Single molecule imaging

Abstract

Single-molecule imaging is a powerful tool to understand working mechanisms of glycoproteins and glycolipids in cell plasma membranes by overcoming intrinsically stochastic and inhomogeneous properties of biological molecular systems. Here, basic techniques for single-molecule imaging microscopy are described. This method can be carried out by total internal reflection fluorescence microscopy or by oblique illumination microscopy with high-sensitive CCD cameras. The densities of fluorescently labeled membrane proteins and lipids must be low (<2 molecules/μm2) in cell plasma membranes because highdensity expression interferes with tracking of individual fluorescent spots. Therefore, single-molecule imaging in cell membranes requires that conditions be optimized for protein expression, fluorescent labeling, and lipid incorporation. In this chapter, methods for observing and detecting colocalization of individual fluorescent spots of proteins or lipids are described. Furthermore, analysis methods for single-molecule imaging, focusing on colocalization of two different fluorescent spots, are explained.