Two-photon fluorescence excitation in detection of biomolecules

Two-photon fluorescence excitation has been found to be a very powerful met hod for enhancing the sensitivity and resolution in far-field light microsc opy. Two-photon fluorescence excitation also provides a substantially backg round-free detection on the single-molecule level. It allows direct monitor ing of formation of labelled biomolecule complexes in solution, Two-photon excitation is created when, by focusing an intensive light source, the dens ity of photons per unit volume and per unit time becomes high enough for tw o photons to be absorbed into the same chromophore. In this case, the absor bed energy is the sum of the energies of the two photons. In two-photon exc itation, dye molecules are excited only when both photons are absorbed simu ltaneously. The probability of absorption of two photons is equal to the pr oduct of probability distributions of absorption of the single photons. The emission of two photons is thus a quadratic process with respect to illumi nation intensity, Thus in two-photon excitation, only the fluorescence that is formed in the clearly restricted three-dimensional vicinity of the foca l point is excited. We have developed an assay concept that is able to dist inguish optically between the signal emitted from a microparticle in the fo cal point of the laser beam, and the signal emitted from the surrounding fr ee labelled reagent. Moreover, the free labels outside the focal volume do not contribute any significant signal. This means that the assay is separat ion-free,The method based on two-photon fluorescence excitation makes possi ble fast single-step and separation-free immunoassays, for example, for who le blood samples. Since the method allows a separation-free assay in very s mall volumes, the method is very useful for high-throughput screening assay s. Consequently we believe that two-photon fluorescence excitation will mak e a remarkable impact as a research tool and a routine method in many field s of analysis.