Concentration dependence of 2-photon-excited fluorescence emission for CM-pyranine and fluorescein.

<p>2-photon-excited fluorescence from solutions of each fluorophore at various concentrations were imaged on a laser-scanning microscope. After background subtraction, the average fluorescence intensity in each image was plotted against concentration (see Experimental Section for details). Each data point is the average of 4–8 replicate measurements; all error bars are smaller than the symbol size. <b>(A)</b> Fluorescence vs. concentration for CM-pyranine. The red line is the linear least-squares fit of the data. Linearity is observed over the entire concentration range examined (up to 20 mM). <b>(B)</b> Fluorescence vs. concentration for fluorescein. The solid red line is the linear least-squares fit of the data up to 2 mM. The dashed red line is a visual aid to show nonlinear behavior at high concentrations of fluorescein.