Quantum dot-based single-molecule microscopy for the study of protein dynamics.

Real-time microscopic visualization of single molecules in living cells provides a molecular perspective of cellular dynamics, which is difficult to be observed by conventional ensemble techniques. Among various classes of fluorescent tags used in single-molecule tracking, quantum dots are particularly useful due to their unique photophysical properties. This chapter provides an overview of single quantum dot tracking for protein dynamic studies. First, we review the fundamental diffraction limit of conventional optical systems and recent developments in single-molecule detection beyond the diffraction barrier. Second, we describe methods to prepare water-soluble quantum dots for biological labeling and single-molecule microscopy experimental design. Third, we provide detailed methods to perform quantum dot-based single-molecule microscopy. This technical section covers three protocols including (1) imaging system calibration using spin-coated single quantum dots, (2) single quantum dot labeling in living cells, and (3) tracking algorithms for single-molecule analysis.