Scanning Tunnelling Method And Atomic Force Microscopy Biology Essay
Abstract: One of the most intensively researched areas today is to make material smaller and smaller. The new catch phrase for the world and the material research community is “small is the new big”. Technology has shrunk from micro to nano to pico sizes. This emphasis has spurred research on equipment that can help in the production and examination of such material. The use of nanotechnology is a promising approach for developing better material. This paper studies the two popular methods of examination of nanomaterial, namely atomic force microscopy (AFM) and scanning tunneling microscopy (STM). An overview of the two techniques and the difference between the two has been presented. The current applications of these technologies have been discussed. However, with further reduction in sizes and growing applications, these two methods are increasingly used complementarily with other methods. From a careful analysis of these technologies, it is clear that a number of challenging issues for examining material size in the range of and current higher end technologies will be addressed in this paper.
“Scanning probe microscopy (SPM) comprises a family of techniques that measure surface topography and properties on the atomic scale.”  The image is obtained when the probe moves mechanically in a rectangular pattern of image capture and reconstruction, i.e., does a raster scan, of the specimen. The probe-surface interaction is recorded as a function of position. The field started with the invention of scanning tunnelling microscope in 1981 and has been followed by numerous inventions ever since.
Scanning probe microscopic techniques include Electrostatic Force Microscopy (EFM), Magnetic Force Microscopy (MFM), Near-Field Scanning Optical Microscopy (NSOM), Scanning Thermal Microscopy (SThM), apart from the most prominent ones, Scanning Tunnelling Microscope (STM) and Atomic Force Microscope (AFM). Most of these methods are capable of achieving atomic level resolution. The methods also differ in the type of interaction they involve including local investigations of forces, evanescent optical waves, acoustic waves, temperature gradients, local potentials, capacitance variation among others.  The techniques also vary according to the type of interaction between the sample and probe such as contact mode, non-contact mode, tapping mode etc. The method chosen for a particular sample depends on a number of reasons such as surface of sample, whether the sample is conducting or not, roughness of the surface, information we require from the sample and so on. It would be impossible to review all of the SPM techniques, so we present a review of the first one which started the trend, i.e, STM and the one that revolutionised research, AFM in the following sections.
SCANNING TUNNELING MICROSCOPY
Scanning tunneling microscopy (STM) opened a new research area of “near field microscopy”, which was based on local investigation of forces.
OPERATING PRINCIPLES OF STM
The operating principles of STM rely on a quantum effect, the tunnel effect.  When two metals are sufficiently close to each other (, overlapping of electron clouds occurs at each electrode. When a bias voltage is applied across Metal-Insulator-Metal (MIM) junction, a current of electrons forms near the Fermi level and the direction is given by sign of the applied voltage. The tunneling current exhibits an exponentially dependence to the interatomic distance d between the two metals. For a simple square barrier geometry, the current is given by
Where, V is the voltage applied, d is the interelectrode distance, is the work function of the metal. B = 4π = 10.25 eV-1/2nm-1
When the values are substituted in the equation (1), it can be shown that is divided by 10, then the distance increases by 0.1 nm. Replacement of planar electrode with a sharp needle acting as a local current probe will therefore be effective for detecting lower atomic resolution. If the tip is ended with one atom, then the different between the top of a surface atom (relative high electron density) and valley between two surface atoms (low electron density) can be detected. Irrespective of the ambient medium (air, liquid or vacuum), high resolutions can be obtained. Piezo-electric ceramics were the first tips to be used in STM. The setup was designed to maintain a constant tunneling current by displacement of tip in the Z direction (perpendicular to the sample) using a feedback loop while scanning the needle in the X and Y direction (parallel to sample). One very important feature is that 5% accuracy in tunneling current suffices to reach 0.01 nm resolution in Z direction.
OPERATING PRINICIPLE OF ATOMIC FORCE MICROSCOPE
G Binning et al proposed Atomic force microscope (AFM) as a method capable of measuring forces as small as 10-18 N and for investigation of surface of insulating materials at the atomic scale. Drawing from the principles of scanning tunneling microscope and stylus profilometer, the probe uses a nondestructive testing method. While Binning et al, reported a lateral resolution of 30 and vertical resolution of 1 Å in air, it has now further reduced due to different developments .
Force monitoring done with monitoring of elastic deformation of springs. The used to measure the motion of a cantilever beam of ultrasmall mass. The displacement range was 10-4 Å and the force required for this would be 10-18N. Leveraging on the small mass involved, the sensitivity enables the interatomic forces between atoms. AFM can be used to study conducting and insulating materials on atomic scale. It can be used to measure not only interatomic forces but electromagnetic forces as well. In a STM, atomic surface is well resolved but equivalent for bulk insulators is missing. Stylus profilometer is powerful microscopic technique – three dimensional images with 1000 Å lateral resolution and 10 Å vertical resolution. Scanning capacitance microscope with 5000 Å lateral resolution and 2 Å vertical resolution. SP and STM are similar in the sense that both scan the surface and sense variations of the sample and generate 3D images. The SP uses a cantilever beam and radius is about 1 micron so can record loads from 10-2 to 10-5 N .
The spring in the AFM is soft and stiff with high resonant frequency in order to minimize the sensitivity to vibrational noise from the building near 100 Hz. The resonant frequency of the system is f0 = (1/2) (k/m0)1/2. Based on the equation, to decrease k, a corresponding decrease in m0 is necessary so that the ratio k/m0 is large. Special springs with mass around 10-100 kg and capable of vibrating at resonant frequency is used in AFM. Displacement of 10-4 Å can be measured with STM when the tunnelling gap is modulated . The force required to produce the displacements is 2 x 10-16 N and with change of Q to 100, this force is reduced by two orders further. AFM images are produced by measuring the force on a sharp tip created by proximity of the surface to the sample. At the time of development, the force was kept constant and controlled using a feedback mechanism.
Fig 1: Schematic of STM and its operation. The tip of microscope as depicted in (a) is scanned over surface of sample S with a piezoelectric tripod (X,Y,Z). The rough positioner brings the sample within reach of the tripod. A vibration filter system P protects the instrument from external vibrations. In constant tunneling current mode of operation, a voltage Vz is applied to the Z piezoelectric element by means of the control unit CU depicted in (b) to keep the tunneling current constant while the tip is scanned across the surface by altering Vx and Vy. The trace of the tip, a y-scan, generally resembles the surface topography. Electronic inhomogeneities also produce structure in the tip trace, as illustrated on the right above two surface atoms having excess negative charge. 
IMPROVEMENTS OF STM
The number of patents found on Scanning Tunneling Microscope since its invention and first patent in 1982 is enormous. While Binning et al , sought to utilize the vacuum tunnel effect to invent an instrument which at ultra-high vacuum and cryogenic temperatures using a fine tip to scan across the surface of a conducting sample. In their device, the vertical separation between the tip and the sample surface is controlled so that the measured variable is constant and proportional to tunnel resistance, i.e. the tunneling current. The tip is controlled by piezo electric drive acting along three coordinate directions and the spatial coordinates are graphically displayed. The drive currents or voltages of the piezo electric drives are hence displayed.
One of the problems with STM is that it has a relatively large mass and relatively low stiffness and hence it has a low resonance frequency, since environmental vibrations also have low frequency, STM are sensitive to vibration and have high resonance frequency. Gimzweski et al , sought to solve this problem by reducing the size of the STM and making it very stiff and this even had a higher resolution. The invention was better also since it was capable of operating even at low voltages as against the earlier STMs which required high-gain voltage amplifiers. The production technique for this STM was based on silicon micromachining techniques.
The next obvious improvement in the STM technology was to produce tip which are replaceable. This was required because in an STM the distance between the tip and sample surface was to be maintained at about 1 nm and sometimes when tip was brought in contact to the sample surface, it resulted in breaking the tip or the sample surface and therefore resulting in unnecessary time delays. Also, sometimes since the sample is cleaned at high temperatures of around 1200oC, the heat or contaminants can damage the tip. Kobayashi et al , invented a tip holder which can be detachably mounted and a sample holder to the piezoelectric drive mechanism. A replacement rod for replacing the tip holder is mounted so as to be able to move across the sample surface during observation and can be moved away from the sample during replacement. One important characteristic is that this can be done in ultra high vacuum condition. Different embodiments of the same patent include a tip holder which is driven by piezoelectric drive mechanism and can be detached, a tip drive unit driving a tip mounted detachably, usage of a transfer rod to achieve the same among others.
Another improvement was proposed by Miyata et al , with a few movement element block having a probe and a fine movement element is disposed removably to a revolver of a microscope and a rough movement mechanism for moving a sample in the direction of the microscope on sample stage of the given microscope.
To keep the STM’s insulated from external vibrations, they were levitated using superconducting magnets and later usage of 2 stage spring systems. Eddy current damping with permanent magnets and vibration elimination with viton dampers is also done for STM.
Fabrication of ultrasharp and stable tips involves very complicated mechanical and chemical preparation techniques and this has received a lot of attention in recent times. Since the sharpness and stability determines the lateral resolution, grinding or etching is the standard method. “Insitu” sharpening of tips and blowing away of whiskers with high voltage application to the tip is also carried out. Since unintentional tip-sample contact can ruin the tip, the geometry of tip is evaluated by imaging of structure with a known surface. The STM image combines image of tip with image of actual surface , for eg, symmetric surfaces imaged with asymmetric tips appear asymmetric.
Many modifications of the STM have been done on the primitive STM invented by Binning et al and these modifications are still in progress.
The next section aims to draw comparisons between STM and AFM and elaborate on some of the challenges of the AFM before proceeding to explain how some of these challenges are being overcome in new AFM devices.
COMPARISON OF STM AND AFM
The significant differences between STM and AFM are summarized in the form of Table 1 . One similarity is that they were both invented by Binning et al who invented STM in 1981 followed by AFM in 1985. AFM can therefore be considered as an improvement to since it can be used to investigate insulating material.
SCANNING TUNNELING MICROSCOPE
ATOMIC FORCE MICROSCOPE
2 Dimensional image of atoms
3 Dimensional surface profile of Nano-objects
Better resolution because of the exponential dependence on current to distance and true atomic resolution; Uses wave phenomenon so possible to get images at level of atoms
A no. of factors affect resolution such as tip shape, force etc and therefore relationship is complex; Uses classical forces so at a coarser level
Components - Tip
Uses a sharp conducting tip
Uses a sharp cantilever tip generally silicon or silicon nitride with radius in the order of nanometers and the probe is used to scan the specimen surface
Components – Spring
No spring present
Tip is attached to spring with small spring constant
Components – Laser
No laser is involved
A laser beam is used to detect the bending of the cantilever
Tip is mounted on scanner
Sample is mounted on scanner
Tunneling current and distance between the tip and sample; the two are dependent on each other
The height between sample and tip, the voltage and frequency of oscillation of spring can be adjusted; the parameters can be controlled independently
Electrical current between tip and surface
Records movement due to electromagnetic force between atoms
Records the tunneling current
Records the small “van der Waals” force between the tip and the surface
Physical contact between tip and substrate
Close proximity but no actual contact
Physical contact present
Faster than AFM, real time low quality scans can be obtained
Requires several minutes
Used to visualize and manipulate atoms
Used to image non-conducting objects such as DNA, proteins etc.
Table 1: Comparison of STM and AFM
CHALLENGES FACED BY AFM WITH RESPECT TO STM
Compared to a STM where the tip has to be scanned across the surface with a precision of a few picometers with a feedback mechanism controlling the height so that the tunneling current is constant and hence a successful realization may seem very difficult, an AFM may seem easier to operate. In order to understand the differences between the two, comparison of the physical observables is to be done. Some of the factors are discussed below.
Non-monotonic imaging signal: While comparing the plot of tunneling current and tip sample force as a function of time, the tunneling current is a monotonic function of the tip-sample distance and increases sharply with decreasing distance as shown in the figure below. This is useful because it allows implementation of a feedback loop as the tunneling current can be fed into a logarithmic amplifier to produce an error signal which is linear with the tip-sample distance. On the other hand, the tip-sample force has long and short range components and is not monotonic. For long distance the force is attractive and for short distances the force between the tip-sample becomes repulsive. So stable feedback can only be got from the monotonic part of the curve . Since tunneling current is monotonic with time and force is not, it is easier to establish the height feedback for STM than AFM.
Fig 2: Plot of tunneling current It and force Fts (typical values) as a function of ditance z between center of front atom and plane defined by the centers of surface atom layer 
Stability: Chemical forces exist along with van der Waals forces between the tip and sample. While van der Waals forces are always attractive, the chemical forces are attractive only if the distance is greater than equilibrium distance. Since the tip is mounted on a spring, approaching the spring, a sudden movement referred to as “Jump to contact” can occur when the stiffness of cantilever is less than a certain value . This occurs in quasi-static mode. So large amplitudes or cantilevers with large spring constants are required for the stability criteria to be satisfied and since amplitudes are related to resonance phenomenon and other operating criteria, stability may be difficult to achieve and should be carefully considered on case by case basis.
Long range forces: Many forces exist between the tip and sample such as electrostatic, magnetic, van der Waals and chemical in vacuum condition. In ambient conditions, meniscus forces also exist. While electrostatic is eliminated by equalizing the potential between tips, magnetic by using non-magnetic tips and meniscus forces are eliminated by carrying out experiment in vacuum, van der Waals forces continue to exist. Since we require only force components that vary at the atomic scale, long range forces are undesirable . In STM, these long range forces are automatically eliminated because the tunneling current naturally blocks contributions of tip atoms that are further from the sample. In static AFM both short and long range force add to the imaging signal while for dynamic AFM, long range forces can be weakened by adopting suitable cantilever oscillation amplitude.
Noise in Imaging signal: The forces are measured by deflection of the spring is accompanied by noise especially at low frequencies (1/f noise). In static AFM, the force is given by deflection of cantilever and the noise component is included, however in dynamic AFM, the low frequency noise can be discriminated if the eigenfrequency is larger than the corner frequency (1/f) and can be removed by adopting a bandpass filter .
Noise in imaging signal, stability and long range force problems can be eliminated by adopting Frequency Modulated Atomic Force Microscopy (FM-AFM) and other methods as described in the next section. Research is still ongoing in resolving the issue of the non-monotonic nature of the force and the tip-sample distance.
ADVANCES IN AFM
Frequency Modulated Atomic Force Microscopy (FM-AFM): In this technique, the cantilever can be used a frequency determining element of a constant amplitude oscillator with the oscillator output being instantaneously modulated in accordance with the force gradient acting on the cantilever [11,26]. The deflection signal which enters the bypass filter is split into 3 branches, one is phase shifted before being sent to analog multiplier and being sent back to cantilever through actuator, and the second is used to calculate the actual oscillation amplitude while the third is used as a feed for the frequency detector.
The simplest case of a FM-AFM is to consider a free cantilever with an eigen frequency f0 = 2π(k/m*)0.5, k is the spring constant and m* is the effective mass of the cantilever. The force between tip and sample change the frequency f = 2π(k*/m*)0.5 with k*=k+kts, where the tip-sample force gradient is given by kts. If kts and constant within the tip’s trajectory to and from the sample, the frequency change is given by Δf =f0 kts / 2k. The force causes a frequency shift and with the feedback circuit adjusts the tip-sample distance such that Δf remains a constant and results in the creation of an image.
The technique depends on a number of operating parameters. The spring constant of the cantilever, the eigenfrequency of the cantilever and the quality factor of the cantilever affect the FM-AFM image and should be suitably selected. The frequency shift of the cantilever (Δf) and oscillation amplitude (A) can be varied and adjusted as required. FM-AFM is used a standard method for atomic imaging of semiconductors, metals, insulators, organic films among others.
Scanning dissipation microscopy: When the drive signal required to maintain constant amplitude of a Frequency Modulated AFM was measured it was found that there were major Ohmic losses of currents that were induced due to variable capacitance due to oscillation of the tip-sample assembly in connection with the tip-sample voltage. The dissipation images thus produced of the heterogenous semiconductors had a feature size of around 10nm. This technique was extended further by Luthi et al  who studied the damping signal of atomic resolution of silicon. A number of other works describe the energy loss in dynamic AFM and even dissipative lateral forces.
Small Amplitude AFM: It has been shown that long range background forces can affect AFM because of resonance issues. In dynamic force microscopy, the various force components Fi with a corresponding range λi is related to the imaging signal of by the cantilever oscillation amplitude A.
For Aλ, the imaging signal
For Aλ, the imaging signal
Thus, for small amplitudes, the imaging signal is proportional to force gradient and the weight of short range forces is much more than that of long range forces. This concept was used by Hoffman et al  in developing “Off resonance technique” where the cantilever is oscillated at frequency much below its resonance frequency. When the cantilever comes near the sample, the resultant amplitude becomes,
Where, kst is the tip-sample stiffness. Though this method is good since the signal to noise ratio is improved, the quality of image is not as good as achieved by classic or small amplitude FM-AFM data. The decrease in quality is attributed to slow scanning speed and thermal drift. Research is still ongoing to test if whether the problem is due to fundamental reasons or technical imperfections.
Stiff cantilever operating in dynamic mode: The two major challenges of an AFM are the instability problem and the 1/f noise problem. These two are eliminated by using a stiff cantilever operating in dynamic mode. Greater sensitivity can be achieved by using with short range forces at small amplitudes. Tests by Giessibl et al showed that when thermal amplitude to enhance short range force contributions, stability issues arose. After a number of trials tests on FM-AFM with very stiff cantilevers, gave good results. The proof of this concept was shown by Giessibl et al , however, manufacturing of devices with k proved to be a challenge and instead k were tested and yielded positive results. AFM images of Silicon with excellent resolution was obtained. For the first time, clear features inside atom was imaged, as shown in Figure below. While accuracy of the subatomic resolution is debated, the small amplitude technique with very stiff cantilever produces excellent images of single atom images with non-trivial internal structures as shown with silicon and non-earth metal atoms. While Eguchi et al  proved experimentally the value of this technique, Huang et al , provided theoretical backing that atomic substructures linked to atomic orbitals are expected to occur when tip and sample reach distances similar to bulk next neighbor spacings.
Fig 3: Image of a Si(111)-(7 X 7) surface imaged with a qPlus sensor. Parameters: k = 1800 N/m, A = 2.5 Å, fo – 14.772 Hz, ∆f = +4Hz, γ = 28fNm-1/2. 
Dynamic Lateral Force Microscopy: Since its invention in 1987 by Mate et al , the lateral force microscope has grown in resolution capacity. The high resolution wear studies conducted on KBr . True atomic resolution was achieved by large-stiffness, small amplitude, lateral frequency modulated AFM by Giessibl et al.  Frequency shift was measured along with the difference in power which was measured as difference in power required for maintaining constant amplitude when the cantilever was close to the sample and the power when cantilever is far away from the sample yielding a connection to friction forces. Experimental data has shown that when cantilever is oscillating laterally over an atom almost no energy dissipation occurs while when approaching the atom from the side, an energy dissipation of few eV per oscillation cycle is measured.
APPLICATIONS OF STM AND AFM
STM and AFM are one of the most exciting discoveries and have spurred nanotechnology research into new realms. Chemistry, biology, physics, materials, they have made a huge impact in different areas. Since a whole book will be needed to explain the different sectors which are making use of AFM and STM, only a few applications are listed in the section below.
The applications of scanning tunneling microscope in the areas of surface science, nanoscience and catalysis are enormous. Books are available on how STM has been utilized in studying the surface of a particular compounds or use to understand a phenomenon such as catalysis. Some applications are outlined below:
Surface structure determination: Before STM was invented; diffraction methods were used to determine the surface structure. The methods provided inaccurate and even contradictory results in some cases and were applicable only to simple and defect free surface devoid of variations. Since the surface determination of Si (111) – 7 x 7 by STM transformed surface determination, a number of surfaces have been evaluated to understand the position of individual atoms on metal surfaces, study of lattice defects at metal surface with atomic resolution and other parameters to evaluate crystal growth .
Local tunneling spectra of superconductors: The Barden-Cooper-Schrieffer (BCS) theory of superconductors is based on tunneling phenomenon, so techniques like STM can be used to probe the local properties of superconductors. One of the experiments conducted used an ideal superconducting material 2H-NbSe2. The material had a charge-density-wave (CDW) temperature of 33K and transition temperature of 7.2 K. The topographic images showed a very clean and Abrikosov flux. When magnetic field of 1T was introduced, the expected spacing between adjacent vortices was expected to be few hundred Å. When the distance between the vortices was investigated, the dI/dV curve reveals a superconducting gap. Near and at the centre of the curve, dI/dV has a pronounced peak near the zero voltage point. This unexpected discovery was followed by theroritical investigation, which showed that peak is due to the trapped state near the centre of the vortex and showed that STM investigations can help understand the hitherto unknown state of material [3, 4].
Surface chemistry: STM can be used to study the chemical phenomenon at atomic level. One of the first studies done using STM was the initial stage of oxidation of Si surface. The site selectivity was also investigated in the study. Si (111) – 7 x 7 was exposed to ~ 0.2 L of oxygen and the STM showed 2 sides. One was a dark site and the other a bright site. The two had different spatial distributions. The bright sides had more corner adatoms than centre in ratio 4:1. (An adatom or an “adsorbed atom” – is atom lying on the surface of a crystal). The faulted half of crystal also showed 8 times more bright sites. The darker sites showed only 50% corner than centre adatoms and only 2 times more in the faulted half of crystal. Often a grayish site is present near the dark sites. To understand the chemical natures of these sites, a correlation of photoemission studies and first principles was done. While bright site is the insertion of oxygen atom into Si-Si bond, the dangling bond essentially empty gives bright appearance. The dark site is difficult to understand however, with increase in oxygen dosage, the dark sites increases steadily while the bright sites remains same and the strong tendency of conversion of bright sites to dark sites indicates the dark sites have an oxygen atom on top of Si adatom that eliminates the dangling bond. Other reactions such as reaction of hydrogen with silicon atom, epitaxial growth of silicon on silicon as disilane among others have been experimentally proved. Experiments like this can show the structure of surface without dispute [3, 4].
Study of organic molecules: Sleator and Tycko, Ohtani et al, Froster et al  showed the organic molecules adsorbed on various conducting substrates such as graphite. Organic molecules adsorbed on crystalline substrates form regular pattern. Smith et al investigated the liquid-crystal molecules through STM study. The molecules were sublimated onto freshly cleaved graphite crystal. The STM image was independent of the distance indicating that the STM tip pushes away the top layer and images the layer directly in contact with the graphite layer. The structure is indicative of the graphite surface.
Applications in Electrochemistry: Electrochemical processes depends dramatically on atomic surface of electrode surface. For example, plating area can vary upto 2 order of magnitude depending on crystallographic variation. STM and AFM which work at solid-liquid interface as well are natural tools for study of electrochemical processes at the molecular level. Sonnenfeld et al  reviewed solid surfaces immersed in electrolytes, the problem in STM is that Faradaic current is added to tunneling current and makes the signal-to-noise ratio worse. The 3 electrodes present in an electrochemical cell are reference electrode, counter electrode and working electrode. The Faradaic current is minimized by setting the tip potential equal to that of reference electrode and covering most of the tip except tiny end. However, a bias exists between the tip and working electrode. Still atomic resolution is obtained and new information regarding the electrodes is obtained. Among experiments, Magnussen et al  revealed in real space the atomic structure of the Cu monolayer on Au (111) and Au (100) surfaces in copper sulfate (CuSO4) solution. Imaging of clean Au surface by STM was also done as reference. The relative position of the Cu atoms on the Au surface are determined form the subsequent STM images.
Applications in Catalysis: STM is one of most useful tools available for probing local surface modifications associated with chemical reactivity at the solid-vacuum interface and mobility of reaction intermediates and final products on surfaces in real time. For selection of suitable model, it is necessary to compare experiment with theory and only method like STM that can the intermediate steps involved at level of atom. Simulations can provide insights about landscape governing the reactions from reactants to final products and STM simulations can aid understanding of image contrast and interpreting particular experimental images. The accuracy of the “ab initio” images recorded
Use of AFM in drug discovery: One of the most important properties of AFM with respect to biological investigation is that fact that it can work in fluid condition; this enables the researcher to run a test under near-physiological condition. Since the AFM can operate in both contact and in tapping mode (where the tip oscillates above the sample), it produces less lateral force and does not affect the delicate nature of the sample . A very important step in the investigation is the drawing of the “force curve”. There are three important steps :
Approaching phase: To construct the curve, the cantilever is held stationary and the tip is lowered towards the surface.
Recording phase: When the tip and substrate make contact, the cantilever is deflected and this is recorded by photomultipliers.
Withdrawal phase: The tip is raised and the probe and substrate are separated. The cantilever is deflected and returning to its original positions and is further deflected as a result of attraction of the tip towards the substrate by adhesion forces which may be chemical or electrostatic.
These adhesion forces can be minimized by adjusting the force curve. Since it provides a measure for the degree of attraction between the probe and the specimen, it can be used to distinguish area in the sample that has differing physical characteristics. A further development is the using specially funtionalized tips that can interact with biological molecules . For example, a tip coated with biotin has been developed and it has been used to study the interaction between biotin and streptavidin.
A special technique known as “single-molecule force spectroscopy” which uses AFM technique is used for studying protein unfolding and nature of interaction between protein molecules. In this method, the AFM tip is attached to the protein of interest and the vertical force exerted on the molecule is manipulated by the cantilever. The force extension produces a ‘sawtooth’ pattern causes by sequential unfolding of the modules within the proteins. This is indicative on the mechanism responsible for the mechanical stability of the proteins. Proteins of modular construction such as titin (responsible for passive elasticity of muscle) and fibronectin (present in extracellular matrix) were investigated by this technique. Single polysaccharide molecules can also be detected . Features such as intermediate and misfolded states can be revealed using single molecule measurements which cannot be observed by other bulk techniques such as X ray, NMR etc.
“Nano-scalpel” technique, where the molecules can be altered and manipulated using an AFM is another important application where the proteins can be used to study the oligomeric structures and then nano-dissection can be applied to break down the oligomers. In the nanodissected form, AFM can be used to identify structural information such as gap junctions.
Some of the applications of AFM in the pharmaceutical sector have helped to investigate biodegradeable polyanhydrides based on aromatic and aliphatic dicarboxylic acids, analysis of adhesive properties of salbutamol particles, usage in aerosol or dry powder inhalers and other carriers for therapeutic application and for protection of DNA from degradation.
The interaction of drugs with receptors is a prominent area in the drug industry. The receptors are most commonly proteins are present in span or intimately connected to plasma membrane. Since the membrane proteins are difficult to isolate and purify and inorder to simulate environments similar to those existing in the cell, they have to be introduced in a lipid environment. 2D crystallization and followed by imaging is an useful technique to study the structural features of the proteins. Radioligand binding is used to study the interaction of new potential drug with protein targets . Qualitative information such as affinity of the binding interaction can be derived which is useful for drug development. However, the structure of the complex formed and effect of the binding on the protein structure cannot be obtained from the technique and this information can be derived from the AFM.
Many modifications have been done in STM for specific applications. For instance, high pressure STM was used to study surface mobility of atoms and molecules.
Applications in Electrochemistry for AFM: One advance AFM technique uses optical beam deflection in repulsive force regime. In this technique a typical fluid-cell is used. The top of cell is made of glass to allow light to enter. Since no tunneling current is involved, using AFM it is easier to achieve atomic resolution. Since the AFM tip is an insulator, it interferes much less in the electrochemical process. Among tests done was one by Manne et al , who studied the underpotential deposition of Cu on Au. The tests unearthed many startling discoveries. For instance, it was found that structure of Cu adlayer on Au electrolyte depends dramatically on the nature of the electrolyte. Several such phenomena are being studied in detail.
Biological applications: AFM was used to study a single living cell infected by virus, with a resolution of 10nm by Haberle et al . This was the first time a single cell was investigated directly without drying or staining as required by the earlier electron microscopy methods. Real time studies under physiological conditions have been carried out for providing nanometer size resolution. Haberle et al  used AFM for imaging for monkey kidney cells cultured in standard growth solution. A series of images starting with dried, uncoated cells, followed by addition of a drop of liquid containing orthopox viruses, thereby infecting the cell. A few minutes after the addition, the cell membrane is softened, a few hours later a large protrusion is observed while after many hours the protrusion increases dramatically. The final stage is disappearance of protrusion leaving a scar which means the virus exited the cell.
Study of Nucleic acid: Among one of the first few studies was the one conducted on DNA. Bustamante et al showed that AFM can be used to study nucleic acids. The study used carbon tips to study DNA in air which was adsorbed to the substrate (mica) due to the presence of Mg2+. The topography showed larger width and lower height than expected. The wider width was attributed to convolution with the radius of tip, increasing the apparent width. The height measurements was attributed to error because of compression of DNA by top and because part of the substrate was covered by magnesium acetate.  The next few experiments was conducted in aqueous solution, observation of DNA degradation by DNase I, analysis of double helix, transcription by RNA polymerase, visualization of DNA bending induced by integration host factor, mechanical properties measurement and their modulation upon the binding of small molecules, and so on so forth. Many papers on the analysis of chromosome, imaging of duplex RNA, and protein binding sites were also written [16, 17, 18, 19].
Study of microbial cell surfaces: Rapid progress has been made in the imaging of microbial cells by AFM. Two-dimensional bacterial protein crystals in aqueous solution with subnanometer resolution are achieved using AFM though resolution of soft microbial cells is still difficult to achieve. Many new developments in sample preparation technique, easier instrumentation methods and dynamic imaging and cryogenic techniques have been recently achieved. Since the biological processes occur very fast dynamic processes with greater time resolution need to be achieved and faster imaging techniques for single biomolecules imaging need to be developed. [20, 21] Physical properties such as adhesion, surface charges, cell-wall elasticity, biomolecular interactions such as single-molecule elasticity and receptor-ligand interactions has also been shown.
Bioprobes development is underway to test specialized biomolecular cells for probing specific cell interactions. Also in future microbiological AFM-based measurements for understanding of the structure, properties and functions of microbial cell surfaces at the molecular and supra-molecular level are underway.  Modifications to facilitate lithographic processing, nanomachining, tips that can rotate individual bonds between molecules, many other exciting possibilities have been opened up bringing in more and more changes in STM and AFM.
STM and AFM are fast becoming the most viable methods for real space imaging of structural, chemical, electronic properties of surfaces. The most important features are its adaptability to different environments and utilization of relatively low electron energies making them very attractive techniques with enormous application in the ever burgeoning field of science and technology. While they were started as imaging techniques, their ability in specimen modification has made them one of the most useful techniques available in world today.
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