Atomic force microscopy (AFM) is a powerful imaging tool and quantitative analysis instrument that studies the surface of a sample using a mechanical probe and provides real time three-dimensional topography mapping with resolution to the order of a sub-nanometer (˜10-9) [7] Unlike its predecessor the scanning tunneling microscope (STM) which only has the ability to generate a topographical map, AFM also has the capability to localize interactions of biological systems spanning scales between cells to molecules. As mentioned, the STM is credible for the advent of AFM; as for speaking of STM’s legacy however the development of AMF is barely striking the surface (pun intended). Development of STM brought about an entire branch of microscopy: scanning probe microscopy (SPM). Gerd Binnig and Heinrich Rohrer of IBM’s Zürich Research Laboratory developed the scanning tunneling microscope in 1981. [5] Analytic information was extracted through the phenomena of electron (or quantum) tunneling. A conducting tip is scanned across a surface (see figure 1), when a voltage difference is applied to both systems, electrons are permitted to tunnel between them. The development of the STM earned its creators the Nobel Prize in Physics in 1986 (the same year Binnig’s review letter for AFM was published). [8] There are several factors that drove STM to obsolescence in comparison to AFM. The mechanical interaction between STM’s conducting tip and the sample nearly ensures damage to the sample. Furthermore, the scope of samples available for analyzation in STM are limited to their conductive properties and STM cannot make determinations in the presence of air or liquid. Tunneling can become unstable when contaminants collect at the tip. Conversely with AMF, force curves (see figure 3) can be constructed for any type of surface and in every environment. [3]
Strengths of AFM include:
AFM is the only tool with capabilities of measuring interactions between surfaces among the order of a nanometer in size. This technology grants the researcher means to compare local forces to the discrete properties of the sample analyte. [3] AFM is an instrument with the capability to reveal functional conformation changes, this is significantly important to biological studies of proteins. [7] Traditional methods of analysis for biochemical models often require extensive preparation of the sample such as purification, state transition for a detectable form of the substance, or treatment for labeling such as the introduction of an enzyme immunoassay used in the ELIZA test. Excessive pretreatment of a sample inevitably results in damage to the natural structure of the molecules. AFM brings with it a process of investigating the behaviors of molecules in their native state. The ability of AFM to perform analysis on samples in both conditions suggests a level of versatility that demonstrates itself a powerful tool in research and analysis. [8]
Weaknesses include:
The topographical mapping of a surface from AFM reflects interaction of the probe to surface as opposed to true sample topography due to tip convolution. [9] In order to withstand the exerted force of the probe the sample must be well attached to an appropriate solid substrate, thus sample preparation in some cases is necessary. [7] With the technology at present AFM takes minutes to record an image, comparatively to the rate at which biological processes occur this is a significantly large differential. However, it should be noted that the development of high-speed AFM in the past decade has improved rendering image time 1000-fold. Because of these developments real-time conformational changes of single biological molecules can be observed through AFM. [12] Interpreting data may sometimes be difficult as many forces are collectively contributed to the curves. [6]
Key considerations
Resolution to the order of a nanometer (~10-9 m)
Equations
Eq. 1 Hooke’s law can be used to describe the relationship that force has to the spring constant (cantilever) and the deflection of the laser off the back of the cantilever:
F=kx
In this equation “k” is the spring constant, “x” is the laser deflection and F is the interaction force. [8]
Eq.2 Resonant frequency, f_0, of the spring system:
f_0=(1/2π) (k/m_0 )^(1/2)
In this equation “k” is the spring constant and m_0 is the effective mass that loads the spring. [8]
Eq. 3 The Lennard-Jones model consists of a repulsive term and attractive term, it approximates the interaction forces acting between the cantilever tip and sample surface
V(r)=A/r^12 -B/r^6
In this equation “V” is the intermolecular potential, “A” and “B” represent the cantilever tip and the sample surface, “r” is the distance of separation between “A” and “B”. (see figure 3)
Diagrams
Figure 1: The sharp tip of the cantilever follows the contour of the surface (B), close range attractive forces acting between the tip and the surface cause the cantilever to bend towards the surface when brought closer, increasing repulsive forces cause the cantilever to bend away.
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Figure 1, taken from [4]
Figure 2: An optical laser is focused upon the back side of the cantilever; the reflected beam strikes a quartered photodiode used for detection. As the cantilever bends the position of the laser striking the photodiode moves to another location on the photodiode. A feedback loop adjusts the Z position of the cantilever to return the laser to its origin. The adjustments of the cantilevers transposition are used to generate the topographic map of the surface.
2, adapted from [1]
Figure 3: A “force curve” or “Lenard Jones potential curve” is generated showing force as a function of position on the Z axis. The dashed curve in the top quadrant shows the repulsive force acted upon the cantilever while the dashed curve in the bottom quadrant shows the attractive force. The solid black curve expresses the total interaction between the two. (see equation 3)
Figure 3, adapted from [2]
Physical/chemical principles
The tip-sample interactive forces calculated through Hooke’s law is a summation of multiple forces dependent on the molecular composition of the sample and the method parameters of analyzation. Possible interacting forces being measured by AFM include: mechanical contact force, meniscus forces, capillary force, intermolecular forces, Casimir forces, solvation forces, electrostatic forces, etc. [6] Because the amount possible contributing forces is so extensive it is important to consider all possibilities relevant to the sample being analyzed. The utmost prevalent contributing forces associated in AFM are van der Waals attractive force and electronic repulsive force. [8]
Method discussion
Principles
AMF operates by raster scanning the oscillating microfabricated tip of a cantilever (suspended by a spring for flexibility) across an analyte’s surface. (see figure 1) The electrostatic and piezoelectric interactions influence the cantilever position. [11] An optical laser is deflected off the top of the cantilever to a position-sensitive photodiode detector, changes in position of the cantilever are monitored by the displacement of the deflected beam upon the photodetector. The resultant values of cantilever position as a function of force are displayed by the generated force curves (see figure 3). Depending on the method of analysis a feedback loop is used to maintain a parameter of constant oscillation, distance, or force.
Analysis with AFM is commonly performed on one of three methods:
Contact Mode – An area of the surface is raster scanned with the tip of the cantilever in-contact with the surface of the sample. variations of deflection off the cantilever are collected by the photodiode. [13]
Non-Contact Mode – The cantilever is oscillated by a piezoelectric transducer as the sample is approached and withdrawn from the axis perpendicular from the surface. In this method resonance frequency of oscillations as a function of the distance between the tip and sample surface are used to determine force curves and a topographical map. [12]
Tapping mode – The cantilever tip intermittently making contact with the sample. Tapping mode is the most recent and efficient method by mitigating risks of deformation from contact mode. The amplitude of the oscillating cantilever is observed and kept constant by adjustment of the cantilever’s position in the vertical axis through a piezoelectric driver in a feedback loop. [4] Displacement of the cantilever to counteract amplitude dampening from interactions between the tip and surface are used to generate force curves and a topographical map. [10]
Method limitations:
Scanning in contact mode brings forth the possibility of damaging the sample due to scratch deformation. [8] Scratching the sample can not only repudiate data in the location of the scratch but the cantilever tip is at risk of damage which would devalue all subsequent collected data. Tapping mode is usually considered as a way of circumventing this limitation
Outlook
Although AFM exhibits much stronger potential due to its versatility compared to its predecessor STM, the arrival of STM brought about a branch of microscopy (scanning probe microscopy) from its derivatives each in its own respect provides distinct analytical insight on the molecular composition of the sample. The original STM model is highly modular that being by changing a few parameters such as the type of detector or how information is extrapolated from it. For example, scanning tunneling spectroscopy (STS) provides information on electron density in a sample by maintaining a fixed height between the tip and the sample, by varying the voltage the electron tunneling current is measured. Another modulation of the traditional AFM mechanism that has established further thresholds for analysis is SMFS. By attaching a ligand or antibody on the tip of the cantilever recognition of individual receptors on the cell surface will be made possible by the resultant force curves, this technique is known as single-molecule force spectroscopy (SMFS). SMFS ability to quantify the unbinding process of individual receptor-ligand pairs brings forth information useful in the mapping of possible conformations of single molecules. [8]
It is unclear where further innovations and variations on AFM or any of the branches of SPM will take our understanding of the sub-atomic world but it is clear that the STM brought about a highly customizable system. Furthermore, as technology and research progresses in this field the limitations of information this family of instruments can provide will grow exponentially small. It only took a decade to make AFM 1000-fold more efficient with respect to time, one can only wonder what enhancements and innovations will be made in the next decade.