Reck-Peterson Samara L, Derr Nathan D, Stuurman Nico
Cold Spring Harb Protoc. 2010 Mar;2010(3):pdb.prot5398. doi: 10.1101/pdb.prot5398.
When performing analysis using total internal reflection fluorescence microscopy (TIRFM), fluorescence intensity depends linearly on excitation intensity, until the dye spends most of its time in the excited state (i.e., saturation is reached). How much light is seen by the dyes in the sample depends not only on the amount of light exiting the objective but also on the area over which it is spread out. The parameter of interest is the power density, measured in W/cm(2). The highest signal-to-noise ratios will be reached when the illumination power just approaches saturation. To determine at what power density a dye is saturated, one should measure fluorescence intensity as a function of power density, as described in this protocol.
使用全内反射荧光显微镜（TIRFM）进行分析时，荧光强度与激发强度呈线性关系，直到染料大部分时间处于激发态（即达到饱和）。样品中染料接收到的光量不仅取决于从物镜出射的光量，还取决于光的扩散面积。感兴趣的参数是功率密度，以W/cm²为单位。当照明功率接近饱和时，将获得最高的信噪比。要确定染料在何种功率密度下达到饱和，应按照本方案所述，测量荧光强度作为功率密度的函数。
