Atomic force microscopy (AFM) or scanning force microscopy (SFM) 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.. 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 research groups reporting reliable data. 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 tipradius 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 astrain 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 z = 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.

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

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 resonant frequency where the amplitude of oscillation is typically a few nanometers (<10 nm). The van der Waals forces, 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 resonant 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 to 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.[1] 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. Amplitude modulation has also been used in the non-contact regime to image with atomic resolution by using very stiff cantilevers and small amplitudes in an ultra-high vacuum environment.

Tapping mode

In ambient conditions, most samples develop a liquid meniscus layer. Because of this, keeping the probe tip close enough to the sample for short-range forces to become detectable while preventing the tip from sticking to the surface presents a major problem for non-contact dynamic mode in ambient conditions. Dynamic contact mode (also called intermittent contact or tapping mode) was developed to bypass this problem.[3] 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 similar to non-contact mode. However, 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, dipole-dipole interaction, electrostatic forces, etc. cause the amplitude of this oscillation to decrease as the tip gets closer to the sample. 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 intermittent contacts of the tip with the sample surface. This method of "tapping" lessens the damage done to the surface and the tip compared to the amount done in contact mode. Tapping mode is gentle enough even for the visualization of supported lipid bilayers or adsorbed single polymer molecules (for instance, 0.4 nm thick chains of synthetic polyelectrolytes) under liquid medium. With proper scanning parameters, the conformation of single molecules can remain unchanged for hours.[2]

AFM cantilever deflection measurement AFM beam deflection detection 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 the 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. 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.[8]

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. advantages and disadvantages The first atomic force microscope Just like any other tool, an AFM's usefulness has limitations. When determining whether or not analyzing a sample with an AFM is appropriate, there are various advantages and disadvantages that must be considered. ]Advantages AFM has several advantages over the scanning electron microscope (SEM). Unlike the electron microscope which provides a two-dimensional projection or a twodimensional 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. ]Disadvantages 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 150150 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[9][10] making the AFM microscope less suited for measuring accurate distances between topographical features on the image. However, several fastacting designs [11][12] were suggested to increase microscope scanning

productivity including what is being termed videoAFM (reasonable quality images are being obtained with videoAFM at video rate: faster than the average SEM). To eliminate image distortions induced by thermal drift, several methods have been introduced.[9][10] AFM images can also be affected by hysteresis of the piezoelectric material[13] 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. 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[13] 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.[13] 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 scanners 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 - non-contact mode

AFM beam deflection detection

Oil immersion
From Wikipedia, the free encyclopedia

Principle of immersion microscopy. Path of rays with immersion medium (yellow) (left half) and without (right half). Rays (black) coming from the object (red) at a certain angle and going through the coverslip (orange, as the slide at the bottom) can enter the objective (dark blue) only when immersion is used. Otherwise, the refraction at the coverslip - air interface causes the ray to miss the objective and its information is lost.

Two Leica oil immersion objective lenses. Oil immersion objective lenses look superficially identical to non-oil immersion lenses.

In light microscopy, oil immersion is a technique used to increase the resolution of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens. Immersion oils are transparent oils that have specific optical and viscosity characteristics necessary for use in microscopy. An oil immersion objective is an objective lens specially designed to be used in this way. Many condensers also give optimal resolution when the condenser lens is immersed in oil.

[edit]Theoretical

background

The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects. This minimum distance is labelled . If two objects are separated by a distance shorter than , they will appear as a single object in the microscope. A measure of the resolving power of a lens is given by its numerical aperture, NA:

where is the wavelength of light. From this it is clear that a good resolution (small ) is connected with a high numerical aperture. The numerical aperture of a lens is defined as NA = nsin0 where 0 is the angle spanned by the objective lens seen from the sample, and n is the refractive index of the medium between the lens and specimen (1 for air). State of the art objectives can have a numerical aperture of up to 0.95. Because sin 0 is always less than or equal to unity, the numerical aperture can never be greater than unity for an objective lens in air. If the space between the objective lens and the specimen is filled with oil however, the numerical aperture can obtain values greater than unity. This is because oil has arefractive index greater than 1.
[edit]Oil

immersion objectives

From the above it is understood that oil between the specimen and the objective lens improves the resolving power by a factor 1/n. Objectives specifically designed for this purpose are known as oil immersion objectives. Oil immersion objectives are used only at very large magnifications that require high resolving power. Objectives with high power magnification have short focal lengths, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. (In inverted microscopes the oil is applied to the objective). The refractive indices of the oil and of the glass in the first lens element are nearly the same, which means that the refraction of light will be small upon entering the lens (the oil and glass are optically very similar). The correct immersion oil for an objective lens has to be used to ensure that the refractive indices are correctly matched. Use of an oil immersion lens with the incorrect immersion oil, or without immersion oil altogether, will suffer from spherical aberration. The strength of this effect depends on the size of the refractive index mismatch.

Oil immersion can generally only be used on rigidly mounted specimens otherwise the surface tension of the oil can move the coverslip and so move the sample underneath. This also happen on inverted microscopes because the coverslip is below the slide.
[edit]Oil

immersion and the condenser

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[edit]Immersion
This section

oil

requires expansion.

Before the development of synthetic immersion oils in the 1940s Cedar tree oil was widely used. Cedar oil has an index of refraction of approximately 1.516. The numerical aperture of cedar tree oil objectives is generally around 1.3. Cedar oil has a number of disadvantages however: it absorbs blue and ultraviolet light, yellows with age, has sufficient acidity to potentially damage objectives with repeated use (by attacking the cement used to join lenses), and diluting it with solvent changes its viscosity (and hence refraction index and dispersion). Cedar oil must be removed from the objective immediately after use before it can harden, since removing hardened cedar oil can damage the lens. In modern microscopy synthetic immersion oils are more commonly used, as they eliminate most of these problems.[1] NA values of 1.6 can be achieved with different oils. Unlike natural oils synthetic ones do not harden on the lens and can typically be left on the objective for months at a time, though dust accumulation can be a problem. Xylene is often used to remove oil from the objective when desired.
In optics and fiber optics, an index-matching material is a substance, usually a liquid, cement (adhesive), or gel, which has an index of refraction that closely approximates that of an optical element or fiber, and is used to reduce Fresnel reflection at the surface of the element

In light microscopy, a water immersion objective is a specially designed objective lens used to increase the resolution of the microscope. This is achieved by immersing both the lens and the specimen in water which has a higher refractive index than air, thereby increasing the numerical aperture of the objective lens.
[edit]Applications

Water immersion objectives are used not only at very large magnifications that require high resolving power, but also of moderate power as there are water immersion objectives as low as 4X. Objectives with high power magnification have short focal lengths, facilitating the use of water. The water is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in water. Sometimes with water dipping objectives, the objective is directly immersed in the solution of water which contains the specimens to look at. Electrophoretic preparations used for instance in the cases of Comet Essay can benefit largely with water objectives. The refractive indices of the water and of the glass in the first lens are different but less than it would be the cases between air and glass as it will be the case with a non immersion objective, which means that the refraction of light will be small upon entering the lens.
[edit]Correction

collar

Unlike oil, water does not have the same or near identitical refractive value as the cover slip glass, so a correction collar is needed to be able to variate for its thickness. Lens without correction collar generally are made for the use of a 0.17 mm cover slip or for use without a coverslip (dipping lens).