Fluorescence spectroscopy is presently enjoying explosive growth due to new experimental capabilities. In this talk we summarize some recent advances in probe chemistry, multi-photon excitation and light quenching.
Evanescent illumination at one wavelength and incident angle, coupled with light quenching at a second wavelength and incident angle, can be used for selective excitation of fluorophores located up to 5000 C 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 observed 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 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 fiuorophores with two different wavelengths, a phenomenon we refer to as two-color two-photon excitation. The emission of several fiuorophores including PPD scintillator, p-terphenyl and indole were observed when the sample was illuminated with both 380 nm and 760 nm pulses from a picosecond dye laser, but only when the picosecond pulses were spatially and temporally overlapped. When illuminated with both wavelengths, the emission depended quadratically on total power, indicating two-photon excitation. When the intensity at one wavelength was attenuated, the signal depended linearly on the power at each wavelength, indicating the participation of one-photon at each wavelength to the excitation process. For 2C2P excitation of PPD the time-zero anisotropy was larger than possible for single-photon excitation. This phenomenon can have numerous applications in the chemical and biomedical sciences, as a method for spatial localization of the measured volume.
We demonstrated from steady state and time-resolved measurements that two-photon excitation of fluorophores in intralipid is due only to the ballistic photons, and not to the diffusely scattered photons. This observation opens the possibility of highly localized excitation in turbid media based on multi-photon excitation.
Luminescence is typically on the ns timescale. The use of metal-ligand complexes allows a range of decay times on the microsecond timescale. This possibility is illustrated by rhenium (I) carbonyl complexes which display a wide range of emission wavelengths, quantum yields, lifetimes and high fundamental anisotropies depending on the chromophoric ligand. These complexes can be further modified by using substituted pyridines as a nonchromophoric ligand. These pyridines can have substitutes (R = NH2, NCS, COOH, for example) that are readily conjugatable to proteins, DNA, and lipids, which can be utilized in the study of the dynamics of these macromolecules on extremely long time scales.