Fluorescence spectroscopy is presently enjoying explosive growth due to new experimental capabilities. In this talk we summarize some recent advances which can have applications in cell biology and imaging.

We show that evanescent illumination at one wavelength and incident angle, coupled with light quenching at a second wavelength and incident angle (Figure 1), can be used for selective excitation of fluorophores located up to 5000 Å into the aqueous phase. The use of combined evanescent wave excitation and evanescent wave quenching could provide selective excitation of fluorophores in the cytoplasmic region of cells. We also reported the first experimental observation of two-photon excitation using the evanescent wave resulting from total internal reflection. Two-photon excitation of the calcium probe Indo-1 was demonstrated by the quadratic dependence on intensity (Figure 2) as well as by the anisotropy and time-resolved fluorescence measurements.

Two-photon excitation is routinely performed using a single wavelength. We recently observed two-photon excitation of organic fluorophores with two different wavelengths, a phenomenon we refer to as two-color two-photon excitation.