In many research areas such as biology, biochemistry, and biophysics, measuring distances or identifying and counting objects can be of great importance. To do this, researchers often need complicated and expensive tools in order to have accurate measurements. In addition, these measurements are often done under nonphysiological settings. X-ray diffraction, for example, gets Angstrom-level structures, but it requires crystallizing a biological specimen. Electron microscopy (EM) has about 10 Å resolution, but often requires frozen (liquid nitrogen) samples. Optical microscopy, while coming closest to physiologically relevant conditions, has been limited by the minimum distances to be measured, typically about the diffraction limit, or ~. 200. nm. However, most biological molecules are <. 5-10. nm in diameter, and getting molecular details requires imaging at this scale. In this chapter, we will describe some of the experimental approaches, from our lab and others, that push the limits of localization accuracy and optical resolution in fluorescence microscopy.