Towards Undistorted Fluorescence for Tissue Diagnostics and Therapy Monitoring

Fluorescence techniques for applications in tissue diagnostics and therapy monitoring were investigated and developed in the work presented in this thesis. The aim was to develop fluorescence diagnostics for the detection of malignant tumors and better delimitation of the tumor margins, using the enhanced contrast in fluorescence between tumor and surrounding healthy tissue. Fluorescence may be generated by either endogenous fluorophores in the living organism, or exogenous fluorophores that target tumors. The contrast in fluorescence enables visualization of the fluorescent objects, such as tumors. The fluorescence spectra may also contain valuable information on tissue structures and functions for early detection and identification of diseased tissues. Compared with the standard histopathological examinations used for diagnosis, fluorescence modalities provide a reliable, noninvasive and sensitive tool, which can be used in vivo and in real-time.

Fluorescence can also be used for treatment monitoring and drug quantification for photodynamic therapy (PDT). PDT has been developed rapidly during the past decades. It is a localized non-ionizing radiation treatment modality used to kill tumor cells, relying on the interactions between visible light and tumor tissues that contain a tumor-targeted photosensitizing drug (the so-called photosensitizer) in the presence of oxygen. Many factors, such as the localization of tumors, and real-time monitoring of treatment, as well as the pharmacokinetics and biodistribution of photosensitizers, play an important role in optimizing the outcome of PDT. These factors can be achieved by fluorescence detection techniques, due to the fact that most photosensitizers emit fluorescence in the visible range.

Fluorescence detection has been performed with fluorescence spectroscopy both in the imaging (using multi-spectral imaging) and point monitoring (using spectroscopic fiber optic probes) mode in this work. Fluorescence imaging provides a wide field of view with a higher spatial resolution, while point monitoring can provide an overall higher spectral resolution, sensitivity, and depth resolution by inserting to probe tip deeper into underlying regions. Both techniques make use of the undistorted fluorescence signal originating directly from the fluorophores, either endogenous or exogenous (i.e. photosensitizers), before it is attenuated by turbid tissue or confounded by other factors. Such undistorted fluorescence is believed to be linearly dependent on the true concentration of fluorophores and thus well correlated to tissue properties. Different approaches, including light transport models in turbid tissue, the dimensionless ratio of spectral bands, and multivariate analysis techniques, have been proposed in this work to obtain the undistorted fluorescence. These approaches have shown significant improvements in the correlation between the detected fluorescence signal and fluorophore concentration.

Upconverting nanoparticles (UCNPs) doped with rare earth ions are an emerging type of fluorescence contrast agent for molecular imaging. Because of their unique features, UCNPs have attracted considerable interest in biomedical applications. UCNPs can be excited in the near-infrared range where tissues have low absorption and autofluorescence. They can thus be utilized for deep-tissue and whole-body imaging of small animals with a high sensitivity. UCNPs can have multiple emission bands from the visible to near-infrared. Surface modication and multifunctionalization of UCNPs facilitate their use as drug delivery vehicles and excitation source for PDT. New techniques have been developed in this work for the synthesis of UCNPs and the characterization of their power-density-dependent quantum yield.