For the observation of fluorescence signals, a confocal laser-scanning microscope system (FV3000, Olympus) was used. For live-cell imaging, a stage chamber (Tokai Hit) was used to maintain the temperature and moisture and CO2 levels. Phenol red-free DMEM supplemented with 10% FBS and 1 µg ml−1 Hoechst 33342 was used to visualize chromosomes if necessary. The images obtained were processed and analysed using MetaMorph (Molecular Devices), Fiji47 (link) or Python 2 or 3 with add-on libraries (Numpy, Scipy, Pandas, Matplotlib, OpenCV and seaborn).
Stage chamber
Manufactured by Tokai Hit
The Stage chamber is a laboratory equipment designed to provide a controlled environment for various experimental applications. Its core function is to create and maintain a stable, isolated environment with precise control over environmental conditions such as temperature, humidity, and atmospheric composition.
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Lab products found in correlation
2 protocols using stage chamber
1
Fluorescence Microscopy of Live Cells
2
Multicolor Confocal Imaging of Mitochondria
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All fluorescent imaging experiments were performed using a confocal laser-scanning microscope (FV1000 IX81; OLYMPUS) with a ×60 oil immersion objective lens (Supplementary Figs S11 , S12 ).
mitAT1.03 and TMRE were excited by a diode laser (440 nm) and a helium-neon laser (559 nm), respectively, through a 20/80 beam splitter. Emitted fluorescence was separated by dichroic mirrors (510 nm and 560 nm), and signals from mseCFP, mVenus and TMRE were detected at 460–500 nm, 510–530 nm and 575–675 nm, respectively, using band pass filters. The widths of the 460–500 nm and 510–530 nm filters were manually adjusted using monochromators to ensure the lowest amount of background and fluorescence leakage from other channels or excitation lights. Width of 575–675 nm was a specified value of a barrier filter in our system.
Images were acquired at regions approximately 0–150 µm from the edge of axons, with a resolution of 0.132 μm/pixel every 10 sec for 10–15 min. During imaging, cells were maintained at 37 °C and in a 5% CO2 atmosphere using a stage chamber (TOKAI HIT).
mitAT1.03 and TMRE were excited by a diode laser (440 nm) and a helium-neon laser (559 nm), respectively, through a 20/80 beam splitter. Emitted fluorescence was separated by dichroic mirrors (510 nm and 560 nm), and signals from mseCFP, mVenus and TMRE were detected at 460–500 nm, 510–530 nm and 575–675 nm, respectively, using band pass filters. The widths of the 460–500 nm and 510–530 nm filters were manually adjusted using monochromators to ensure the lowest amount of background and fluorescence leakage from other channels or excitation lights. Width of 575–675 nm was a specified value of a barrier filter in our system.
Images were acquired at regions approximately 0–150 µm from the edge of axons, with a resolution of 0.132 μm/pixel every 10 sec for 10–15 min. During imaging, cells were maintained at 37 °C and in a 5% CO2 atmosphere using a stage chamber (TOKAI HIT).
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