Control of two-photon fluorescence of common dyes and conjugated dyes

We present a comprehensive study of the selective excitation of two-photon fluorescence from various pairs of dyes and dyes in different conjugation states with tailored pulse shapes found with a genetic algorithm (GA). We investigate a number of biologically important dyes, and include dyes conjugated to trastuzumab (Herceptin(R)) and to a poly(amidoamine) dendrimer. We consider in detail the ability of tailored pulse shaping to discriminate dyes with significant spectral overlap. Our procedure for adaptive pulse shaping includes power-law and chirp-scaling checks to prevent trivial convergences. The GA uses a multiplicative fitness parameter in a graded search method that converges on pulse shapes that not only differentiate two-photon processes, but do so in a high signal regime. We consider the results in terms of not only the absolute maximum ratio of discrimination achieved, but also present the evolutionary course of the GA and compare the improvement to a quantitative measure of the noise level. We also implement a time-domain acousto-optic measurement of two-photon excitation cross-section spectra. The results show that the ability to discriminate dyes is determined almost entirely by their differences in two-photon excitation cross section.