Biophysical applications of correlative scanning probe and super-resolution microscopy

Imaging live cells in their native environment is crucial for the understanding of complex biological phenomena. Modern optical microscopy methods such as fluorescence super-resolution microscopy are increasingly combined with complementary, label-free techniques to put the high-resolution fluorescence information into cellular context. Common high-resolution imaging approaches used in combination with fluorescence imaging such as electron microscopy and atomic force microscopy (AFM) were originally developed for solid-state material characterization and thus are not straightforwardly applicable to image live cells for sustained periods of time. In this thesis, I am focusing on advancing state-of-the-art correlative scanning ion-conductance microscopy (SICM) combined with novel super-resolution imaging techniques. The work presented in this thesis demonstrates various technological advancements for both imaging modalities as well as experimental proof-of-principle studies. The first part of the work focuses on a novel experimental combination of SICM and super-resolution optical fluctuation imaging for single-cell imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. We employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction resolution. In order to increase the imaging depth for thick samples, a novel remote focusing modality based on an adaptive optics device was developed. Here, we use a deformable mirror to acquire multiple image planes of the labelled cells and we process the data using a single-molecule localization routine and/or computed spatiotemporal correlations, demonstrating subdiffraction resolution within a whole imaging volume. Second, we demonstrate the advantages of a new SICM probe manufacturing method allowing the batch production of glass nanopipettes used for SICM imaging and sensing. Finally, we show a new application of SICM tailored for single-molecule characterization based on electrical signatures while performing controlled translocations of surface-immobilized DNA molecules. The method is developed to be compatible with single-molecule fluorescence imaging and was applied for the detection of various analytes in a correlative manner. We believe that a synergy between fundamentally different imaging modalities is going to become necessary in an age of computational microscopy to ensure an unbiased data interpretation in the rapidly progressing field of biological research.