In this paper we present the first application of a new spectral technique, differential laser-induced perturbation spectroscopy (DLIPS), to the in vivo detection of epithelial pathology in an animal model.22,23 The DLIPS sensing scheme incorporates three complementary techniques to improve upon previous fluorescence-based biosensing strategies: laser-induced fluorescence emission, ultraviolet (UV) laser perturbation of tissue, and difference spectroscopy.23 In this instance, fluorescence is used to measure the response of tissue fluorophores before and after the tissue is laser-perturbed. The perturbation pulses from the deep-UV excimer laser (193 nm, 6.4 eV) are strongly absorbed by biological tissue and used to cleave molecular bonds within the extracellular matrix (ECM) as shown schematically in Fig. 1. Irradiation of biological matrices at 193 nm can cause photoionization, including strand breakage, locally denatured sites, interstrand cross-linking, reactions via photo-hydrates, -dimers, and other products.24 In the current work, despite being well below the intensity threshold for tissue ablation, permanent alteration of the underlying tissue structure is induced, with resulting changes realized within the fluorescence spectrum, specifically with respect to photoreactive biomolecules, as made apparent with the DLIPS scheme. We note here that while no direct ablation is realized, a single photon of 193 nm radiation exceeds nearly all bond energies in the biological matrix; hence permanent photochemistry is induced despite being below the critical photon flux to affect material removal. Additionally, because the pre- and post-perturbation spectra are combined [see Eq. (1)] into a difference spectrum, the DLIPS technique mitigates unwanted contributions from unperturbed tissue fluorophores, broadband fluorescence, and importantly, variations in fluorescence emission bands, which are unique to the patient, but not necessarily to the targeted pathology. Equation (1) shows the DLIPS spectral response, namely: Display Formula
(1)where and represent the fluorescence emission intensity recorded at each wavelength before (pre) and following (post) perturbation by the UV excimer laser, respectively. As defined, a negative DLIPS signal corresponds with a reduction in fluorescence intensity following the photo-perturbation step, which is generally attributed to the destruction of a corresponding fluorophore. In contrast, a positive DLIPS signal corresponds to an increase in fluorescence intensity following perturbation, which may indicate destruction of a fluorescence-quenching species and/or the destruction of a concomitant absorbing compound, thereby allowing more light to reach the actual fluorophore. Overall, the complexity of the local fluorescence environment provides the opportunity for the perturbing UV radiation to affect a unique change to the resulting fluorescence response. Therefore, this combination of fluorescence, photochemical perturbation, and differential spectroscopy creates a completely unique spectral signature from targeted tissue. The result is a technique that specifically couples to important photosensitive tissue biomarkers of early pathological changes and that has promise to mitigate the apparent noise sources due to inter-patient variations.