Stained cells were visualized with a Nikon laser scanning multiphoton confocal microscope. Each experiment was repeated at least three times, and representative images from 15 high-power fields are shown. Intracellular localization was quantified using the Cell function in Imaris software. Segmentation algorithm involved nuclei detection using Hoechst channel and cell body detection using the red channel with adjacent cell splitting based on one nucleus per cell and cell body signal intensity. For each cell, the sum of each channel intensity in cytoplasm and nucleus was exported to Microsoft Excel to then calculate the cytoplasm-to-whole cell intensity ratios. Statistical analysis and graphing were performed in GraphPad Prism software.
Laser scanning multiphoton confocal microscope
Manufactured by Nikon
The Laser scanning multiphoton confocal microscope is a specialized laboratory instrument designed for high-resolution imaging of biological samples. It utilizes a focused laser beam to excite fluorescent molecules within the sample, enabling the capture of detailed three-dimensional images. The device combines the principles of multiphoton excitation and confocal microscopy to provide enhanced depth penetration and reduced background noise, allowing for the visualization of living cells and tissues with exceptional clarity.
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2 protocols using laser scanning multiphoton confocal microscope
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Confocal Microscopy Image Analysis
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Confocal Microscopy Image Analysis
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Stained cells were visualized with a Nikon laser scanning multiphoton confocal microscope. Each experiment was repeated at least three times, and representative images from 15 high-power fields are shown. Intracellular localization was quantified using the Cell function in Imaris software. Segmentation algorithm involved nuclei detection using Hoechst channel and cell body detection using the red channel with adjacent cell splitting based on one nucleus per cell and cell body signal intensity. For each cell, the sum of each channel intensity in cytoplasm and nucleus was exported to Microsoft Excel to then calculate the cytoplasm-to-whole cell intensity ratios. Statistical analysis and graphing were performed in GraphPad Prism software.
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