Dyes and nanoparticles for bioimaging: linear photophysical and nonlinear optical study

Abstract

This thesis is performed in the framework of a collaborative project Nanochemistry of molecular materials for two-photon functional application (Nano2Fun), funded by the EU commission. The main aim of Nano2Fun is to develop optimized materials and methods for specific applications of two-photon absorption towards bioimaging and nanofabrication. In this context, the contribution of this work is an extensive spectroscopic study to address the linear and nonlinear photophysics of organic dyes and of fluorescent organic nanoparticles of interest for two-photon induced processes. Nonlinear optical studies include two-photon absorption and two-photon excited anisotropy, stimulated emission depletion and amplified spontaneous emission. Essential-state models were applied to describe linear and nonlinear optical response of dyes with particular focus on the role of molecular symmetry, solvent effects and vibrational coupling in two-photon absorption and two-photon excited anisotropy spectra. We describe fluorescent properties, two-photon absorption response and photochemical stability of nanoparticles which were prepared using two different approaches. The first one is based on one-step solvent exchange reprecipitation method and the second approach relies on using cholesterol-rich small unilamellar vesicles – quatsomes (QS) – as dye carriers prepared by a partner unit within Nano2Fun, using supercritical fluid technology. We demonstrate the potential of dye-loaded QS as FRET-based multicolor probes and in bioimaging. Fluorene-based QS show good specificity for tracking lysosomes and were applied for bioimaging of HCT 116 cancer cells. Cyanine-loaded QS exhibit appealing properties as probes for sub-diffraction stochastic optical reconstruction microscopy and they were successfully imaged in HeLa cells. Depending on the encapsulate dye cyanine-loaded QS show different staining patterns.