**1. Introduction**

Even though we have several techniques, represented by the electron microscopy, to obtain images of single molecules, in this chapter, we use 'single-molecule imaging' (SMI) for a lim‐ ited means—that is, imaging of fluorescently labeled biological molecules at work for ana‐ lyzing their behaviors. To observe biological molecules at work, imaging in aqueous conditions is essential. Therefore, optical microscopy is the main technology in SMI. Fluores‐ cence labeling is good to use for imaging in optical microscopy, because it allows high con‐ trast and selective imaging of molecules that we are interested in. SMI provides information of dynamics and kinetics of molecular reactions. In 1995, two groups firstly and independ‐ ently realized SMI of biological molecules in aqueous conditions [1,2]. In the early days, SMI was used mainly for the *in vitro* studies of protein motors [1,2] and metabolic enzymes [3]. Detection of enzymatic reaction (reaction kinetics) [1,3] and detection of protein dynamics (lateral and rotational movements) [2] have been the two main usages of SMI since the first development of this technology. After that, the application of SMI has been extended, and in 2000, SMI became to be used in living cells [4,5].

Probably, for the readers, the most familiar usage of SMI is the measurement of molecular dynamics, including conformational changes and lateral and rotational movements. Howev‐ er, kinetic analysis is one of the original applications of SMI as mentioned above, and in many cases, it relates more closely to the higher-order biological functions than does dy‐ namics analysis. This chapter focuses on the kinetic analysis of protein-protein interactions, including molecular recognitions and enzymatic reactions in living cells. As the application of SMI, we introduce a ligand-receptor interaction on the cell surface, and a process of pro‐ tein activation occurs in a ternary protein complex beneath the plasma membrane. Because

© 2013 Hibino et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Hibino et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the main subject of this chapter is the technical issue, these two examples of applications are chosen to explain how to analyze single-molecule data to understand kinetics of molecular interactions quantitatively in living cells. At the present time, we have several textbooks spe‐ cialized for the technologies and applications of SMI in a wide field [6-8]. Please refer to these books for information lacking in this chapter.

these metastable points in various timescales. post-translational modifications, such as phos‐ phorylation, may stabilize one or some of the metastable structures, according to each sin‐ gle molecule. SMI measurements are good to detect distributions and fluctuations of reactions and structures caused by static and dynamic polymorphisms of biological macromole‐ cules [3,9-11]. In some cases, non-random reactions of proteins have been detected using

Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells

http://dx.doi.org/10.5772/52386

435

In a cellular context, it is difficult to perform long time-series analyses of the reaction on the same single molecules due to high density and lateral movements of the molecules. Howev‐ er, SMI measurements still have advantages over conventional ensemble average measure‐ ments. SMI allows *in situ,* non-destructive quantitative measurements. SMI measurements provide absolute values of the kinetic and dynamic parameters of the molecular reactions and dynamics, including number, density, reaction rate constants, lateral diffusion coeffi‐ cient, and transport velocity, with the smallest disruption of the living cell systems [8,12,13]. As mentioned above, for the time-series analyses, SMI measurements do not require physi‐ cal synchronization, which is indispensable in ensemble molecule measurements. Synchro‐ nization, like concentration or temperature jumps, generally alters the condition of cell cultures, possibly affecting many cellular reactions in addition to the subject of the experi‐ ment. Therefore, it sometimes becomes ambiguous if the changes of the measured value re‐ flect the reaction kinetics itself or the cellular dynamics triggered by the changed culture conditions. SMI measurements allow kinetic analysis in steady state, avoiding changes of culture conditions. For example, Hibino et al. [14] measured dissociation kinetics between

two protein species, Ras and RAF, using SMI in quiescent cells in a steady state.

SMI measurements can detect small numbers of reactions in a limited volume inside living cells, because they are based on imaging with spatial resolution and possess the extreme sensitivity to detect single molecules. Using SMI measurement, Ueda et al. [15] measured the numbers and rate constants of the reactions between cAMP and its receptor, comparing the front and rear halves of a single *Dictyostelium* amoeba during chemotaxis. Tani et al. [16] analyzed reaction kinetics at the growth cone of nerve cells. These works have revealed that kinetic parameters of the same reactions diverged according to the positions in single cells. In addition, SMI measurements have directly revealed that cellular responses, such as neu‐ rite elongation [16] and calcium response [17], are caused by signaling of tens or hundreds

The range of application of SMI measurements is broad, covering various fields of biological sciences of cells, including not only basic biochemistry and biophysics but systems biology, pathology of genetic diseases, action points analyses in pharmacology, and toxinology. SMI measurements provide absolute values of reaction parameters, which can be substituted in‐ to the reaction models described using mathematical equations. Since these values are deter‐ mined in live cell conditions, SMI measurements are good to use in combination with mathematical modeling constructed to explain and predict dynamics of complex intracellu‐

SMI [3,9,11].

of protein molecules.

lar reaction networks.

**2.2. Single molecule measurements in living cells**
