Abstract
Fluorescence microscopy offers a non-invasive way to image biological species in real time. The phenomenon of diffraction, however, restricts the resolution of the conventional fluorescent microscopes to submicrometer dimensions in both the horizontal and vertical directions. This limitation can be overcome with the aid of photoactivatable fluorophores. Photoactivatable fluorophores switch from a nonemissive state to an emissive one under irradiation at an activation wavelength and then emit light in form of fluorescence upon illumination at an excitation wavelength. Such a concatenation of activation and excitation events translates into the possibility to switch fluorescence on within a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence offers the opportunity to monitor dynamic processes in real time as well as to reconstruct images with resolution at the nanometer level. As a result, these photoresponsive molecular switches are becoming invaluable analytical tools to probe the structures and dynamics of a diversity of materials relying on the noninvasive character of fluorescence imaging. In this context, I explored ways to photoactivate fluorescence based on 2-nitrobenzyl derivatives. To begin with, fluorescence photoactivation of a coumarin fluorophore with the aid of an appended halochromic auxochrome was designed and its photophysical and photochemical properties were investigated in organic and aqueous environments. Using this strategy, we were able to activate fluorescence in liquid solutions, within rigid matrices and inside micellar assemblies. Furthermore, it is possible to imprint fluorescent patterns on polymer films, to monitor proton diffusion within such materials in real time on a millisecond timescale and acquire images with spatial resolution at the nanometer level. Besides, a mechanism was devised to open the ring of an oxazine auxochrome irreversibly under the influence of light acting on the 2-nitrobenzyl group. The photochemical and electrochemical properties of this compound were also explored. To improve the brightness of the photoactivatable probes employed to obtain super-resolution images, strategies for fluorescence activation of borondipyrromethene (BODIPY) chromophores were explored. In addition, a protocol was designed to study the guest exchange between dynamic supramolecular assemblies inside cells.