Single-molecule imaging of fluorescent proteins expressed in living cells

This chapter focuses on single-molecule imaging (SMI) in living cells using green fluorescent protein (GFP) or its related fluorescent protein tags (GFPs). Use of GFPs is a convenient technique to achieve molecular imaging of most proteins in living cells. However, because of difficulties in preparing samples suitable for SMI and the instability of fluorescence signals, special care is required for SMI using GFPs in living cells. Techniques for vector preparation, protein expression, sample preparation, microscopy, and image processing for SMI of GFPs in living cells are discussed in this chapter, along with examples of imaging applications. Double labeling of single molecules and single-pair fluorescent resonance energy transfer (spFRET) are possible in living cells using GFP and YFP as fluorescent tags. The limitations of SMI using GFPs are also discussed.