Cells were imaged at room temperature exposed to ambient atmosphere on either a Nikon Diaphot 300, a Nikon Eclipse TiE, or a Zeiss TIRF Axiovert. Fluorescence images were acquired with high numerical aperture oil immersion objectives, using either wide field illumination or total internal reflection excitation. Transmitted light images were obtained using phase-contrast optics with both oil and air immersion objectives. Images were collected on 1k back-thinned cooled EM-CCD cameras, Andor DU888 (Andor) or Hamamatsu ImageEM (Hamamatsu). Long tracks of cells were obtained from cells replated into an Ibidi μ-Dish 35 mm I high (Ibidi) sealed with Parafilm and imaged across a 4×4 grid of images with each position sampled every minute. A red filter was applied to the transmitted light illuminating cells to be imaged over a long period of time in blebbistatin.
Eclipse ti e
Manufactured by Zeiss
The Eclipse Ti-E is an inverted research microscope system designed and manufactured by Zeiss. It provides high-resolution imaging capabilities for a variety of microscopy techniques, including brightfield, phase contrast, and fluorescence. The Eclipse Ti-E is equipped with advanced optics and control systems to enable precise and accurate observation and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Du 888, by Oxford Instruments (1 mentions)
Imageem, by Hamamatsu Photonics (1 mentions)
Diaphot 300, by Nikon (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
Synergy 2 multi mode reader, by Agilent Technologies (1 mentions)
Discovery hr 2 rheometer, by TA Instruments (1 mentions)
Inova 500 nmr spectrometer, by Agilent Technologies (1 mentions)
Lsm780 spectral microscope, by Zeiss (1 mentions)
Vevo 2100, by Fujifilm (1 mentions)
4 protocols using eclipse ti e
1
Live-Cell Imaging of Cells
2
Photoinduced Rac1 Activation Assay
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To detect ERK activation upon protrusion generation by photoactivation, cells were transfected with ERKKTR-GFP along with mCherry-PA-Rac130 (link). Cells were starved in Leibovitz’s L-15 medium containing 1% FBS overnight prior to imaging. Time-lapse videos were captured on either a Nikon Eclipse Ti-E or Zeiss LSM780 microscope. Protrusions were induced with repeated blue light irradiation. For Nikon Eclipse Ti-E, the whole viewfield under 100X objective was irradiated with 440 nm laser at 3% power for 5 s every 20 s. For Zeiss LSM780, the whole viewfield under 63X objective was irradiated with 458 nm laser at 40% power for 10 iterations (~25 s) every 1 min.
3
Fluorescence Imaging of Subcellular Organelles
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For GFP-FYVE2 puncta assay, U2OS GFP-FYVE2 cell line was cultured on coverslip and fixed by 4% paraformaldehyde for 20 min at room temperature for imaging. For endogenous Rab5, EEA1, EGFR, Lamp1, and Rab7, cells were fixed by 4% paraformaldehyde and blocked by 2.5% BSA and then incubated with appropriate primary antibodies for 12 hours at 4°C. Cells were then incubated with Alexa Fluor–conjugated secondary antibodies. For LC3 of muscle tissue, skeletal muscle was isolated from C57BL/6 J mice and then sectioned with a Leica VT1200 S Fully automated vibrating blade microtome. Muscle sections were maintained in phosphate-buffered saline (PBS) and then stained with LC3 primary antibodies and Alexa Fluor-conjugated secondary antibody for imaging. All fluorescence was detected using a laser scanning confocal microscope (Nikon Eclipse Ti-E and Zeiss LSM 510).
4
Multimodal Characterization of Materials
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Proton nuclear magnetic resonance ( 1 H NMR) was performed on a Varian Inova 500 NMR Spectrometer. Fourier transform infrared spectroscopy (FTIR) was performed on a Nicolet 6700 FTIR Spectrometer and samples were run on polyethylene infrared sample cards. Scanning electron microscope (SEM) images and elemental analysis measurements by energy-dispersive spectroscopy (EDS) were taken using a JEOL (Peabody, MA) JSAM-6010la. Rheological characterization was performed using a TA Instruments Discovery HR-2 rheometer. ELISA color development was monitored with an ELISA plate reader (BioTek Synergy 2 Multi-Mode Reader) at 405 nm with wavelength correction set at 650 nm. Echocardiographic measurements were obtained using FUJIFILM VisualSonics Vevo 2100 equipped with a 30 MHz transducer. Tissue was sectioned using a CryoStar NX70 Cryostat. Brightfield images were taken using a Nikon Eclipse Ti-E. Confocal images were taken using a Zeiss LSM 780 spectral microscope.
Zeiss ZEN blue software was used to quantify different parameters for image analysis from confocal images.
Zeiss ZEN blue software was used to quantify different parameters for image analysis from confocal images.
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