Atomic force microscopy (AFM) can probe single living cells and single native membrane proteins in natural fluid environments with label-free high spatial resolution. It has thus become an important tool for cellular and molecular biology that significantly complements traditional biochemical and biophysical techniques such as optical and electron microscopy and X-ray crystallography. Imaging surface topography is the primary application of AFM in the life sciences. Since the early 1990s, researchers have used AFM to investigate morphological features of living cells and native membrane proteins with impressive results. Steady improvements in AFM techniques for imaging soft biological samples have greatly expanded its applications. Based on the authors' own research in AFM imaging of living cell morphologies, a review of sample preparation procedures for single-cell and single-molecule imaging experiments is presented, along with a summary of recent progress in AFM imaging of living cells and native membrane proteins. Finally, the challenges of AFM high-resolution imaging at the single-cell and single-molecule levels are discussed.