The evolution of the 3D morphology is at the center of many relevant biological processes ranging from cellular differentiation to cancer invasion and metastasis. Microscopy techniques, such as electron microscopy (EM), super-resolution (SR) optical microscopy, and atomic force microscopy (AFM), have been applied to image the structure of cells in great detail. The major challenge is to obtain 3D information at nanometer resolution without affecting the viability of the cells and avoiding interference with the process. The thesis presents the development of a time-resolved scanning ion conductance microscope (SICM), from concept to prototype, capable of resolving spatiotemporal biological processes with unprecedented resolution and imaging speed. By integrating advances in nanopositioning, controls theory, microelectronics, and nanopore fabrication, the time-resolved SICM system enabled sub-5 nm resolution, performed high-speed imaging of 0.5 s per pixel, and allowed large imaging volumes. Moreover, the stability of the system enabled performing live cell imaging over 48 h without perturbations. We applied time-resolved SICM to dynamic processes on cell membranes that included: Structural evolution of circular dorsal ruffles (CDRs), shedding light on their mechanisms of formation; Morphological changes in human melanoma cells upon treatment with a drug known to reduce resistance to immunotherapy; And mechanisms of bacteria-host infection in the human cell membrane. Furthermore, we combined time-resolved SICM with super-resolution fluorescence optical fluctuation microscopy (SOFI). By optimizing the SOFI computational approach and fluorophore's properties, high-speed correlative 3D imaging with mitigated phototoxic effects was achieved. The complementary capabilities of time-resolved SICM and SOFI provided comprehensive information on cell membrane morphology and cytoskeleton protein architecture, offering sub-diffraction resolution in living cells. This combined method holds promise as a routine tool for studying membrane processes. In addition to imaging, time-resolved SICM was successfully adapted for single-molecule spectroscopy using nanopores, creating a new technique called scanning ion conductance spectroscopy (SICS). SICS overcame limitations in nanopore technology by controlling the nanopores' position and the translocation speed of individual molecules. The ability to control the speed and average multiple readings of the same molecule has resulted in two orders of magnitude increase in signal-to-noise ratio compared to conventional free translocation. In our glass nanopore experiments, we detected a 3.4 angstroms single nucleotide missing in a long strand of dsDNA. Moreover, we utilized SICS to successfully identify and analyze intricate topological features within complex DNA structures, including DNA-dCas9 complexes, hairpins, molecular rulers, and dsDNA gaps. The increased detection capability with SICS has the potential to be transferable to other solid-state and biological nanopore methods, significantly improving diagnostic and sequencing applications.