Instrumentation for fluorescence microscopy with picosecond time resolution.
Instrumentation for fluorescence microscopy with picosecond time resolution.
复制标题
具有皮秒时间分辨率的荧光显微镜仪器。
DOI：
10.1111/j.1751-1097.1985.tb01620.x
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发表时间：
1985
影响因子：
3.3
通讯作者：
Firey,PA
中科院分区：
文献类型：
--
作者：
Rodgers,MA;Firey,PA
使用主动锁模染料激光器激发和时间相关单光子计数分析的时间分辨荧光显微镜被证明是获得高度空间和时间分辨率的有效方法。显微镜的成像能力使最佳的仪器响应功能，即使是廉价的光电倍增管。迄今为止，受限于（由激光源）长波长可见光激发，优异的光收集和成像，加上单光子计数的灵敏度，使得可见光染料的弱得多的U-V二次谐波非常可能是可用的。当然，使用锁模c.w. Nd：YAG激光器，或来自锁模连续波的基本谱线。离子激光器作为激发源将增强该技术。然而，只有用可见光吸收染料才能激发叶绿素、卟啉、氧杂蒽（玫瑰红、赤藓红B）、藻胆蛋白、噻托溴铵染料、溴化乙锭等荧光染料。此外，该技术可以直接扩展到偏振光测量，从而可以测定荧光染料在细胞和细胞器中的旋转扩散。扩展到可变温度的情况是容易设想的。除了用于细胞和亚细胞实体的检查外，所述设备还可以在空间分辨率可以提供额外信息的任何地方有利地使用，例如粉末和表面和界面结构的研究。
Time‐resolved fluorescence microscopy using excitation by actively mode‐locked dye lasers and analysis by time‐correlated single photon counting is shown to be an effective way of obtaining a high degree of spatial and temporal resolution. The imaging capabilities of the microscope make for optimal instrument response functions even with inexpensive photomultiplier tubes. Thus far limited (by the laser source) to long wavelength visible excitation, the excellent light collection and imaging, coupled with the sensitivity of single photon counting make it highly probable that the much weaker U‐V second harmonics of the visible dyes will be useable. Certainly the potential of using the third harmonic line (355 nm) of a mode‐locked c.w. Nd: YAG laser, or fundamental lines from mode‐locked c.w. ion lasers as excitation sources will enhance the technique. Nevertheless, with visible‐absorbing dyes only it is possible to excite such fluorochromes as chlorophylls, porphyrins, xanthenes (rose bengal, erythrosin B), phycobiliproteins, thionine dyes, ethidium bromide, and so on. Furthermore, this technique can be straightforwardly extended for polarized light measurements thereby allowing determinations of rotational diffusion of fluorochromes in cells and organelles. The extension to variable temperature situations is easy to conceive. In addition to its use for examination of cellular and sub‐cellular entities, the equipment described can be profitably employed wherever spatial resolution may provide extra information, such as studies of powders and the structures of surfaces and interfaces.