Types of Imaging, Part 3: Atomic Force Microscopy

Authors


This article is the third in a series on types of microscopy. The atomic force microscope (AFM) has become useful for biologists because surface detail can be resolved to the atomic level. As well as in high-resolution scanning, the AFM is able to use nanoindentation of live cells, allowing local mechanical properties to be directly correlated with cytoskeletal structures. The AFM offers new insight into a wide range of tissues and cells, as well as biological events that affect physiological processes involved in disease. Binnig et al. (1986) first reported this new type of microscope. Most microscopes use radiation, which is emitted and recorded using lenses. The resolution power of AFMs is affected by damage and/or diffraction to the biological sample caused by the illuminating beam.

However, the first type of microscopy to depend on an effect that only occurs in the area immediately surrounding the sample and a physical probe is the scanning tunneling microscope (STM) (Amrein, 2007). STM involves an electrically conductive needle approaching the sample until flow of current. The STM is of interest to scientists owing to its considerable power of resolution and lack of damage by radiation. However, the use of STM in biological sciences is restricted by insufficient electrical conductivity of many biological samples; consequently, acceptable images can only be achieved by coating specimens with metal.

The basic ideas behind the STM were applied in developing new microscopes, including the scanning probe microscope (SPM), which was developed for material science applications. In the SPM, a physical probe scans samples at nanometer resolution. Properties of samples are mapped by a minimal interaction volume in the near field of the probe and sample. The AFM is a variant of the SPM.

THE AFM

The AFM is a fairly simple apparatus that has fractions of a nanometer resolution (Kirmizis and Logothetidis, 2010), but requires a relatively high level of technical expertise. This resolution is greater than 1,000 times the optical diffraction limit. The AFM comprises a rectangular or “V”-shaped cantilever. In biological applications, the cantilever is made from silicon nitride, it is 100–200 mm in length, and has a thickness of less than 1 μm. The end of the cantilever has a sharp tip (probe), which is often composed of a fractured diamond fragment. The AFM functions by a laser-tracking deflection of this probe, while the tip of the probe scans the surface of a biological sample and the topographic structure of the surface is mapped. AFM is dependent on an accurate scanner, which can be attached to either the sample or the probe. This enables scanning of the sample, with the stylus in the plane of the sample (x plane and y plane). The probe's and sample's height (the z direction) are adjusted with precision at the subatomic level (Amrein et al., 1997). The scanner in an AFM is composed of voltage-driven piezoceramic elements. Deflections of the cantilever are measured to record small repulsive tracking forces (10−6 to 10−9 N) between the sample and the tip. The deflection of the cantilever is used to accurately measure the topology of the sample. This also enables good manipulation of the loading force of the stylus onto the sample, which is important for biological applications of AFM. A photograph of an AFM is shown in Fig. 1.

Figure 1.

Photograph showing an AFM (Digital Instruments Multimode SPM) at the University of Auckland, which has the capability to conduct AFM in contact and noncontact modes.

When there is physical contact between the tip and the sample, the sample deforms until there is a sufficient increase in the contact area so that there is accommodation of the load. This deformation determines the reliability and resolution of AFM images.

IMAGING MODES

The AFM can be operated in various modes that depend on different combinations of force detection and the feedback mechanism (Meyer, 1992; Nikiforov and Bonnell, 2008). The terminology can be confusing because there is no standard nomenclature among microscope suppliers. These different modes of AFM imaging depend on the interaction between the cantilever deflection detector and the servo-system that adjusts the height of the cantilever. The types of imaging modes are as follows:

  • 1The most commonly used imaging mode is the contact mode. In the situation where the AFM is run in a mode for sensing repulsive features between the tip and the sample, the tip contacts the sample. The tip traces over individual atoms. However, forces generated on the cell surface by the probe can cause damage to the sample. In the contact mode, ionic repulsion forces enable topographical information of the surface to be determined at high resolution. This is the best mode for imaging molecular structure.
  • 2Another mode of operation detects the attractive forces between the sample and the tip. A feedback system stops the tip from coming into contact with and damaging the sample (noncontact AFM). Experiments using this type of mode are usually performed in liquid or a high vacuum to prevent water condensation between the tip and the sample surface. However, this mode of operation results in reduced lateral resolution (Hansma et al., 1988). In this noncontact mode, forces—including electrostatic, van der Waals, magnetic, or capillary forces—are detected and provide information regarding magnetic domain wall structure, surface topography, distributions of charges, or distribution of liquid film. Although noncontact imaging is the most delicate mode for objects that are extremely deformable or not well immobilized, it is not widely used in biological studies because high-resolution stable mapping of an interface of a biological sample cannot usually be achieved.
  • 3Damage to a sample can be minimized by a technique called intermittent contact AFM (dynamic force microscopy), which has the advantages of contact and noncontact AFM. In this mode, the oscillating cantilever tip approaches closely to the sample and hardly touches or “taps” the sample. Intermittent contact AFM provides topographic and internal structural information, and in molecular samples, it has a resolution of ∼1 nm (Figs. 2 and 3).
  • 4Phase imaging is advantageous because it can be simultaneously performed with topographic imaging using the intermittent mode. Therefore, phase and topographic images are achieved with a single scan (Alonso and Goldmann, 2003). The phase of the sinusoidal oscillation of the cantilever is recorded in relation to the driving signal that is applied to the cantilever to cause the oscillation. Recording this phase shift during the intermittent mode results in phase images. Phase imaging is used to study components of polymers related to their stiffness or regions of varying hydrophobicity in hydrogels that are submerged in saline solution.
  • 5The AFM is also used to investigate surface adhesion or elastic properties by generating force curves (see below for applications). Besides the loading force produced by the cantilever probe, other forces act between the sample and the stylus. These forces may be attractive or repulsive. A force that is attractive increases the effective load of the tip onto the sample more than expected from only bending of the cantilever. A force that is repulsive acting before contact decreases the load of the tip onto the sample. The spring constant of the cantilever and deflection of the cantilever affect the total loading force of the tip. Evaluation of these interactions is achieved by obtaining force versus distance curves (also called “force spectroscopy”).
Figure 2.

Tapping mode image in air showing Pseudomonas chlororaphis O6 bacterial lawn. A BS-TAP-300 cantilever was used with a Digital Instruments Bioscope IIIa AFM. Scale bar = 1 μm. The image was kindly provided by Dr. David Britt, Department of Biological Engineering, Utah State University.

Figure 3.

Tapping mode image in air showing bacteria flagella and/or pilli, as well as possible exopolysaccharide nodules on the surface (2 μm × 2 μm). A BS-TAP-300 cantilever was used with a Digital Instruments Bioscope IIIa AFM. The image was kindly provided by Dr. David Britt, Department of Biological Engineering, Utah State University.

USE OF THE AFM WITH OTHER TYPES OF MICROSCOPES

AFM can be combined with other types of microscopy. AFM and confocal laser scanning microscopy can be used together to image biological systems (Doak et al., 2008). The combination of AFM with light microscopy, such as differential interference contrast microscopy and confocal laser scanning microscopy, have enabled determination of the organization of adhesion molecules on the cell surface and their correlation with structures involved in binding (Poole et al., 2004). The entire AFM can be put on the stage of an inverted light microscope for simultaneously viewing cells, including fluorescence microscopy, where the probe of the AFM is shifted relative to an immobile sample. AFM can be integrated with fast-spinning disc confocal and total internal reflection fluorescence microscopy to determine cellular behavior at the subcellular level, thereby providing high temporal and spatial resolution (Trache and Lim, 2009). The following combinations of AFM with other types of related microscopes are also used: AFM/magnetic force microscope, AFM/friction force microscope, and AFM/STM (Meyer, 1992).

PREPARATION OF SAMPLES

Minimum preparation of samples is required for experiments using AFM. Mica, glass, and gold surfaces are used for sample immobilization, which allows biomolecules to be strongly bound (Engel et al., 1999). Samples coated with gold increase reflectivity, which is useful for experiments on cell mechanics where the laser intensity might be reduced by phenol red, which is a component of culture medium. These methods are used for filamentous structures or preparing small particles. However, simple adsorption to hydrophilic substrates is sufficient for immobilizing planar samples, including two-dimensional protein crystals and membranes. The success of this method is dependent on adjusting the ionic strength of the adsorption buffer in relation to the sample's surface charge and substrate using monovalent or divalent ions. AFM provides the ability to visualize materials in nonvacuous environments (air or liquid) without a requirement for staining, coating, or freezing of samples, while still producing images comparable with scanning electron microscopy and tunneling electron microscopy (Francis et al., 2010).

ACQUISITION OF IMAGES

Samples can be dehydrated and scanned in air (Engel et al., 1999). The tapping mode is best when imaging in air (Figs. 2 and 3). When imaging is carried out in air, a water bridge often occurs between the stylus and the sample. The force of this meniscus may be great (e.g., from 10 to 7 N) and can result in the tip being pulled onto the sample. As a result, the resolution may be poor and the sample and stylus can be damaged. The choice of imaging mode (e.g., use of the intermittent mode) may offset the problems of imaging in air. Ideally, aqueous buffer solutions should be used for imaging of samples because this enables optimal preservation of biological structure and precise control of interactions between the tip and sample; however, this method is more difficult than scanning in air. Most cellular or macromolecular samples require maintenance in aqueous surroundings to investigate their native structure and study their function.

APPLICATIONS OF THE AFM IN BIOLOGY

AFM has wide a range of applications, from imaging crystal surfaces at atomic resolution to manipulating or imaging entire cells. Most of the knowledge in biology that has been obtained by AFM has come from investigating macromolecular complex structures or single macromolecules (Amrein, 2007). For studying the structure of macromolecules, AFM is comparable with transmission electron microscopy, although the latter is better than AFM for determining the three-dimensional structure of macromolecules because AFM only reveals topography. The high signal-to-noise ratio at the molecular level in AFM can be useful in the situation where a molecular complex is not greatly structurally defined and individual units substantially vary from each other. AFM enables a flexible analytical approach, which provides considerable membrane (fixed cell) and intracellular (live cell) detail, thereby presenting advantages over other techniques, such as scanning electron microscopy. Biological applications of AFM are discussed below.

Surface Structure

Cells membranes form biological interfaces where complex interactions occur. Therefore, it is necessary to understand the cell surface and the effect that exogenous substances have on their surrounding microinterface. AFM is effective for imaging the surface structure of membrane proteins incorporated in a lipid bilayer, outer membranes of Gram-negative bacteria, and other small molecules (Fotiadis et al., 2002). Rigid fibrils (e.g., collagen) are easily imaged by the microscope. AFM has many potential clinical applications, for example, AFM can be used in the characterization of modified and unmodified surfaces for application in implantable structures and regenerative medicine (Schneider et al., 2007).

Live-Cell Imaging

AFM can be used to image living cells (Dvorak, 2003). Each type of imaging mode of the AFM obtains unique information on live cells. AFM enables a flexible analytical approach that can provide intracellular detail in live cells, which has distinct advantages over other microscopic techniques such as scanning electron microscopy (Francis et al., 2010). AFM maps the elastic properties of live cells, which has provided insight into many physiological cell processes. Currently, AFM imaging of live mammalian cells is limited to resolutions ranging from 50 to 100 nm (Müller and Dufrêne, 2011). AFM is useful for measuring the constrained diffusion of elements in plasma membranes in living cells, providing information that has greatly contributed to an understanding of the nature of lipid rafts. Adhered cells are imaged more easily with AFM than suspended cells, which need to be immobilized for imaging. Cells are most effectively imaged with AFM combined with light microscopy. As successful imaging of living cells is affected by many variables, there are still obstacles that must be overcome before it can be widely applied in biology.

Roughness of the Cell Surface

The roughness of a cell surface can be calculated, thus providing information on the effect of external stimulation and developmental stage on membrane topography (Girasole et al., 2007). Surface roughness data could be useful for novel cell surface biomarkers, which, when combined with other conventional biomarkers (e.g., histology and immunohistochemistry), could be applied to making clinical decisions.

Molecular Biology

AFM can be applied to experiments at the molecular level. Molecular images can be achieved in which single macromolecules are recognized and then the individual molecules are focused with the stylus of the AFM. Approximately 30% of the human genes code for membrane proteins, which are difficult to crystallize (Francis et al., 2010). By phase imaging, AFM is able to image such assembled molecular structures, because resolution can reach 50–150 nm, thus enabling subunit structure to be characterized. Currently, AFM is used to investigate RNA, DNA, chromosomes, nucleic acid–protein complexes, and ligand–receptor binding.

Dynamics of Force Interactions

AFM can use a combination of imaging modes that are sensitive to various sample properties, and it measures forces and interactions (Amrein, 2007). A unique advantage of AFM is its ability to locally manipulate and probe samples, as well as imaging. Force mapping involves an imaging and force probing, which creates indentations in an array spanning an area of interest on the sample and creating an isoforce image from the z-position, where the probe achieves a preset constant deflection (Radmacher et al., 1996). However, images may be complicated by variable cell topography (e.g., living cells). Therefore, better accuracy is achieved by analyzing the indentation data and producing an image that is directly representative of the elastic properties acquired at each pixel location. This method is termed AFM elastography (Kirmizis and Logothetidis, 2010). AFM has been used in recent years for measuring various mechanical properties of different cell types, including fibroblasts and endothelial cells, in different areas within the same cell and in diverse conditions. Examples of applications include investigating the forces in a collagen matrix and unfolding DNA and proteins and separating molecular binding partners. This has provided information on DNA and protein folding, as well as conformation on the character of binding pockets.

Acknowledgements

The author thanks Dr. David Britt from the Utah State University for generously providing the images and Dr. Bryony James from the University of Auckland for assistance for photographing the atomic force microscope. Ellen C. Jensen* The Anatomical Record

Ancillary