified His-GFP-JRAB/MICAL-L2 with or without His-mCherry-Rab8ADA. The mixture was placed on a glass-bottom dish. Images were acquired using an A1 confocal laser scanning microscope (Nikon) equipped with an oil objective lens (PlanApo VC × 60, NA = 1.4).
5
Liposome Preparation and Membrane Protein Visualization
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Liposomes were prepared using L-α-phosphatidylcholine (brain PC, porcine) and L-α-phosphatidylserine (brain PS, porcine) purchased from Avanti Polar Lipids (Alabaster, AL, USA); these lipids were used without further purification. Each lipid was solubilized in chloroform at a concentration of 10 mM to make a stock solution. Then, the stock solutions of brain PC and brain PS were mixed and diluted in chloroform to 0.7 and 0.3 mM, respectively. The solvent of the lipid mixture was evaporated under a flow of nitrogen gas for 1 h. The dried lipids were suspended in 0.3 M sucrose at 37 °C for 1 h, followed by vortexing (final concentration: 1 mM). The liposome suspensions were diluted 20-fold with hypotonic buffer L (20 mM HEPES-NaOH, pH 7.5, 100 mM KCl, 1 mM DTT) to obtain large liposomes (typically 1–10 μm in diameter), and then mixed with purified His-GFP-JRAB/MICAL-L2 and His-mCherry-Rab8ADA (final concentration: 150 nM). The mixture was placed on a glass-bottom dish, covered to avoid evaporation, and observed using an A1 confocal laser scanning microscope (Nikon) equipped with an oil objective lens (PlanApo VC ×60, NA = 1.4).
6
Quantifying RNA-Binding Protein Localization in Neuronal Granules
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Neurons expressing GFP- and mRFP1-tagged RBPs were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min. After washing with PBS, the specimens were mounted in Mowiol (Sigma-Aldrich, Burlington, MA). Fluorescence images were acquired using an A1 confocal laser scanning microscope equipped with a Ti-E inverted microscope (Nikon, Tokyo, Japan) and a PlanApo VC 60 × water immersion objective. The fluorescence intensity of the images was measured using ImageJ. To measure the accumulation of RBPs in the granules in dendrites, the images were smoothed with a mean filter using ImageJ. Then, fluorescence intensity within a single granule was measured along a line parallel to the dendrite across the fluorescence peak of the granule to draw a fluorescence intensity curve. The difference in fluorescence intensity between two adjacent pixels at half the maximum fluorescence intensity of the curve was calculated as the slope (Fig. 1 B, right schematic). The granule/cytoplasm partition coefficient of RNG105 was calculated as (Fg-Fb)/(Fc-Fb), where Fg, Fc, and Fb are the fluorescence intensities of RNG105 in granules, cytoplasm, and extracellular areas (background), respectively.
7
Optically Triggered GUV Manipulation
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Imaging was conducted using an inverted microscope (Nikon Ti-U), equipped with a water immersion objective (Nikon Plan APO VC, 60×, NA = 1.2) and LED EPI illumination (CoolLED pE-300), enabling rapid alternation between transmitted and EPI illumination modalities. The transmitted mode was employed to assess the integrity of GUV membranes, while fluorescence illumination served to quantify the cargo content within the GUVs. Optical tweezers (Tweez, Aresis d.o.o., Slovenia, Nd:YAG laser, λ = 1064 nm) with acousto-optic deflectors (AOD) for beam steering were integrated into the microscope. The laser power was measured by a power meter Coherent PM USB PS10.
Shockwaves were triggered by focusing the laser on optically opaque polystyrene microparticles (Dynabeads M-270 Streptavidin, ThermoFisher Scientific, nominal diameter dp = 2.8 μm), which had a streptavidin coating to enable binding to biotin molecules in the GUV membrane. A high-speed camera (Photron SA-Z type 2100K-M-64GB) was used for evaluation of the microparticle response to laser illumination.
Shockwaves were triggered by focusing the laser on optically opaque polystyrene microparticles (Dynabeads M-270 Streptavidin, ThermoFisher Scientific, nominal diameter dp = 2.8 μm), which had a streptavidin coating to enable binding to biotin molecules in the GUV membrane. A high-speed camera (Photron SA-Z type 2100K-M-64GB) was used for evaluation of the microparticle response to laser illumination.
8
Multiphoton Excited Fluorescence Spectroscopy
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Multiphoton excited fluorescence spectra were recorded by exciting a solution of E-CA4CN in dimethylsulfoxide (DMSO) (10 mM) at wavelengths between 800 to 960 nm using the output from the femtosecond pulsed Ti-sapphire laser at laser powers ranging from ∼0.1 to 6 mW at the sample.
The excitation light was delivered to the sample through the Nikon Plan Apo VC 60× water immersion objective (N.A. 1.2, WD 0.31 to 0.28 mm) without scanning the laser beam.
The spectrograph (Acton 275) for the measurement of twoand three-photon excited emission spectra was calibrated using a Hg-lamp. Spectral detection was achieved using an Andor charge-coupled device camera.
The excitation light was delivered to the sample through the Nikon Plan Apo VC 60× water immersion objective (N.A. 1.2, WD 0.31 to 0.28 mm) without scanning the laser beam.
The spectrograph (Acton 275) for the measurement of twoand three-photon excited emission spectra was calibrated using a Hg-lamp. Spectral detection was achieved using an Andor charge-coupled device camera.
9
Time-lapse Imaging of Cytoskeletal Dynamics
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Time-lapse images were captured with an inverted microscope (IX71; Olympus) equipped with a single-chip color CCD camera (DP70; Olympus) and an objective lens (LCPlanFl 20/0.40 NA; UPlanApo 60/0.90 NA; UPlanFl 100/1.30 NA Oil; Olympus).
During observation, cells were warmed on a thermoplate set to 37°C (MATS-U55R30; Tokai Hit). Images were captured every 5 min and analyzed using Lumina Vision version 2.4.2 software (Mitani Corporation). Images of cells expressing mCherry-NMHC-IIA and EGFP-NMHC-IIB were captured using an inverted microscope (Ti-E; Nikon) equipped with an oil-immersion objective lens (Plan Apo-VC 60/1.40 NA; Nikon). During observation, cells were warmed in an incubation chamber heated to 37°C (INUBG2H-TIZB; Tokai Hit). Images were captured and analyzed using NIS-Elements C software (Nikon).
During observation, cells were warmed on a thermoplate set to 37°C (MATS-U55R30; Tokai Hit). Images were captured every 5 min and analyzed using Lumina Vision version 2.4.2 software (Mitani Corporation). Images of cells expressing mCherry-NMHC-IIA and EGFP-NMHC-IIB were captured using an inverted microscope (Ti-E; Nikon) equipped with an oil-immersion objective lens (Plan Apo-VC 60/1.40 NA; Nikon). During observation, cells were warmed in an incubation chamber heated to 37°C (INUBG2H-TIZB; Tokai Hit). Images were captured and analyzed using NIS-Elements C software (Nikon).
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