Practical confocal microscopy and the evaluation of system performance
Practical confocal microscopy and the evaluation of system performance
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DOI：
10.1006/meth.1999.0812
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发表时间：
1999-08-01
期刊：
影响因子：
--
通讯作者：
Price, OT
中科院分区：
文献类型：
--
作者：
Zucker, RM;Price, OT
激光扫描共聚焦显微镜在生物学的许多领域具有巨大的应用潜力。目前，共聚焦显微镜的性能评估带有主观性，主要是通过用户实验室提供的特定测试载玻片来评估系统。为了从设备中获得更好的性能，有必要运行一系列测试以确保光学机器正常工作。我们在徕卡TCS-SP和TCS-4D系统上设计了这些方法。通过测量视场照度、透镜清晰度、激光功率输出、二向色功能、光谱对准、轴向分辨率、激光功率稳定性、机器性能和系统噪声的测试，对徕卡激光扫描共聚焦显微镜系统进行了测试。这些测试也应该适用于其他制造商的系统。光电倍增管（PMT）电压、激光功率和直径为10 μ m的测试珠平均之间的关系表明，随着光电倍增管（PMT）的增加，图像中的噪声（珠强度变化系数，CV）增加。因此，增加PMT设置会导致噪声增加。为了获得理想的图像质量，似乎最好降低PMT设置并增加激光功率，因为高PMT设置产生的噪声对图像质量的降低远远超过高激光功率引起的漂白。平均可以用来改善高PMT值的图像，只要样品没有被激光的重复通过漂白。
The laser scanning confocal microscope has enormous potential in many fields of biology. Currently there is a subjective nature in the assessment of a confocal microscope's performance by primarily evaluating the system with a specific test slide provided by the user's laboratory. To achieve better performance from the equipment, it is necessary to run a series of tests to ensure that the optical machine is functioning properly. We have devised these methods on the Leica TCS-SP and TCS-4D systems. Tests measuring field illumination, lens clarity, laser power output, dichroic functioning, spectral alignment, axial resolution, laser power stability, machine performance, and system noise were derived to test the Leica laser scanning confocal microscopy system. These tests should be applicable to other manufacturers' systems as well. The relationship between photomultiplier tube (PMT) voltage, laser power, and averaging using a 10-mu m-diameter test bead has shown that the noise (coefficient of variation of bead intensity, CV) in an image increases as the PMT increases. Therefore increasing the PMT setting results in increased noise. For ideal image quality, it appears that it is better to decrease the PMT setting and increase laser power, as noise generated by high PMT settings will reduce the image quality far more than the bleaching caused by higher laser power. Averaging can be used to improve the image at high PMT values, provided the sample is not bleached by repeated passes of the laser.