Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems
Performance comparison between the high-speed Yokogawa spinning disc confocal system and single-point scanning confocal systems
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DOI：
10.1111/j.1365-2818.2005.01473.x
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发表时间：
2005-05-01
影响因子：
2
通讯作者：
Dunn, KW
中科院分区：
文献类型：
--
作者：
Wang, E;Babbey, CM;Dunn, KW
活细胞动态的荧光显微镜对显微镜成像系统提出了特殊的挑战，同时需要高空间分辨率和高时间分辨率，但照明水平足够低以防止荧光团损伤和细胞毒性。我们比较了高速横河CSU 10旋转光盘共聚焦系统与几个传统的单点扫描共聚焦（SPSC）显微镜，使用图像信噪比和荧光团光漂白之间的关系作为系统效率的指标。这些研究表明，CSU 10的效率始终超过SPSC系统。CSU 10的高效率意味着可以在低得多的照明水平下收集高质量的图像; CSU 10能够在仅产生SPSC系统光漂白速率的1/15的照明水平下实现SPSC系统的最大信噪比。虽然CSU 10系统的一些相对效率可能归因于使用CCD而不是光电倍增管检测器系统，但我们的分析表明，SPSC系统的高速成像受到荧光饱和度的限制，因为在高帧速率下经常需要高水平的照明来收集图像。高速、高效和不受荧光饱和度影响的联合收割机使CSU 10能够有效地对活细胞进行扩展成像，其速率能够捕获每秒移动数微米的内体的三维运动。
Fluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high-speed Yokogawa CSU10 spinning disc confocal system with several conventional single-point scanning confocal (SPSC) microscopes, using the relationship between image signal-to-noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal-to-noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high-speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three-dimensional motion of endosomes moving up to several micrometres per second.