Project Summary My research takes a unique approach in which the development of next-generation microscopy methods progresses in parallel with fundamental discoveries in cell biology. On the method-development front, besides earlier success in achieving sub-10 nm resolution for super-resolution microscopy, my recent work has pioneered the concept of multidimensional and multifunctional super-resolution microscopy, in which intracellular functional parameters, including local chemical polarity, pH, diffusivity, and protein activity, are mapped out at nanometer resolution and single-molecule sensitivity. Empowered by such capabilities, my lab has been highly successful in unveiling hidden subcellular structures and processes, as well as their underlying biophysical rules, for diverse systems ranging from the mammalian cytoskeleton, intracellular transport, organelle morphology and biogenesis, to membrane biology. Our future research continues to push forward the synergy between method development and biological discoveries. Major directions include charge-modulated protein interactions and effects on diffusion inside the organelle lumen, superdiffusion and subdiffusion in the living cell, organelle pH dynamics and role in protein trafficking, and the structure and physical properties of the ER exit site in relationship with the biogenesis of transport carriers. Moreover, by integrating super-resolution microscopy with FIB-SEM, we will obtain holistic pictures of the unusually thin tubular organelles we recently discovered and further substantiate their functions and biogenesis. Separately, we are developing a new tool, single-molecule electrophoresis microscopy, to quantify protein charge states at the super-resolution level. Together, through the continued development of empowering microscopy tools and their tactical application to fundamental biological questions, we will continue shifting the paradigms of how we understand the complex, dynamic behavior of the cell.