1. Introduction

Microscopy has laid the foundation for many revolutions in biology since Leeuwenhoek first glimpsed ‘animicules’ through a glass lens. Richard Feynman saw microscopy of single molecules as the key to the problems of modern molecular biology. His famous 1959 lecture There’s plenty of room at the bottom (Feynman 1992) put it this way:

It is very easy to answer many of these fundamental biological questions; you just look at the thing! You will see the order of the bases in the chain; you will see the structure of the microsome. Unfortunately the present microscope sees at a level which is just a bit too crude. Make the microscope a hundred times more powerful, and many problems of biology would be made very much easier.

The all-powerful microscope of Feynman’s dreams is not here yet, but progress in scanning probe microscopy since the first edition of this book (Lindsay 1993) has been remarkable. The atomic force microscope (AFM) is now used routinely as a diagnostic probe of biomaterials. Fundamental new structural information is being revealed as AFM imaging becomes a routine complement to transmission electron microscopy (TEM) and new types of measurements on single molecules utilize the scanning probe microscope (SPM) as a local probe. These developments have been accompanied by similar advances in optical methods for studying single molecules, and we are beginning to learn that the world is a very different place when looked at one molecule at a time (Weiss 1999).

Advances in instrumentation have been rapid. The atomic force microscope can now reproduce the sort of atomic detail on clean surfaces once thought to be the exclusive domain of the scanning tunneling microscope (Giessibl 1995; Giessibl 1997) and this level of performance is being approached in liquids (Ohnesorge and Binnig 1993; Ohnesorge 1999). The scanning tunneling microscope, which featured so heavily in the first edition of this book, has almost been retired from the scene, though it merits a short postscript here.

Some quite remarkable resolution has been achieved (Shao and Yang 1995; Shao, Yang et al. 1995; Yang and Shao 1995; Engel, Schoenberger et al. 1997) even in solution (Bustamante, Rivetti et al. 1997) where movies of enzyme activity have been recorded (Kasas, Thomson et al. 1997; Stokstad 1997). Cryo-AFM (Shao and Zhang 1996) is beginning to produce spectacular results. A new field is emerging in which the probe is used as a chemical or force sensor or manipulator rather than as an imaging device (Rief, Gautel et al. 1997; Rousso, Khachatryan et al. 1997).

The field is too dynamic for a comprehensive review. A number of recent articles are helpful. They provide a key to the (exponentially expanding) literature (Bustamante, Keller et al. 1993; Bustamante, Erie et al. 1994; Hansma and Hoh 1994; Bustamante and Keller 1995; Shao and Yang 1995; Shao, Yang et al. 1995; Bustamante and Rivetti 1996; Hansma 1996; Ushiki, Hitomi et al. 1996; Bustamante, Rivetti et al. 1997; Engel, Schoenberger et al. 1997; Miles 1997; Muller, Schoenberger et al. 1997). The laboratories of Hansma and Bustamante have been particularly active in developing techniques for imaging important processes as they occur, for example the real-time binding of protein to DNA (Guthold, Bezanilla et al. 1994) the nuclease digestion of DNA (Bezanilla, Drake et al. 1994) and the action of RNA polymerase (Kasas, Thomson et al. 1997). News of further exciting developments will undoubtedly appear in the literature in the near future (Stokstad 1997). The best that this review can do in the face of such rapid growth is to provide some names on which to base a search. We have chosen to focus the bulk of this chapter on the physical and instrumental aspects of AFM in fluids because we believe that this will be the most useful approach. This material fills a gap in the current literature, but we expect that instrumental developments will continue apace, so be warned!

In order to make this chapter self-contained we begin with a very brief introduction to the physics of the AFM. We then discuss the factors that control resolution and interaction forces. We then turn to a rather extensive discussion of dynamic force microscopy in fluids. In the dynamic force microscope (DFM), interactions are sensed via the change in amplitude or phase of an oscillated tip. There have been important developments in the field not pulled together in another review, and this section forms the bulk of the present chapter. Our goals are to explain the physics of the imaging mechanism and operating criteria as well as to describe the instrumentation. We then consider methods of sample preparation. Many new approaches have become possible as a result of the flexibility of the SPM, and the impact of different preparation methods can be explored in a systematic manner. We then show some examples of images (a number of which are included separately as color plates). We turn finally to measurements on individual molecules such as elastic properties, unfolding, chemical bonding and charge transport. We end with discussion of the STM and conducting AFM.