AFM operation under liquid

The imaging of biologically relevant polymers must be performed in most cases under liquids in order to reduce the forces between the scanned probe tip and the sample surface or, more importantly, to ensure that the polymers of interest retain their integrity, shape, and biological function when studied by AFM approaches. This Chapter will provide an introduction to AFM operation under liquid and will elucidate the peculiarities of force microscopy operation in conjunction with a liquid cell. Finally, ex situ and in situ studies of biopolymeric specimens will be highlighted. 

As mentioned, in AFM studies of biopolymers the use of a suitable liquid cell is indispensable in many cases. On the one hand, biopolymers or even living cells may be studied in vitro under natural conditions (pH, temperature, salt, etc.) and variations of these conditions is often possible during the experiment [71-74], on the other hand excessive normal and lateral forces can be reduced to a minimum, which still allows one to image and study the biopolymeric samples non-invasively [79-81], Hence we will first provide an introduction to the use of the mentioned liquid cells and then treat contact mode AFM and intermittent contact mode AFM operation under liquid. The procedures and operation principles discussed can of course be readily extended to the study of non-biological polymers (see e.g. Chap. 4). 

Fig. 3.37 Schematic of AFM operation under liquid without rubber ring. A water drop, which is spanned between liquid cell and sample, encloses the imaged area incl. tip and cantilever completely...

For TM-AFM operation under liquid, the system is set up similar to the contact mode in liquid. By contrast, the cantilevers used are typically much softer than for operation in air. Often conventional contact mode levers (spring constant of 0. 3-few N/m) are used single beam cantilevers work in some cases better than triangular levers. The tuning of the lever is performed after the crude approach has... 

As with STM, the AFM can be operated in air, in vacuum or under liquids, providing a suitable cell is provided. Liquid cells (figure Bl.19.20) are particularly usefiil for the examination ofbiological samples. 

In contrast to many other surface analytical techniques, like e. g. scanning electron microscopy, AFM does not require vacuum. Therefore, it can be operated under ambient conditions which enables direct observation of processes at solid-gas and solid-liquid interfaces. The latter can be accomplished by means of a liquid cell which is schematically shown in Fig. 5.6. The cell is formed by the sample at the bottom, a glass cover - holding the cantilever - at the top, and a silicone o-ring seal between. Studies with such a liquid cell can also be performed under potential control which opens up valuable opportunities for electrochemistry [5.11, 5.12]. Moreover, imaging under liquids opens up the possibility to protect sensitive surfaces by in-situ preparation and imaging under an inert fluid [5.13]. 

The choice of operation modes and, if applicable, suitable imaging environments depend on many factors, including the type of polymer system to be analyzed and the type of information that is required. Biologically relevant materials or effects that are intrinsic to the liquid—solid interface, for instance, require, of course, AFM under liquid. For a number of experiments, these almost trivial considerations dictate the choice and we refer to the hands-on sections in the corresponding chapters for more detailed information. 

One central concern with routine AFM on polymers is the presence of shear forces that occur in CM. These forces are a result of friction between AFM probe tip and the polymer sample and may deform and plastically modify the polymer surface. This has been observed even for glassy materials, such as PS, when imaged at ambient conditions (see Sect. 3.2.3 in Chap. 3 Fig. 3.16). In addition to sample damage, the tip may be affected by adhering particulates or, even worse, by wear. These phenomena limit the resolution dramatically and may result in unwanted artefacts (excessive tip imaging). Thus, minimized imaging forces are essential, and this may require the operation under a suitable liquid to eliminate capillary forces. 

For the operation of contact mode AFM under liquid there are only few details that differ from operation in air. The imaging forces can often be controlled much more precisely if the adhesion is lower due to the absence of capillary forces. Hence the adjustment of the setpoint requires more attention and can be done with much more precision. The adjustment can be based on acquired force-displacement curves (see below, Fig. 3.39). Setpoint deflection values close to the out of contact deflection yield minimized normal forces [82-84],... 

Non-contact AFM is performed in liquid, under vacuum and in air [90]. In the non-contact regime, the water film present on a sample in air will typically not be penetrated. If for a particular sample this is a problem, and contact mode also happens to be a problem (for example a soft sample could be damaged by a dragging tip) then a tapping mode operation of the cantilever in the intermittent regime may be the best choice. 

Since the AFM is commonly used under ambient conditions, it must be borne in mind that the sample is likely to be covered with multilayers of condensed water. Consequently, as the tip approaches the surface, a meniscus forms between tip and surface, introducing an additional attractive capillary force. Depending on the tip radius, the magnitude of this force can be equal to or greater than that of the van der Waals forces and is observed clearly in the approach curve [98], In fact, this effect has been exploited for the characterization of thin liquid lubricant films on surfaces [95], The capillary forces may be eliminated by operation in ultrahigh vacuimi, provided both tip and sample are baked, or, most simply, by carrying out the experiment under a contamination-free liquid environment, using a liquid cell [99],... 

Since the AFM does not use tunneling currents to sense the deflection of the cantilever and does not require a vacuum environment for operation, it has been used very successfully under water and other liquids. Figure 74 B shows the type of liquid cell employed by Hansma and coworkers [239]-[243]. The tip with its mirror is attached to the underside of a strip of poly-(methyl methacrylate) that also carries a circular groove for a small O-ring. Once the sample has made contact with the O-ring. the cell is sealed, but the O-ring material is sufficiently flexible to allow movement of the sample under the tip in the usual way. A recent modification [244] of the design allows the fluid in the cell to be circulated in order to simulate flow conditions, while inserted electrodes allow cyclic voltammetry to be performed. 

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