Time-Resolved Fluorescence Microscope System

在过去的五年里，稳态荧光显微镜以其高度的灵敏度和特异性使光学显微镜发生了革命性的变化。稳态荧光显微镜用于测量化学或分子参数时，时间分辨率一直在几秒到几百毫秒的范围内。根据探测器的不同，荧光和磷光的寿命可以达到从纳秒到秒的时间分辨率，并可用于延长测量的时间分辨率。此外，荧光或磷光探头的化学和物理环境会影响探头激发态的寿命。因此，激发脉冲产生的发光衰减是化学和物理环境的特征，因此时间分辨显微镜可以用来测量细胞中的化学和分子变化。测试显微镜系统的结果证明了该方法的可行性，我们正在寻求商业上可用的组件来组装一个完整的系统。这些应用包括表征新开发的染料的寿命，测量细胞内离子环境的变化，以及测量标记大分子的旋转扩散系数。后一种测量将允许表征活细胞中大分子的组装和结合过程。时间分辨显微镜将成为稳态显微镜的补充工具，并将成为许多必须最小化总激发剂量的应用中的选择方法。事实上，旋转扩散测量所需的较低的总激发剂量和较高的灵敏度可能会使其成为目前流行的荧光光漂白重分布技术在许多应用中的选择方法。

In the past five years, steady state fluorescence microscopy has revolutionized light microscopy due to the high degree of sensitivity and specificity. The time resolution of steady state fluorescence microscopy when used to measure chemical or molecular parameters has been in the seconds to hundreds of milliseconds range. Fluorescence and phosphorescence lifetimes can attain time resolutions from nanoseconds to seconds depending on the probe and can be used to extend the temporal resolution of measurements. In addition, the chemical and physical environment of a fluorescent or phosphorescent probe influences the lifetime of the excited state of the probe. Thus the decay of the luminescence produced by an excitation pulse is characteristic of the chemical and physical environment so time resolved microscopy can be used to measure chemical and molecular changes in cells. Results with a test microscope system have demonstrated the feasibility of the approach and we are asking for the commercially available components to assemble a complete system. The applications include characterizing the lifetimes of newly developed dyes, measuring changes in ionic environments in cells to measuring the rotational diffusion coefficients of labeled macromolecules. This latter measurement will allow the characterization of assembly and binding processes of macromolecules in living cells. Time resolved microscopy will become a complementary tool to steady state microscopy and will be the method of choice in many applications where the total excitation dose must be minimized. In fact the lower total excitation dose required and the higher sensitivity of rotational diffusion measurements will probably make this the methods of choice over the now popular fluorescence photobleaching redistribution technique in many applications.