Live imaging of cells was performed using a LCV100 (Olympus) equipped with a UAPO ×40/340 objective lens (Olympus), a LED light source, a DP30 camera (Olympus), differential interference contact (DIC) optical components, and interference filters. Live-cell imaging of MLC2-EGFP or Lifeact-EGFP was obtained using the spinning-disc laser confocal microscope IX71 (Olympus) equipped with CSU-X1 (Yokogawa), a Yokogawa YOKO R485/561 filter, a Neo sCMOS camera (ANDOR), and a UPLSAPO ×60/1.35 oil lens. The cells were set on a ChamlideTC CO2 incubator (Live Cell Instrument) at 37 °C. Live-cell imaging of Raichu probes was performed using a spinning-disc laser confocal microscope IX81 (Olympus) equipped with CSU-W1 (Yokogawa), an ImagEM-1K camera (Hamamatsu), and a PLANAPO N ×60/1.42 oil lens. Images were processed using MetaMorph (Molecular Devices) and ImageJ (NIH).
Lcv100
Manufactured by Olympus
The LCV100 is a compact and durable laboratory centrifuge designed for general-purpose applications. It features a maximum rotational speed of 4,000 rpm and can accommodate sample volumes up to 100 mL. The LCV100 is equipped with a fixed-angle rotor and provides a straightforward user interface for easy operation.
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Lab products found in correlation
Orca flash 4, by Hamamatsu Photonics (3 mentions)
Dp30 camera, by Olympus (2 mentions)
40 uapo 340 objective lens, by Olympus (2 mentions)
Csu x1, by Yokogawa (2 mentions)
Uapo 40 340 n a 0, by Hamamatsu Photonics (2 mentions)
Brightline single band filter set, by IDEX Corporation (2 mentions)
Metamorph 6, by Molecular Devices (2 mentions)
Imagem 1k camera, by Hamamatsu Photonics (1 mentions)
Neo scmos camera, by Oxford Instruments (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Csu w1, by Yokogawa (1 mentions)
Uapo 340, by Olympus (1 mentions)
Csu x1, by Olympus (1 mentions)
Metamorph, by Universal Imaging (1 mentions)
Fv500, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
6 protocols using lcv100
1
Live Imaging of Cellular Dynamics
2
Visualizing Wound Healing and Cell Migration
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For analysis of wound healing and cell migration by live imaging, we used a LCV100 (Olympus) equipped with a UAPO 40×/340× objective lens (Olympus), an LED light source, a DP30 camera (Olympus), and DIC optical components and interference filters, except Video 2 , for which we used an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and electron-multiplying charge coupled device (EMCCD; iXon+, Andor Technology). To observe actin dynamics, we isolated stable lines of wild-type and αEcat KO Caco-2 cells expressing LifeAct-RFP. A wild-type line of these transfectants was additionally transfected with Nap1-GFP in a transient way. These cells were observed using an inverted fluorescence microscope (IX-81, Olympus) equipped with a spinning disk confocal imaging unit (CSU-X1, Yokogawa), a 40×/1.35 oil-immersion objective (UApo/340, Olympus), and a 561-nm laser (Sapphire LP, Coherent) for RFP excitation or a 488-nm laser (Sapphire LP, Coherent) for GFP excitation. We took fluorescence images with multiple z-stacks by EMCCD (iXon+, Andor Technology) at the specified time intervals and then made maximum-intensity Z projections.
3
Time-lapse Imaging of Fluorescent Proteins
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Cells were grown on 35-mm glass-bottom dishes in phenol red-free DMEM containing 10% FBS. Cells were subjected to long-term, time-lapse imaging using a computer-assisted fluorescence microscope (Olympus, LCV100) equipped with an objective lens (Olympus, UAPO 40×/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CMOS camera (Hamamatsu Photonics, ORCA-Flash4.0), differential interference contrast (DIC) optical components, and interference filters. The halogen lamp was used with BrightLine® single-band filter set (Semrock): “FITC-2024B” for observing the h2-3 fluorescence, and “mCherry-C” for observing the AxaleaB5 fluorescence. For DIC imaging, the red LED was used with a filter cube containing an analyzer. Image acquisition and analysis were performed using MetaMorph 6.37 and 7.10 software (Molecular Devices), respectively. See Table II for details.
4
Time-Lapse Imaging of Cultured Cells
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Imaging studies were conducted as described previously20 (link)21 (link). Briefly, cells cultured on a 35-mm glass-bottom dish in DMEM/10%FBS were subjected to time-lapse imaging by LCV100 (Olympus) with an objective lens (X40). An image was acquired every 15 minutes until the end of the observation. Image acquisition and analysis were performed with MetaMorph (Universal Imaging). The cells were cultured in starvation medium for 24 hr before imaging, if required.
5
Time-lapse Imaging of Fluorescent Cells
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Cells were grown on 35-mm glass-bottom dishes in phenol red-free DMEM containing 10% FBS. Cells were subjected to long-term, time-lapse imaging using a computerassisted fluorescence microscope (Olympus, LCV100) equipped with an objective lens (Olympus, UAPO 40×/340 N.A. = 0.90), a halogen lamp, a red LED (620 nm), a CMOS camera (Hamamatsu Photonics, ORCA-Flash4.0), differential interference contrast (DIC) optical components, and interference filters. The halogen lamp was used with BrightLine® single-band filter set (Semrock): "FITC-2024B" for observing the h2-3 fluorescence, and "mCherry-C" for observing the AxaleaB5 fluorescence. For DIC imaging, the red LED was used with a filter cube containing an analyzer. Image acquisition and analysis were performed using MetaMorph 6.37 and 7.10 software (Molecular Devices), respectively. See Table 2 for details.
6
Quantitative Microscopy Techniques for Neuron Analysis
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Dependency of polarization angle of excitation light was measured with an inverted microscope (IX71, Olympus) equipped with a polarizer (Sigma-Koki), a standard 75-W xenon lamp, and a CMOS camera (Orca-Flash4.0; Hamamatsu Photonics). A 40 3 objective lens with a moderately low numerical aperture (NA 0.75) was used to minimize aperture effect. Intrinsic angle dependency of the system (g-factor) was calibrated using an isotropic solution of fluores- cein. Long-term live imaging was performed using an incubation imaging system (LCV100, Olympus). The acquired images were reconstructed using the tiling function of MetaMorph software (Molecular Devices). Photoconversion experiments were performed using a laser scanning microscope (Olympus FV500 for Figure 1C; FV1000 for Movie S1) equipped with 405, 488, and 543 nm lasers and a 60 3 objective lens (NA 1.35). Culture of primary rat hippocampal neurons and transfection were performed as described elsewhere (Hama et al., 2004) . A confocal laser scanning microscope (FV1000, Olympus) was used to acquire high-resolution fluorescence images with a large field of view (Figure S3).
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