# 3D single molecule and super-resolution fluorescence microscopy of polymer systems

In the past few decades, single molecule and super-resolution fluorescence microscopy techniques have gained much importance in biological and material sciences. With single molecule fluorescence microscopy, one can significantly observe individual events, such as the heterogeneities inside polymer films, unlike ensemble measurements that otherwise average these exciting events. By surpassing a significant barrier of the diffraction limit of light, super-resolution techniques have enabled imaging of nanostructures with a resolution of few nanometers. But, along the axial direction, the standard point spread function (PSF) exhibits limited resolution. However, with the advent of 3D imaging techniques, this limitation has also been overcome. With the advancement in nanoscience, polymer nanostructures have become potential structures for a wide range of applications in material and biological sciences. However, to understand the full potential of these polymeric structures, a thorough investigation of their structure and environmental conditions must be carried out. The present thesis consists of three projects which utilize the power of 3D super-resolution and 3D single molecule fluorescence imaging to analyze the structures of polymeric materials. The first project of this thesis focuses on the investigation of the 3D structure and the polarity conditions of soft colloidal structures called microgels. For this purpose, a super-resolution technique called points accumulation for imaging in nanoscale topography (PAINT) with a possibility of 3D imaging via astigmatic imaging was employed. Additionally, to investigate the compartmental polarity inside the microgels, the solvatochromic behavior of Nile Red fluorophore was utilized without the need for covalent labeling. This technique gives a detailed understanding of altering the structural and polarity properties of temperature-sensitive microgels when changing the temperature beyond the volume phase transition. The second project of this thesis focuses on the 3D translational diffusion of single molecules embedded in thin polymer films, mainly focusing on the effect of interfaces namely polymer-air and polymer-substrate interfaces on the translational diffusion of single molecules. Heterogeneous diffusion of single molecules was observed at 30 °C beyond the T$_g$ of the PBMA polymer, which was the polymer under investigation. The fast diffusing single molecules were always of lower intensity than the slow diffusing single molecules, which is attributed to the closer proximity to the oxygen-containing air of molecules in the topmost layer of the polymer thin film. However, the exact relation between the axial position and the heterogeneous diffusion could not be determined due to limiting factors like mislocalizations caused due to dipole moment orientation of single dye molecules. The third project of this thesis focuses on the 3D imaging of single QDs embedded in PVA film via a 3D imaging technique called double-helix PSF imaging. These experiments were carried out to demonstrate the proof of principle of the new idea of multimodal super-resolution imaging, which combines the cumulant analysis from Super-resolution Optical Fluctuation Imaging (SOFI) with the imprinting of three-dimensional, spectral or other information into peculiar Point-Spread Function (PSF) patterns. This new concept allows for encoding multidimensional or multimodal information into a single image plane and extracting this information by an appropriate spatio-temporal correlation analysis of emitter fluctuations.