Two-photon excitation microscopy of tryptophan-containing proteins.

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.