Tissue morphology and mechanics are crucial to the regulation of organ function. Investigating the exceptionally complex tissue of the brain at the sub-micron scale is challenging due to the complex structure and softness of this tissue, despite the large interest of biologists, medical engineers, biophysicists, and others in this topic. Atomic force microscopy (AFM) both as an imaging and as a mechanical tool provides an excellent opportunity to study soft biological samples such as live brain tissues. Here we review the principles of AFM, the performance of AFM in tissue imaging and mechanical mapping of cells and tissues, and finally opening the prospects and challenges of probing the biophysical properties of brain tissue using AFM.