1. Atomic Force Microscopy

2. ~ Introduction ~ Principle ~ How it works? ~ AFM Resolution ~ Vibration Isolation ~ Piezoelectric scanners ~ AFM Modes *Contact mode *Non contact mode *Tapping mode ~ Comparison of AFM with other techniques ~ Advantages / disadvantages ~ Biological Applications CONTENTS:

3. INTRODUCTION: The atomic force microscope (AFM) or scanning force microscope (SFM) is a very high-resolution type of scanning probe microscopy, with demonstrated resolution of fractions of a nanometer. scanning probe microscopynanometer It is more than 1000 times better than the optical diffraction limit.optical diffraction limit Binnig, Quate and Gerber invented the first AFM in 1986.QuateGerber The AFM is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale.nanoscale The atomic force microscope (AFM) is one of the most powerful tools for determining the surface topography of native biomolecules at subnanometer resolution.

4. INTRODUCTION: AFM allows biomolecules to be imaged not only under physiological conditions, but also while biological processes are at work.AFM allows biomolecules to be imaged not only under physiological conditions, but also while biological processes are at work. By using AFM one can not only image the surface in atomic resolution but also measure the force at nano-newton scale.By using AFM one can not only image the surface in atomic resolution but also measure the force at nano-newton scale. Measures attractive or repulsive forces between tip and sampleMeasures attractive or repulsive forces between tip and sample Detection apparatus measures the vertical deflection for heightDetection apparatus measures the vertical deflection for height Can achieve resolution of up to 10 pmCan achieve resolution of up to 10 pm Unlike electron microscopes, can measure samples in air and liquidUnlike electron microscopes, can measure samples in air and liquid

5. PRINCIPLE The AFM works in the same way as our fingers which touch and probe the environment when we cannot see it. By using a finger to "visualize" an object, our brain is able to deduce its topography while touching it. The resolution we can get by this method is determined by the radius of the fingertip.

6. PRINCIPLE To achieve atomic scale resolution, a sharp stylus (radius ~1-2 nm) attached to a cantilever is used in the AFM to scan an object point by point and contouring it while a constant small force is applied to the stylus. With the AFM the role of the brain is taken over by a computer, while scanning the stylus is accomplished by a piezoelectric tube. This simple technique provides a peep into the microscopic world, and it enables us to understand how the "smallest bricks" (i.e., the biomolecules) of biological systems might function.

7. Figure 1. Principle of the AFM. (a) A fine stylus is mounted on a cantilever spring and scanned over the surface. At sufficiently small forces the corrugations of the scanning lines represent the surface topography of the sample. (b) The vertical deflection of the cantilever is detected by reflecting a laser beam onto a 2-segment photodiode. The photodiode signal is used to drive a servo system which controls the movement of the piezo xyz- translator. In this manner the applied force between the stylus and the sample can be kept constant within some tens of a piconewton. The imaging process can be performed in a liquid cell filled with buffer solution. This ensures that the biomolecules remain hydrated. (c) Atomic resolution of a mica surface recorded in aqueous solution. The distance between adjacent protrusions is 5.4 Å.

8. HOW IT WORKS? An atomically sharp tip is scanned over a surface with feedback mechanisms that enable the piezo- electric scanners to maintain the tip at a constant force (to obtain height information), or height (to obtain force information) above the surface. Tips are typically made from Si3N4 or Si, and extended down from the end of a cantilever. The nanoscope AFM head employs an optical detection system in which the tip is attached to the underside of a reflective cantilever. A diode laser is focused onto the back of a reflective cantilever. As the tip scans the surface of the sample, moving up and down with the contour of the surface, the laser beam is deflected off the attached cantilever into a dual element photodiode.

9. HOW IT WORKS? The photo detector measures the difference in light intensities between the upper and lower photo detectors, and then converts to voltage. Feedback from the photodiode difference signal, through software control from the computer, enables the tip to maintain either a constant force or constant height above the sample. In the constant force mode the piezo-electric transducer monitors real time height deviation. In the constant height mode the deflection force on the sample is recorded. The latter mode of operation requires calibration parameters of the scanning tip to be inserted in the sensitivity of the AFM head during force calibration of the microscope.

10. HOW IT WORKS? Some AFM's can accept full 200 mm wafers. The primary purpose of these instruments is to quantitatively measure surface roughness with a nominal 5 nm lateral and 0.01nm vertical resolution on all types of samples. Depending on the AFM design, scanners are used to translate either the sample under the cantilever or the cantilever over the sample. By scanning in either way, the local height of the sample is measured. Three dimensional topographical maps of the surface are then constructed by plotting the local sample height versus horizontal probe tip position.

11. AFM Resolution The concept of resolution in AFM is different from radiation based microscopies because AFM imaging is a three dimensional imaging technique. The ability to distinguish two points on an image is the standard by which lateral resolution is defined. The images resolved by wave optics is limited by diffraction and images resolved by scanning probe techniques is limited primarily by apical probe geometry and sample geometry. The radius of curvature significantly influences the resolving ability of the AFM. Images of DNA made by the sharper tip have shown dramatic improvements in resolution widths. Even greater improvements in resolution have been attained with Tapping mode but contact imaging still is capable of high resolution imaging.

12. AFM Resolution Here we can see that it is the radius of curvature significantly influences the resolving ability of the AFM. Images of DNA made by the sharper tip have shown dramatic improvements in resolution widths.

13. Vibration Isolation In order to obtain good AFM results, the vibration isolation platform is needed. The vibration isolation consists of a large mass attached to bungy cords firmly anchored to the building. Damping of the oscillation is believed to result from rubbing of the rubber fibres inside of the bungy cord against the outside lining material. Between the low resonance frequency of the bungy cord system and the high resonance frequency of the microscope hardware itself (> 10 kHz ), the AFM effectively comprises a band pass filter. This allows the microscopists to safely image their samples in the intermediate range of about 1 - 100 Hz and obtain atomic resolution.

14. AFM modes Contact mode High scan speed Handles extreme vertical variations Lateral forces can distort features Scraping and damage to soft mat. Low stiffness cantilevers, 'snap-in' to the surface Tapping mode High lateral resolution No scraping and less damage to soft materials Slower scan speed Stiff cantilevers offering stability near the surface Non-contact mode Measures Van der Waals forces Almost non-destructive

15. CONTACT MODE The contact mode where the tip scans the sample in close contact with the surface is the common mode used in the force microscope. Force on the tip is repulsive with mean value of 10-9 N. This force is set by pushing the cantilever against the sample surface with a piezoelectric positioning element. In contact mode AFM the deflection of the cantilever is sensed and compared in a DC feedback amplifier to some desired value of deflection. If the measured deflection is different from the desired value the feedback amplifier applies a voltage to the piezo to raise or lower the sample relative to the cantilever to restore the desired value of deflection. The voltage that the feedback amplifier applies to the piezo is a measure of the height of features on the sample surface.

16. NON-CONTACT MODE The non-contact mode which is used in situations where tip contact might alter the sample in subtle ways. In this mode the tip hovers 50 - 150 Angstrom above the sample surface. Attractive Van der Waals forces acting between the tip and the sample are detected, and topographic images are constructed by scanning the tip above the surface.

17. NON-CONTACT MODE Unfortunately the attractive forces from the sample are substantially weaker than the forces used by contact mode. Therefore the tip must be given a small oscillation so that AC detection methods can be used to detect the small forces between the tip and the sample by measuring the change in amplitude, phase, or frequency of the oscillating cantilever in response to force gradients from the sample. For highest resolution, it is necessary to measure force gradients from Van der Waals forces which may extend only a nanometer from the sample surface.

18. TAPPING MODE In tapping mode the cantilever is driven to oscillate up and down at near its resonance frequency by a small piezoelectric element mounted in the AFM tip holder. The amplitude of this oscillation is greater than 10 nm, typically 100 to 200 nm. Due to the interaction of forces acting on the cantilever when the tip comes close to the surface, Van der Waals force or dipole-dipole interaction, electrostatic forces, etc cause the amplitude of this oscillation to decrease as the tip gets closer to the sample. Van der Waals forceVan der Waals force An electronic servo uses the piezoelectric actuator to control the height of the cantilever above the sample. The servo adjusts the height to maintain a set cantilever oscillation amplitude as the cantilever is scanned over the sample. A tapping AFM image is therefore produced by imaging the force of the oscillating contacts of the tip with the sample surface

19. Comparison of AFM and other imaging techniques 1. AFM versus STM:  In some cases, the resolution of STM is better than AFM because of the exponential dependence of the tunneling current on distance.  The force-distance dependence in AFM is much more complex when characteristics such as tip shape and contact force are considered.

20.  STM is generally applicable only to conducting samples while AFM is applied to both conductors and insulators.  In terms of versatility, needless to say, the AFM wins.  Furthermore, the AFM offers the advantage that the writing voltage and tip-to-substrate spacing can be controlled independently, whereas with STM the two parameters are integrally linked. 2. AFM versus SEM:  Compared with Scanning Electron Microscope, AFM provides extraordinary topographic contrast direct height measurements and unobscured views of surface features (no coating is necessary).

21. information than the two dimensional profiles available from cross-sectioned samples. 4. AFM versus Optical Microscope:  Compared with Optical Interferometric Microscope (optical profiles), the AFM provides unambiguous measurement of step heights, independent of reflectivity differences between materials. 3. AFM versus TEM:  Compared with Transmission Electron Microscopes, three dimensional AFM images are obtained without expensive sample preparation and yield far more complete

22. Piezoelectric scanners 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.

23. Piezoelectric scanners 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. hysteresis 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 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 we can expect large changes in sensitivity. As the scanner ages, the sensitivity changes less with time and the scanner seldom requires recalibration.

24. ADVANTAGES The AFM has several advantages over the scanning electron microscope (SEM). scanning electron microscope scanning electron microscope Unlike the electron microscope which provides a two- dimensional projection or a two-dimensional image of a sample, the AFM provides a true three-dimensional surface profile. Unlike the electron microscope which provides a two- dimensional projection or a two-dimensional image of a sample, the AFM provides a true three-dimensional surface profile.

25. ADVANTAGES Samples viewed by AFM do not require any special treatments 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 living organisms. vacuum AFM can provide higher resolution than SEM. It has been shown to give true atomic resolution in ultra-high vacuum (UHV) and in liquid environments.

26. Figure 2. Surface and sub-surface of fresh articular cartilage from a bovine humeral head. (a) The most superficial layer, typically 200-500 nm thick, consists of acellular and non-fibrous tissue (bottom). Occasionally, this exhibits local discontinuities where the underlying network of collagen fibrils oriented parallel to the surface becomes visible (top). Treatment with chondroitinase AC removes the majority of the articular surface and exposes the superficial collagen fibrils (inset). (b) Higher magnification view of the collagen fibrils which exhibit a mean diameter of ~35nm. Tangential fibrils reveal a characteristic periodic banding pattern of 60±5nm (inset).

27. DISADVANTAGES A disadvantage of AFM compared with the scanning electron microscope (SEM) is the image size. The SEM can image an area on the order of millimetres by millimetres with a depth of field on the order of millimetres. The AFM can only image a maximum height on the order of micrometres and a maximum scanning area of around 150 by 150 micrometres. scanning electron microscopemillimetresdepth of fieldscanning electron microscopemillimetresdepth of field Another inconvenience is that an incorrect choice of tip for the required resolution can lead to image artifacts. Traditionally the AFM could not scan images as fast as an SEM, requiring several minutes for a typical scan, while a SEM is capable of scanning at near real- time (although at relatively low quality) after the chamber is evacuated.

28. 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. 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. hysteresis Due to the nature of AFM probes, they cannot normally measure steep walls or overhangs. Specially made cantilevers can be modulated 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 and additional artifacts. DISADVANTAGES

29. Figure 3. AFM topographs of purple membrane from Halobacterium salinarium. Purple membrane consists of 25% lipids and 75% bacteriorhodopsin. This light driven proton pump comprises 7 transmembrane a-helices which surround the photoactive retinal. (a) Imaged at forces of about 3x10 -10 N two of the three loops connecting the a-helices are visible on the cytoplasmic surface (inset). (b) When the applied force is reduced during imaging (from 3x10 -10 N at the beginning (bottom) to 1x10- 10 N at the end (top) of the scan), the proteins undergo a conformational change. (c) The most prominent loop connecting the a-helices E and F is imaged at 1x10 -10 N, but is bent toward the membrane surface at higher forces.

30. Biological Applications of AFM One of the advantages of AFM is that it can image the non-conducting surfaces. So it was immediately extended to the biological systems, such as analyzing the crystals of amino acids and organic monolayers. Applications of AFM in the biosciences include: DNA and RNA analysis; Protein-nucleic acid complexes; Chromosomes; Cellular membranes; Proteins and peptides; Molecular crystals; Polymers and biomaterials; Ligand-receptor binding. Little sample preparation is required for bioimaging with the AFM. In most cases it is as simple as spotting a few microliters of solution on mica or glass. One area of significant progress is the imaging of nucleic acids. The ability to generate nanometer-resolved images of unmodified nucleic acids has broad biological applications.

31. Figure 4. Conformational change of the hexagonally packed intermediate (HPI) layer of Deinoccocus radiodurans. The HPI layer consists of units which display a large central pore and are assembled from six protomers. The function of the surface layer is not understood, but it is thought to protect the cell from hostile factors of the environment, and might also be responsible for the uptake and release of nutrients and cellular signals. (a) Single pores of the inner surface occur in "open" (circled) and "closed" (boxed) conformations. (b) Imaging the same surface area after 5 min, some of the pores have changed their conformation. This conformational change is fully reversible and can be observed over hours. (c) Averages of open and closed pores as depicted in (a) and (b) together with a calculated difference image.

32. Biological Applications of AFM Cell biologists have applied the AFM's unique capabilities to study the dynamic behavior of living and fixed cells such as red and white blood cells, bacteria, platelets, cardiac myocytes, living renal epithelial cells, and glial cells. AFM imaging of cells usually achieves a resolution of only 20-50 nm, not sufficient for resolving membrane proteins but still suitable for imaging other surface features, such as rearrangements of plasma membrane or movement of sub-membrane filament bundles. There has been recent success imaging individual proteins and other small molecules with the AFM such as collogen. Smaller molecules that do not have a high affinity for common AFM substrates have been successfully imaged by employing selective affinity binding procedures.

33. Figure 5. (a) High resolution topograph of the perisplasmic surface of 2-D OmpF porin crystals. The average shown was calculated from 25 translationally aligned subframes. To allow comparison, the electron density based on the X-ray structure of the OmpF trimer was rendered at a lateral resolution of 15 Å. Zebra-like contours (dark and light blue) on the overlaid transparent AFM topograph mark zones of identical altitude, with a height difference between contours of 1 Å. (b) Atomic model of protein-protein and lipid-protein interactions based on the X-ray structure of the porin trimer.

34. Biological Applications of AFM Countless biological processes - DNA replication, protein synthesis, drug interaction, and many others - are largely governed by intermolecular forces. AFM has the ability to measure forces in the nanonewton range. This makes it possible to quantify the molecular interaction in biological systems such as a variety of important ligand-receptor interactions. It images or quantifies electrical surface charge. The dynamics of many biological systems depends on the electrical properties of the sample surface. In addition to measuring binding forces and electrostatic forces, the AFM can also probe the micromechanical properties of biological samples. Specifically, the AFM can observe the elasticity and, in fact, the viscosity of samples ranging from live cells and membranes to bone and cartilage.

35. THE END OF PRESENTATION

36. GROUP MEMBERS: 26) Imran Waris 27) Prachi Singh 28) Diksha Singh 29) Atul Singh 30) Rishikant Pandey SECTION – ‘C’