Atomic Force Microscope Physics Assignment

Atomic Force Microscope Physics Assignment Group Members: İbrahim Mert DARICI Syed Arslan Afzal HASHMI Ali ZAREI Sudhakar Murthy MOLLI Materials Processing 2006 PHYSICS ASSIGNMENT 1

Content 1 Introduction... 3 1.1 General Definitions... 4 1.2 Applications of AFM... 5 2 Atomic Force Microscope... 5 2.1 Construction and Working... 5 2.2 Modes of Operation... 7 2.2.1 Contact Mode... 7 2.2.2 Non-Contact Mode... 8 2.2.3 Dynamic Mode... 8 2.3 Lateral Force Microscopy... 8 2.4 Force Modulation... 9 2.5 Deflection Mode... 9 2.6 Phase Imaging... 9 2.7 Frequency Modulation... 9 2.8 Tapping Mode... 10 2.9 Lift Mode... 10 2.10 Advantages... 11 2.11 Disadvantages... 11 2.12 Type of Tips... 12 2.13 Dimensions and Magnification... 13 3 Examples of AFM Applications... 14 3.1 Study of DNA-Hydrolyzing Activity of Antibodies to DNA... 14 3.2 Contact Lens Manufacturing... 15 4 Conclusion and Discussion... 18 5 References... 19 5.1 Books... 19 5.2 Journals... 19 5.3 Websites... 19 PHYSICS ASSIGNMENT 2

1 Introduction Typically, when we think of microscopes, we think of optical or electron microscopes. Such microscopes create a magnified image of an object by focusing electromagnetic radiation, such as photons or electrons, on its surface. Optical and electron microscopes can easily generate two-dimensional magnified images of an object's surface, with a magnification as great as 1000X for an optical microscope, and as large as 100,000X for an electron microscope. Although these are powerful tools, the images obtained are typically in the plane horizontal to the surface of the object. Such microscopes do not readily supply the vertical dimensions of an object's surface, the height and depth of the surface features. Figure 1 An example of a surface profiler made in 1929. The atomic force microscope (AFM), developed in the mid 1980's, uses a sharp probe to magnify surface features. With the AFM, it is possible to image an object's surface topography with extremely high magnifications, up to 1,000,000X. Further, the PHYSICS ASSIGNMENT 3

magnification of an AFM is made in three dimensions, the horizontal X-Y plane and the vertical Z dimension. 1.1 General Definitions The atomic force microscope (AFM), which is also known as scanning force microscope (SFM), was invented in 1986 by Binnig, Quate and Gerber. Like all other scanning probe microscopes, the AFM utilizes a sharp probe moving over the surface of a sample in a raster scan. In the case of the AFM, the probe is a tip on the end of a cantilever which bends in response to the force between the tip and the sample. The small probe-sample separation (on the order of the instrument's resolution) makes it possible to take measurements over a small area. To acquire an image the microscope raster-scans the probe over the sample while measuring the local property in question. The resulting image resembles an image on a television screen in that both consist of many rows or lines of information placed one above the other. Figure 2 (a) A schematic illustration of the method of operation of an atomic force microscope (b) An example of a three-dimensional image of the surface topography of an osteoclast that was cultured on a glass coverslip. PHYSICS ASSIGNMENT 4

Unlike traditional microscopes, scanned-probe systems do not use lenses, so the size of the probe rather than diffraction effects generally limits their resolution. The atomic force microscope measures topography with a force probe. 1.2 Applications of AFM AFM is being used to solve processing and materials problems in a wide range of technologies affecting the electronics, telecommunications, biological, chemical, automotive, aerospace, and energy industries. The materials being investigating include thin and thick film coatings, ceramics, composites, glasses, synthetic and biological membranes, metals, polymers, and semiconductors. The AFM is being applied to studies of phenomena such as abrasion, adhesion, cleaning, corrosion, etching, friction, lubrication, plating, and polishing. The publications related to the AFM are growing speedily since its birth. Figure 3 AFM 2 Atomic Force Microscope 2.1 Construction and Working The AFM consists of a cantilever with a sharp tip at its end, typically composed of silicon or silicon nitride with tip sizes on the order of nanometers. The tip is brought into close proximity of a sample surface. The Van der Waals force between the tip and the sample leads to a deflection of the cantilever according to Hooke's law, where the spring constant of the cantilever is known. Typically, the deflection is measured using a laser spot reflected from the top of the cantilever into an array of photodiodes. However a laser detection system can be expensive and bulky; an alternative method in determining cantilever deflection is by using piezoresistive AFM probes. These probes are fabricated with piezoresistive elements that act as a strain gage. Using a Wheatstone bridge, strain in PHYSICS ASSIGNMENT 5

the AFM probe due to deflection can be measured, but this method is not as sensitive as the laser deflection method. In an AFM a constant force is maintained between the probe and sample while the probe is raster scanned across the surface. By monitoring the motion of the probe as it is scanned across the surface, a three dimensional image of the surface is constructed. The constant force is maintained by measuring the force with the "light lever" sensor and using a feedback control electronic circuit to control the position of the Z piezoelectric ceramic. See Following Figure. Figure 4 This figure illustrates the primary components of the light lever atomic force microscope. The X and Y piezoceramics are used to scan the probe over the surface. The motion of the probe over the surface is generated by piezoelectric ceramics that move the probe and force sensor across the surface in the X and Y directions. PHYSICS ASSIGNMENT 6

2.2 Modes of Operation Over the years several modes of operation have been developed for the AFM. The primary modes of operation are Contact mode Non-contact mode, and Dynamic contact mode. 2.2.1 Contact Mode Contact mode is the most common method of operation of the AFM. As the name suggests, the tip and sample remain in close contact as the scanning proceeds. By "contact" we mean in the repulsive regime of the inter-molecular force curve in figure 5. The repulsive region of the curve lies above the x-axis. Figure 5 Force-Separation Graph One of the drawbacks of remaining in contact with the sample is that there exist large lateral forces on the sample as the drip is "dragged" over the specimen. Figure 6 Examples of images generated using contact mode AFM are shown in the above Figure. A freshly cleaved surface of mica was imaged with a BioScope1 AFM to reveal the atomic structure of mica in Figure a. Figure b illustrates the use of the wet cell in contact mode. PHYSICS ASSIGNMENT 7

2.2.2 Non-Contact Mode In the non-contact mode, the cantilever is externally oscillated at or close to its resonance frequency. The oscillation gets modified by the tip-sample interaction forces; these changes in oscillation with respect to the external reference oscillation provide information about the sample's characteristics. Because most samples develop a liquid meniscus layer, keeping the probe tip close enough to the sample for these inter-atomic forces to become detectable while preventing the tip from sticking to the surface presents a major hurdle for non-contact mode in ambient conditions. 2.2.3 Dynamic Mode Dynamic contact mode was developed to bypass this problem. In dynamic contact mode, the cantilever is oscillated such that it comes in contact with the sample with each cycle, and then enough force is applied to detach the tip from the sample. There are also some other modes of operation as described below. 2.3 Lateral Force Microscopy LFM measures frictional forces on a surface. By measuring the twist of the cantilever, rather than merely its deflection, one can qualitatively determine areas of higher and lower friction. Figure 7 Working of Lateral Force Micropscopy PHYSICS ASSIGNMENT 8

2.4 Force Modulation Force modulation refers to a method used to probe properties of materials through sample/tip interactions. The tip (or sample) is oscillated at a high frequency and pushed into the repulsive regime. The slope of the force-distance curve is measured which is correlated to the sample's elasticity. The data can be acquired along with topography, which allows comparison of both height and material properties (hence alternate name is height mode). 2.5 Deflection Mode If the feedback electronics are switched off, then the microscope is said to be operating in constant height or deflection mode. This is particularly useful for imaging very flat samples at high resolution. Often it is best to have a small amount of feedback-loop gain, to avoid problems with thermal drift or the possibility of a rough sample damaging the tip and/or cantilever. Strictly, this should then be called error signal mode. 2.6 Phase Imaging In phase mode imaging, the phase shift of the oscillating cantilever relative to the driving signal is measured. This phase shift can be correlated with specific material properties that effect the tip/sample interaction. The phase shift can be used to differentiate areas on a sample with such differing properties as friction, adhesion, and viscoelasticity. The techniques are used simultaneously with DFM mode, so topography can be measured as well. 2.7 Frequency Modulation Schemes for non-contact and dynamic contact mode operation include frequency modulation. In frequency modulation, changes in the oscillation frequency provide information about a sample's characteristics. PHYSICS ASSIGNMENT 9

2.8 Tapping Mode In amplitude modulation (better known as intermittent contact or tapping mode), changes in the oscillation amplitude yield topographic information about the sample. Additionally, changes in the phase of oscillation under tapping mode can be used to discriminate between different types of materials on the surface. Figure 8 Examples of images acquired by TappingMode are shown in the above Figure. A 2µm TappingMode image of fibrillar collagen is shown in Figure a. Resolution of a dspacing (the spacing between crystalline lattice planes) of 70 nm confirms measurements made with a transmission electron microscope; the latter requires tedious and timeconsuming specimen preparation versus the minimal sample preparation for the AFM. Figure 4b is an image of both normal and sickled human red blood cells. 2.9 Lift Mode Several techniques in AFM rely on removing topographical information from some other signal. Magnetic force imaging and electrostatic force imaging work by first determining the topography along a scan line, and then lifting a pre-determined distance above the surface to re-trace the line following the contour of the surface. In this way, the tip-sample distance should be unaffected by topography, and an image can be built up by recording changes which occur due to longer range force interactions, such as magnetic forces. PHYSICS ASSIGNMENT 10

Figure 9 Shown above are the height (left) and magnetic force (right) images of a 100 µm piece of floppy disc 2.10 Advantages The AFM has several advantages over the electron microscope. Unlike the electron microscope which provides a two-dimensional projection or a twodimensional image of a sample, the AFM provides a true three-dimensional surface profile. Additionally, samples viewed by an AFM do not require any special treatment that would actually destroy the sample and prevent its reuse. While an electron microscope needs an expensive vacuum environment for proper operation, most AFM modes can work perfectly well in an ambient or even liquid environment. This makes it an excellent tool for studying live biological samples. Imaging of conducting and non-conducting surfaces down to sub-nanometer resolution without the need for any additional information. Imaging in air and liquid, allowing in-situ measurements and real time imaging of biological and chemical processes. AFM can be used to measure and localize many different force including adhesion strength, magnetic forces and mechanical properties. 2.11 Disadvantages The main disadvantage that the AFM has compared to the scanning electron microscope (SEM) is the image size. The SEM can show an area on the order of millimeters by millimeters and a depth of field on the order of millimeters. The AFM can only show a maximum height on the order of micrometers and a maximum area of around 150 by 150 micrometers. Additionally, the AFM cannot scan images as fast as an SEM. It may take PHYSICS ASSIGNMENT 11

several minutes for a typical region to be scanned with the AFM, however an SEM is capable of scanning at near real-time (although at relatively low quality). 2.12 Type of Tips One of the most important factors influencing the resolution which may be achieved with an AFM is the sharpness of the scanning tip. The first tips used by the inventors of the AFM were made by gluing diamond onto pieces of aluminum foil. Commercially fabricated probes are now universally used. The best tips may have a radius of curvature of only around 5nm. The need for sharp tips is normally explained in terms of tip convolution. This term is often used (slightly incorrectly) to group together any influence which the tip has on the image. The main influences are broadening compression interaction forces aspect ratio Most AFM available are with cantilevers with their attached tips from commercial vendors, who manufacture the tips with a variety of microlithographic techniques. A close enough inspection of any AFM tip reveals that it is rounded off. Therefore force microscopists generally evaluate tips by determining their "end radius." In combination with tip-sample interaction effects, this end radius generally limits the resolution of AFM. As such, the development of sharper tips is currently a major concern. a b c Figure 10 Three common types of AFM tip. (a) normal tip (3 µm tall); (b) supertip; (c) Ultralever (also 3 µm tall). Electron micrographs by Jean-Paul Revel, Caltech. Tips from Park Scientific Instruments; supertip made by Jean-Paul Revel. PHYSICS ASSIGNMENT 12

Force microscopists generally use one of three types of tip. 1. The "normal tip" (figure 10a) is a 3 µm tall pyramid with ~30 nm end radius. 2. The electron-beam-deposited (EBD) tip or "supertip" (figure 10b) improves on this with an electron-beam-induced deposit of carbonaceous material made by pointing a normal tip straight into the electron beam of a scanning electron microscope. Especially if the user first contaminates the cantilever with paraffin oil, a supertip will form upon stopping the raster of the electron beam at the apex of the tip for several minutes. The supertip offers a higher aspect ratio (it is long and thin, good for probing pits and crevices) and sometimes a better end radius than the normal tip. 3. Finally, Park Scientific Instruments offers the "Ultralever" (figure 10c), based on an improved microlithography process. Ultralevers offers a moderately high aspect ratio and on occasion a ~10 nm end radius. 2.13 Dimensions and Magnification An atomic force microscope is optimized for measuring surface features that are extremely small, thus it is important to be familiar with the dimensions of the features being measured. An atomic force microscope is capable of imaging features as small as a carbon atom and as large as the cross section of a human hair. A carbon atom is approximately.25 nanometers (nm) in diameter and the diameter of a human hair is approximately 80 microns (µm) in diameter. The common unit of dimension used for making measurements in an atomic force microscope is the nanometer. A nanometer is one billionth of a meter: 1 meter = 1,000,000,000 nanometers 1 micron = 1,000 nanometers Another common unit of measure is the Angstrom. There are ten angstroms (Е) in one nanometer: 1 nanometer = 10 Angstroms Magnification in an atomic force microscope is the ratio of the actual size of a feature to the size of the feature when viewed on a computer screen. Thus when an entire cross PHYSICS ASSIGNMENT 13

section of a human hair is viewed on a 500 MM computer monitor (20 inch monitor) the magnification is: Magnification = 500 mm/.08 mm = 6,250 X In the case of extremely high resolution imaging, the entire field of view of the image may be 100 nanometers. In this case the magnification on a 500 mm computer screen is: Magnification = 500 mm/(100 nm*1 mm/1,000,000 nm)=5,000,000 X 3 Examples of AFM Applications As it is stated early in the paper, AFM is used in problems which are in wide range of technologies affecting many different industries. In this section, some proper examples are introduced and one among these examples is explained in details. AFM of biological and organic objects is difficult because the mechanical rigidity of these objects is low in comparison to the surface of hard objects. 3.1 Study of DNA-Hydrolyzing Activity of Antibodies to DNA In this example biological particles was studied which are some antibodies (ABs) exhibit enzymatic activity. Such antibodies were referred to as abzymes (from antibody enzyme), or catalytic ABs. Natural abzymes were found in blood serum of patients with autoimmune diseases. However, the origination, biological role, mechanism, nature, and specific properties of catalysis performed by abzymes remain to be determined. The antibodies to DNA that exhibit DNase activity are of special interest among the natural catalytic ABs because they may be directly involved in pathogenesis of these diseases. In this work, involved scientist used atomic force microscopy to study the interaction of DNA-hydrolyzing ABs to DNA with the super-circular pbr-322 plasmid DNA in systemic lupus erythematosus one of widespread autoimmune human diseases of unknown etiology. PHYSICS ASSIGNMENT 14

Figure 11 AFM images of(a) molecules of the pbr-322 plasmid after (a) incubation at 37ºC without antibodies, (b) AB molecules to ndna The results obtained indicate that AFM is a highly sensitive technique for detection and analysis of intermolecular interactions and that it can be used for fundamental and practical studies of immunity reactions. 3.2 Contact Lens Manufacturing Figure 12 TappingMode in saline solution images of a fresh, out-of-the-box, commercially available contact lens. (a) 47µm, (b) 10µm, (c) 4µm scans. Figure 10 shows three TappingMode AFM images of a brand new commercial soft contact lens under saline. The prominent linear feature that appears in these three images was a surprised finding. The detailed three-dimensional structure is visible in PHYSICS ASSIGNMENT 15

progressively smaller scans, and the features can be measured for their in plane and outof-plane (vertical) size. It is not fully known what size and type of defects or features on the contact lens surfaces are critical in prompting unfavorable responses by the eye. The adhesion and entrapment of protein and contaminants between the lens and the cornea are believed responsible for promoting the growth of bacteria. To help understand how and where protein molecules and contaminants adhere to the lens, the topography of new and used lens surfaces can be mapped in great detail with AFM (Figures 10, 11). Figure 13 TappingMode image of a 1µm x 1µm area of the same type of contact lens as in Figure 2, also immersed in saline. The RMS roughness is 3.5nm for the area shown. A surface defect or pit, clearly seen in the lower right of the image, measures 170nm in width and 150nm in depth and, therefore, is large enough to trap proteins or contaminants. Figure 12 shows a TappingMode AFM image of a contact lens in saline solution, which was made using a diamond-turned mold. The diagonal cross-section reveals the short and long range variation in height. The periodicity of the surface grooves is a 1.5µm. The grooves resulted from the manufacturing process. When combined with clinical studies, this type of information can help clarify the effect of different size grooves. PHYSICS ASSIGNMENT 16

Figure 14 TappingMode AFM measurements on the grooves of a hydrogel lens in saline solution. The grooves originate from the diamond lathed mold. Figure 13 is an AFM image of another hydrogel lens that was made with diamond-turned molds. The lathe grooves run diagonally from bottom left to top right. The periodic occurrence of a defect on the lens surface, approximately every fourth groove, strongly suggests that the origin of these types of defects is traceable back to the mold lathing process. Figure 15 TappingMode AFM image of a hydrogel lens in saline. One way to improve the production yield of contact lenses is to detect and characterize defects early in the production cycle, i.e., on the production molds. AFM is a fast and easy-to-use tool for imaging the molds and accurately measuring feature dimensions. Unlike optical microscopy, AFM provides additional quantitative information about the nature and size of the surface features. PHYSICS ASSIGNMENT 17

AFM is a useful new tool for the contact lens industry, as well as for biomaterials R&D in general. AFM measurements help in evaluating surface finish quality, manufacturing processes, protein adsorption and build-up, lens cleaner efficiency, and materials properties in air or liquids. These types of measurements on lenses and/or molds are very useful for greatly enhancing quality control capabilities. Furthermore, this type of information will help to speed the development of superior polymers and coatings, and new or improved manufacturing processes. It is also demonstrated that AFM can be useful in clinical studies to identify with more confidence the underlying causes of contact lens related discomfort. 4 Conclusion and Discussion The fact that AFM senses small chemical or mechanical forces point-by point by directly contacting the natural sample surface distinguishes it from other surface analysis techniques. AFM complements and improves upon other types of microscopy. Three key advantages of AFM over conventional microscopic techniques are, (1) surfaces can be analyzed with nanometer-level resolution in three dimensions, (2) the analysis can be performed in ambient air or in liquids, and (3) sample preparations and imaging environments known to generate artifacts are eliminated (e.g., dehydration, fixation, freezing, staining, coating, etc). Unfortunately, AFM cannot image all samples at atomic resolution. The end radii of available tips confines atomic resolution to flat, periodic samples such as graphite. In addition, because biological structures are soft, the tip-sample interaction tends to distort or destroy them. With its good properties which allows AFM to be used in many different areas, though its obvious restrictions, it is known as a powerful tool for magnifying materials and particles in nano-scale. PHYSICS ASSIGNMENT 18

5 References 5.1 Books Q. Zhong, D. Innis, K. Kjoller, V.B. Elings, Surf. Sci. Lett. 290, L688 (1993). 5.2 Journals V. G. Vinter, T. A. Nevzorova, O. A. Konolova, M. Kh. Slakhov, (2005), Study of DNA-Hydrolyzing Activity of Antibodies to DNA Using Atomic Force Microscopy, Doklady Biochemistry and Biophysics, Vol. 405, 2005, pp. 414 416. 5.3 Websites http://en.wikipedia.org/wiki/atomic_force_microscope http://stm2.nrl.navy.mil/how-afm/how-afm.html http://spm.phy.bris.ac.uk/techniques/afm/ http://www.che.utoledo.edu/nadarajah/webpages/whatsafm.html http://www.sst.ph.ic.ac.uk/photonics/intro/afm.html http://www.lot-oriel.com/site/site_down/pn_afmhistory_deen.pdf http://www.veeco.com/appnotes/an22_contactlens.pdf PHYSICS ASSIGNMENT 19