Measurement of two ‐photon‐absorption spectra through nonlinear fluorescence produced by a line‐shaped excitation beam

We present a newly conceived experimental technique to measure the two‐photon‐absorption spectra of fluorescent materials. The proposed method is based on the two‐photon fluorescence, but it permits to overcome the typical drawback of such a technique, consisting of the very low signal level, which usually is maintained in a regime of photon counting. In our proposed method the optical excitation of the nonlinear fluorescence is produced by employing a line‐shaped laser beam. This configuration, which permits to maintain the intrinsic high selectivity on two‐photon‐absorption signal which is typical of fluorescence‐exploiting techniques enables to simplify the detection apparatus and to increase the signal allowing to not use a photon counter as a detector, which is usually necessary in such a kind of experiments. In the paper we discuss the principia which our system is based on, the experimental procedure, and, finally, we present test measurements carried out on fluorescein isothiocyanate (FITC) sample, a colorant whose two‐photon‐absorption spectrum is known in literature. The proposed procedure can be classified as an ”indirect method” for the two‐photon absorption detection, because it exploits the phenomenon of two‐photon‐induced fluorescence emission. The employed line‐shaped illumination, with an optical configuration similar to what recently proposed in the nonlinear microscopy domain, is demonstrated to provide the advantage of requiri...
Source: Journal of Microscopy - Category: Laboratory Medicine Authors: Tags: Original Article Source Type: research