| Over the past decade, sophisticated improvements in solid-state tunable high-repetition rate femtosecond lasers have made multi-photon excitation fluorescence (MPEF) experiments more accessible to the biophysical scientist. MPEF methods offer great potential toward the direct analysis of clinical samples in that the inducible nature of two-photon absorption allows the use of exciting light within the “transparent” window of biological tissues (680–100 nm). However, while MPEF has become a mainstay among microscopists, its application toward direct bioanalysis has lagged somewhat behind.; In this thesis, I will illustrate the power of MPEF measurements over classic one-photon excitation schemes for making static and dynamic fluorescence measurements. The thesis is divided into three main areas of discussion: (a) the construction and performance of a new system that allows the rapid acquisition of multifrequency time-resolved intensity and anisotropy decay data sets under one-, two-, and three-photon excitation, (b) results for a two-photon excited fluorescence polarization immunoassay (FPIA), and (c) two-photon excited fluorescence detection of several analytes present at normal and supernormal clinical levels based on ion-responsive molecular probes. |
