Since the invention of atomic force microscope (AFM) two decades ago (Binnig et al., 1986), it has been extensively used in almost every branch of science and engineering. is oers an advantage of having the ability to convey nanometer spatial resolution in three dimensions in the absence of vacuum and contrast reagent. Furthermore, it is compatible with chemical and physical environment control in the liquid state. It provides an alternative for researchers to explore cell and tissue dynamics with molecular resolution under a controlled environment, for example, pH, ion strength, temperature, and signaling molecules. is chapter begins with the working principles and suitable applications of dierent imaging modes, followed by emerging technology, such as quantitative nanomechanical properties measurement of biomaterials, molecular recognition on a substrate or cell membrane, and so on. An uprising trend in characterization techniques is to integrate or tandem dierent instruments to get information from dierent aspects. e correlation of dierent information from a combination of multiple systems provides insight into the underlying mechanism. AFM is not an exception, it has been integrated with advanced uorescence techniques, including confocal uorescence microscopy, total internal reection uorescence microscopy, and electrophysiological means, for example, patch clamp and ion conductance measurement. e applications and challenges of the integration between AFM and optical techniques will also be discussed in this chapter.