Abstract
Fluorescence microscopy in combination with photoactivatable fluorophores offers the opportunity to image noninvasively the biological samples in real time with subdiffraction resolution. Photoactivatable fluorophores switch from nonemissive state to emissive state upon irradiation with activation wavelength and then emit light after excitation of product at suitable excitation wavelength. The concatenation of a photochemical reaction (activation) with a photophysical process (fluorescence) is therefore responsible for the operating principles of these photoresponsive compounds to switch fluorescence on within a defined region of space at a particular interval of time. In turn, such a spatiotemporal control permits the monitoring of dynamic events in real time and the visualization of samples with subdiffraction resolution. The identification of viable operating principles to photoactivate the fluorescence of organic chromophores is, therefore, essential for the further development of these promising imaging techniques. In this context, we designed two mechanisms for the activation of the fluorescence of anthracene derivatives based on photoinduced retro-cycloadditions and photodecarbonylation. Furthermore, mechanism to photoactivate fluorescence by photodecarbonylation was designed to proceed by autocatalytic photochemical pathway where the photoproduct can sensitize its own formation from the reactant, under illumination at a wavelength capable of exciting both species. The amphiphilic polymer can encapsulate diversity of hydrophobic guest into their hydrophobic core and transport them into the aqueous phase, thus, can be a valuable delivery vehicle for diversity of biomedical applications. These supramolecular nanocarriers along with covalent integration of fluorophores in their molecular backbone can be useful to probe them directly into the intracellular space. In this context, we synthesized amphiphilic polymer by covalent integration of either donor or acceptor in their macromolecular backbone. Their photophysical properties both in organic and aqueous phase along with imaging experiment in Hela cells were performed. We further. optimized the brightness of these polymer by keeping compact dimension, with systematic integration of number of fluorophores in their macromolecular backbone, as a result, these polymers are significant brighter than the model monomers. Such a high brightness level is maintained even after injection of the macromolecular probes in living nematodes, allowing their visualization with a significant improvement in signal-to-noise ratio, relative to the model monomer, and no cytotoxic or behavioral effects. Further, the bioconjugate of this polymer with secondary antibody does not hinder the targeting ability of the former and also can be exploited to stain the tubulin structures of model cells to enable their visualization with optimal signal-to-noise ratios. We envisaged the protocol to imprint fluorescent pattern on photoresponsive polymer film with mild illumination condition to overcome limitation imposed by conventional photobleaching method. We investigated the photophysical and photochemical properties of this polymer in organic solvent along with fabrication of microscaled fluorescent patterns in polymer film.