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Determining single-molecule intensity as a function of power density.

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.