Using The Atomic Force Microscope Biology Essay


Atomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit. The precursor to the AFM, the scanning tunneling microscope, was developed by Gerd Binnig and Heinrich Rohrer in the early 1980s at IBM Research - Zurich, a development that earned them the Nobel Prize for Physics in 1986. Binnig, Quate and Gerber invented the first atomic force microscope (also abbreviated as AFM) in 1986. The first commercially available atomic force microscope was introduced in 1989. The AFM is one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale. The information is gathered by "feeling" the surface with a mechanical probe. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable the very precise scanning. In some variations, electric potentials can also be scanned using conducting cantilevers. In newer more advanced versions, currents can even be passed through the tip to probe the electrical conductivity or transport of the underlying surface, but this is much more challenging with very few groups reporting reliable data

Basic principles of ATM

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The AFM consists of a cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically silicon or silicon nitride with a tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever according to Hooke's law. Depending on the situation, forces that are measured in AFM include mechanical contact force, van der Waals forces, capillary forces, chemical bonding, electrostatic forces, magnetic forces (see magnetic force microscope, MFM), Casimir forces, solvation forces, etc. Along with force, additional quantities may simultaneously be measured through the use of specialized types of probe (see scanning thermal microscopy, scanning joule expansion microscopy, photothermal microspectroscopy, etc.). Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes. Other methods that are used include optical interferometry, capacitive sensing or piezoresistive AFM cantilevers. These cantilevers are fabricated with piezoresistive elements that act as a strain gauge. Using a Wheatstone bridge, strain in the AFM cantilever due to deflection can be measured, but this method is not as sensitive as laser deflection or interferometry.

If the tip was scanned at a constant height, a risk would exist that the tip collides with the surface, causing damage. Hence, in most cases a feedback mechanism is employed to adjust the tip-to-sample distance to maintain a constant force between the tip and the sample. Traditionally, the sample is mounted on a piezoelectric tube, that can move the sample in the z direction for maintaining a constant force, and the x and y directions for scanning the sample. Alternatively a 'tripod' configuration of three piezo crystals may be employed, with each responsible for scanning in the x,y and z directions. This eliminates some of the distortion effects seen with a tube scanner. In newer designs, the tip is mounted on a vertical piezo scanner while the sample is being scanned in X and Y using another piezo block. The resulting map of the area s = f(x,y) represents the topography of the sample.

The AFM can be operated in a number of modes, depending on the application. In general, possible imaging modes are divided into static (also called contact) modes and a variety of dynamic (or non-contact) modes where the cantilever is vibrated

File:Atomic force microscope block diagram.svg

Imaging modes

The primary modes of operation for an AFM are static mode and dynamic mode. In static mode, the cantilever is "dragged" across the surface of the sample and the contours of the surface are measured directly using the deflection of the cantilever. In the dynamic mode, the cantilever is externally oscillated at or close to its fundamental resonance frequency or a harmonic. The oscillation amplitude, phase and resonance frequency are modified by tip-sample interaction forces. These changes in oscillation with respect to the external reference oscillation provide information about the sample's characteristics

Contact mode

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In the static mode operation, the static tip deflection is used as a feedback signal. Because the measurement of a static signal is prone to noise and drift, low stiffness cantilevers are used to boost the deflection signal. However, close to the surface of the sample, attractive forces can be quite strong, causing the tip to "snap-in" to the surface. Thus static mode AFM is almost always done in contact where the overall force is repulsive. Consequently, this technique is typically called "contact mode". In contact mode, the force between the tip and the surface is kept constant during scanning by maintaining a constant deflection.

Non-contact mode

In this mode, the tip of the cantilever does not contact the sample surface. The cantilever is instead oscillated at a frequency slightly above its resonance frequency where the amplitude of oscillation is typically a few nanometers (<10 nm). The van der Waals force, which are strongest from 1 nm to 10 nm above the surface, or any other long range force which extends above the surface acts to decrease the resonance frequency of the cantilever. This decrease in resonance frequency combined with the feedback loop system maintains a constant oscillation amplitude or frequency by adjusting the average tip-to-sample distance. Measuring the tip-to-sample distance at each (x,y) data point allows the scanning software to construct a topographic image of the sample surface.

Non-contact mode AFM does not suffer from tip or sample degradation effects that are sometimes observed after taking numerous scans with contact AFM. This makes non-contact AFM preferable to contact AFM for measuring soft samples. In the case of rigid samples, contact and non-contact images may look the same. However, if a few monolayers of adsorbed fluid are lying on the surface of a rigid sample, the images may look quite different. An AFM operating in contact mode will penetrate the liquid layer to image the underlying surface, whereas in non-contact mode an AFM will oscillate above the adsorbed fluid layer t image both the liquid and surface.

Schemes for dynamic mode operation include frequency modulation and the more common amplitude modulation. In frequency modulation, changes in the oscillation frequency provide information about tip-sample interactions. Frequency can be measured with very high sensitivity and thus the frequency modulation mode allows for the use of very stiff cantilevers. Stiff cantilevers provide stability very close to the surface and, as a result, this technique was the first AFM technique to provide true atomic resolution in ultra-high vacuum conditions.

In amplitude modulation, changes in the oscillation amplitude or phase provide the feedback signal for imaging. In amplitude modulation, changes in the phase of oscillation can be used to discriminate between different types of materials on the surface. Amplitude modulation can be operated either in the non-contact or in the intermittent contact regime. In dynamic contact mode, the cantilever is oscillated such that the separation distance between the cantilever tip and the sample surface is modulated.

AFM cantilever deflection measurement

Laser light from a solid state diode is reflected off the back of the cantilever and collected by a position sensitive detector (PSD) consisting of two closely spaced photodiodes whose output signal is collected by a differential amplifier. Angular displacement of cantilever results in one photodiode collecting more light than the other photodiode, producing an output signal (the difference between the photodiode signals normalized by their sum) which is proportional to the deflection of the cantilever. It detects cantilever deflections <10 nm (thermal noise limited). A long beam path (several centimeters) amplifies changes in beam angle.

AFM beam deflection detection

(Atomic Force Microscope) A device used to image materials at the atomic level. AFMs are used to solve processing and materials problems in electronics, telecom, biology and other high-tech industries. Invented by IBM in 1986, it uses a ceramic or semiconductor tip one atom wide positioned at the end of a cantilevered bar. As the tip is moved over the material, it either continously touches or periodically taps the surface and bends as it is repelled or attracted to the structure. A laser picks up the deflections

Force spectroscopy

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Another major application of AFM (besides imaging) is force spectroscopy, the direct measurement of tip-sample interaction forces as a function of the gap between the tip and sample (the result of this measurement is called a force-distance curve). For this method, the AFM tip is extended towards and retracted from the surface as the deflection of the cantilever is monitored as a function of piezoelectric displacement. These measurements have been used to measure nanoscale contacts, atomic bonding, Van der Waals forces, and Casimir forces, dissolution forces in liquids and single molecule stretching and rupture forces. Furthermore, AFM was used to measure in aqueous environment dispersion force due to polymer adsorbed on the substrate. Forces of the order of a few piconewton can now be routinely measured with a vertical distance resolution of better than 0.1 nanometers. Force spectroscopy can be performed with either static or dynamic modes. In dynamic modes, information about the cantilever vibration is monitored in addition to the static deflection.

Problems with the technique include no direct measurement of the tip-sample separation and the common need for low stiffness cantilevers which tend to 'snap' to the surface. The snap-in can be reduced by measuring in liquids or by using stiffer cantilevers, but in the latter case a more sensitive deflection sensor is needed. By applying a small dither to the tip, the stiffness (force gradient) of the bond can be measured as well.

Identification of individual surface atoms

The AFM can be used to image and manipulate atoms and structures on a variety of surfaces. The atom at the apex of the tip "senses" individual atoms on the underlying surface when it forms incipient chemical bonds with each atom. Because these chemical interactions subtly alter the tip's vibration frequency, they can be detected and mapped. This principle was used to distinguish between atoms of silicon, tin and lead on an alloy surface, by comparing these 'atomic fingerprints' to values obtained from large-scale density functional theory (DFT) simulations.

The trick is to first measure these forces precisely for each type of atom expected in the sample, and then to compare with forces given by DFT simulations. The team found that the tip interacted most strongly with silicon atoms, and interacted 23% and 41% less strongly with tin and lead atoms, respectively. Thus, each different type of atom can be identified in the matrix as the tip is moved across the surface.

Such a technique has been used now in biology and extended recently to cell biology. Forces corresponding to (i) the unbinding of receptor ligand couples (ii) unfolding of proteins (iii) cell adhesion at single cell scale have been gathered.

Piezoelectric scanners

AFM scanners are made from piezoelectric material, which expands and contracts proportionally to an applied voltage. Whether they elongate or contract depends upon the polarity of the voltage applied. The scanner is constructed by combining independently operated piezo electrodes for X, Y, and Z into a single tube, forming a scanner which can manipulate samples and probes with extreme precision in 3 dimensions.

Scanners are characterized by their sensitivity which is the ratio of piezo movement to piezo voltage, i.e., by how much the piezo material extends or contracts per applied volt. Because of differences in material or size, the sensitivity varies from scanner to scanner. Sensitivity varies non-linearly with respect to scan size. Piezo scanners exhibit more sensitivity at the end than at the beginning of a scan. This causes the forward and reverse scans to behave differently and display hysteresis between the two scan directions. This can be corrected by applying a non-linear voltage to the piezo electrodes to cause linear scanner movement and calibrating the scanner accordingly

The sensitivity of piezoelectric materials decreases exponentially with time. This causes most of the change in sensitivity to occur in the initial stages of the scanner's life. Piezoelectric scanners are run for approximately 48 hours before they are shipped from the factory so that they are past the point where they may have large changes in sensitivity. As the scanner ages, the sensitivity will change less with time and the scanner would seldom require recalibration.


AFM has several advantages over the scanning electron microscope (SEM). Unlike the electron microscope which provides a two-dimensional projection or a two-dimensional image of a sample, the AFM provides a three-dimensional surface profile. Additionally, samples viewed by AFM do not require any special treatments (such as metal/carbon coatings) that would irreversibly change or damage the sample. While an electron microscope needs an expensive vacuum environment for proper operation, most AFM modes can work perfectly well in ambient air or even a liquid environment. This makes it possible to study biological macromolecules and even living organisms. In principle, AFM can provide higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum (UHV) and, more recently, in liquid environments. High resolution AFM is comparable in resolution to scanning tunneling microscopy and transmission electron microscopy.


A disadvantage of AFM compared with the scanning electron microscope (SEM) is the single scan image size. In one pass, the SEM can image an area on the order of square millimeters with a depth of field on the order of millimeters. Whereas the AFM can only image a maximum height on the order of 10-20 micrometers and a maximum scanning area of about 150Ã-150 micrometers. One method of improving the scanned area size for AFM is by using parallel probes in a fashion similar to that of millipede data storage.

The scanning speed of an AFM is also a limitation. Traditionally, an AFM cannot scan images as fast as a SEM, requiring several minutes for a typical scan, while a SEM is capable of scanning at near real-time, although at relatively low quality. The relatively slow rate of scanning during AFM imaging often leads to thermal drift in the image making the AFM microscope less suited for measuring accurate distances between topographical features on the image. However, several fast-acting designs were suggested to increase microscope scanning productivity including what is being termed videoAFM (reasonable quality images are being obtained with video AFM at video rate: faster than the average SEM). To eliminate image distortions induced by thermal drift, several methods have been introduced.

AFM images can also be affected by hysteresis of the piezoelectric material and cross-talk between the x, y, z axes that may require software enhancement and filtering. Such filtering could "flatten" out real topographical features. However, newer AFMs utilize closed-loop scanners which practically eliminate these problems. Some AFMs also use separated orthogonal scanners (as opposed to a single tube) which also serve to eliminate part of the cross-talk problems.

As with any other imaging technique, there is the possibility of image artifacts, which could be induced by an unsuitable tip, a poor operating environment, or even by the sample itself. These image artifacts are unavoidable however, their occurrence and effect on results can be reduced through various methods.

Due to the nature of AFM probes, they cannot normally measure steep walls or overhangs. Specially made cantilevers and AFMs can be used to modulate the probe sideways as well as up and down (as with dynamic contact and non-contact modes) to measure sidewalls, at the cost of more expensive cantilevers, lower lateral resolution and additional artifacts