Abstract
The atomic force microscope (AFM) is a characterization tool used to image sample surfaces. The AFM operates under many modes and can be manipulated to be used under many different circumstances. Previously, the main applications of the AFM included analyzing surfaces of materials and surfaces forces. More recently, applications of AFM have come to include analyzing living cells as well. By changing the type of tip used to polymer ones, living cells can be scanned without damaging them. Also, because AFM can be done in a liquid environment and dehydrating or coating the sample is not necessary, it aids in creating more accurate image analysis. Some modifications of the AFM that can be made besides changing the material of the tip include using the cantilever without the tip, using different cantilevers, and analyzing samples in different environments. These changes allow the AFM to be used to monitor many different living organisms as will be further described in depth later. The AFM has advantages over both TEM and SEM in that living organism samples are not destroyed during preparations. One limitation of AFM includes the fact that scanning one sample can take several minutes while analysis of an SEM sample occurs in near real-time. Applications of AFM will continue to expand and improvements to the technique will continue to be made.

Introduction to AFM
Atomic force microscopy (AFM) is an imaging technique that can be used in a number of different scientific fields and under many different conditions. The basic concept of this technique is that a 3-D image is mapped out using a sharp probe tip on the end of a cantilever. A laser that is directed to the cantilever is reflected off towards a photodiode which is attached to a detector. As the cantilever “vibrates” over the sample, the detector records the height changes. AFM has many advantages, the main one being that it does not have to done in a vacuum; therefore, making analysis of living organisms possible. Also, a variety of imaging modes are available depending on what it is that needs to be analyzed [1].

The theoretical basis of AFM is that there is a cantilever with a sharp probe at the very end of it. When the tip is near the sample’s surface, there are a magnitude of forces that act for/against it causing the cantilever to deflect. Some of these forces include: capillary forces, electrostatic forces, van der Waals forces, etc. This deflection of the cantilever is tracked by a laser that is directed at it. The laser is then reflected to photodiode detectors. There are many variations of how an AFM actually works, but these are the basic principles of how one operates [1]. The first of three major imaging modes is the contact mode. In contact mode, the tip is in constant contact with the sample. Here, the change in the force applied, depending on the spring constant of the cantilever, to the tip is measured and developed into an image. Softer tips would be used on softer samples, but even then, there are many disadvantages to this method. The tip during contact mode can easily break off if it runs into a sudden artifact on the sample. Also, because attractive forces are so strong, the tip can be pulled into the sample itself, especially biological ones. With that said, contact mode is normally done when the overall forces are repulsive between sample and tip [2]. Another method is the non-contact mode. In this mode, the tip of the probe never comes into contact with the surface of the sample. Instead, the cantilever oscillates at a specific frequency just nanometers above the surface. By doing so and allowing the forces from above the surface to act against the probe tip, an image of the sample’s surface can be measured using the distance between the tip and sample at each data point. There are many advantages to this method. For example, neither the tip or the sample will suffer and damage especially in the case of softer samples [2].

The last of the three major modes is the tapping mode. By using a small piezoelectric chip mounted in the tip of the AFM, the cantilever can be made to oscillate really close to its resonance frequency. With all the forces listed above happening at once, the oscillations decrease as the tip of the cantilever approaches the surface of the sample. The piezoelectric element then acts to maintain the oscillations. The image is produced by recording the intermittent “contacts” the the sample and the tip [2].

Potential applications of the AFM are enormous. The main ones include studying the surface of samples. Using height and deflection mode, one can get a clear image of samples on a nanoscale. Also, the AFM can be used to study surface forces using the force-distance curve. Because new materials are being made and perfected on the microscopic level, the need for AFM is rising. The AFM can also be used to image single atoms and because each atom possesses its own individual fingerprint, you can distinguish between atoms as well [3].

The AFM can also be used to image and analyze biological samples and living organisms. By adjusting the material of the tip or using AFM in a liquid environment instead, one can make observations of organisms without altering its properties or harming it in any way [1]. Even when it comes to imaging living cells, there are still a variety of ways one alter the AFM to suit the experiment at hand even better. Below are five different experiments that utilize the AFM in their studies yet each possess a different technique.Methods
Atomic force microscopy is said to be a development of the scanning tunneling microscope (STM). It is currently being used for a variety of applications by many research groups worldwide. Some of these applications include studying surface characteristics of living organisms as well as biological properties of cells under certain conditions. The following are examples of how this instrument is being used in different labs.

AFM can be used to study the structures of Mycobacterium tuberculosis. In the paper titled ‘Growth and cell-division in extensive (XDR) and extremely drug resistant (XXDR) tuberculosis strains: transmission and atomic force observation,’ four different types of cell division were studied between three different categories of drug-resistant tuberculosis structures. Contact mode was used for this experiment. The cantilever was a micro-fabricated Si3N4 that has been oxide-sharpened. A cell suspension was prepared and then mounted onto the liquid cell of the AFM using tape. The deflection and height was subsequently recorded [4].

In another paper titled ‘Rheology of Human Neutrophils Investigated Using Atomic Force Microscopy’, the AFM was used to study rheological properties of the different sections of neutrophils. For these experiments, modifications were made to the AFM. A polystyrene bead replaced the tip on cantilevers, this increases the contact area as needed and reduces stress that would be caused by the sharper tips. The probe was used to analyze the edges of neutrophils and then compared to the center section of neutrophils [5].

In yet another paper titled ‘Deciphering the Structure, Growth and Assembly of Amyloid-Like Fibrils Using High-Speed Atomic Force Microscopy,’ AFM is utilized again for biological studies. This time, high speed AFM was used in liquid to monitor the growth of fibrillar structures. These images were taken at a rate of 1 image per second using cantilevers with extremely sharp tips, spring constants of 200mN/m, and with water resonance frequencies of 1.2MHz. The fluid cell consisted of 10mM Tris, 100mM NaCl, and at a buffer pH of 8 [6].

Biological studies are only a small fraction of what an AFM can do, yet the list continues on the number of applications possible. Another example would be examining the mechanical properties of three different endothelial cell lines after they have undergone TNF-α stimulation as was done in the paper ‘Probing the mechanical properties of TNF-α stimulated cell with atomic force microscopy.’ Here, AFM is used parallel to a fluorescence microscope to image these cells. The cantilever was V-shaped and made out of silicon nitride, and at the tip was a silica particle probe 5 um in diameter. This is to be considered a large tip and is used to create a large contact area which in turn leads to pressures applied to be more evenly distributed. Also, this limits the depth of penetration upon contact resulting in the information profile wanted [7].

AFM can also be used in more unconventional situations as well. In the following experiments from ‘Noncontact estimation of intracellular break force using a femtosecond laser impulse quantified by atomic microscopy,’ the cantilever of an AFM is used without the tip, to measure the force of a laser pulse. This impulsive force was used to to detach individual cells from each other as leukocytes from endothelial cells [8]. The schematics of the AFM setup is shown below.

The AFM is an extremely diverse tool. It can be manipulated in many ways to function in a way on deems to use it. Above are only a few examples of how it can be used, and only focusing on biological samples. Because the instrument can be used in a liquid environment, dehydrating and/or coating living organisms in not necessary like it is for the SEM. This allows for more accurate images as shrinkage of the cells does not happen.

Results
The results of the above studies would not have been possible without the AFM. Although all studies were done on biological molecules and/or living cells, techniques varied across the board of how the AFM was used. Because the AFM is so versatile is what it makes it such an amazing instrument for research. The AFM is capable of producing results that no other microscopy method can which is why it is such an important and necessary instrument for today’s research.

In the first experiment discussed above regarding the cell division of drug-resistant isolates of Mycobacterium tuberculosis, it was found that the bacteria was able to survive the hostile environments presented to them by changing morphologically. Under the AFM, this was visible. A mother cell that was rod shaped produced round shaped daughter cells. Other shapes that were also produced include V, Y, oval, and multi-branching filaments. Below are images from AFM and TEM. All of them depict Mycobacterium tuberculosis undergoing multi-branching cell division, which is how the bacilli coped with hostile environments. TEM is one of the microscopy methods that has a huge disadvantage when compared to AFM. To prepare a sample for TEM, an ultra thin slice of the specimen is needed which in turn means that the biological sample is no longer functional. However, the dividing cells that were observed by the AFM, can continue through it’s cycle and be imaged again at a later stage.

In the paper studying rheological properties of neutrophils, the researchers found that different sections of an adherent neutrophil responded differently. The regions that are closer to the leading edge and tail are similar and less stiff than the central regions of neutrophils. The neutrophils displayed a dynamic viscoelastic behavior. The leading edges display some elastic property and have a lower elastic moduli which results in favorable conditions for the fluctuations of actin filaments. The image below represents the two sections that were tested using AFM. This describes the role of polymerization of actin filaments that lead to protrusions during an immune response.

In the study using high-speed AFM, very sharp tips were needed to image the protofibrils at a high resolution. AFM allowed them to see that a very high elongation velocity was observed (shown below). Also, they were able to see that the end of one protofibril was associating with the beginning of another fibril forming what they called a false branching point. All in all, researchers were able to gather enormous amounts of information using AFM which leads them to conclude that there is a whole new side to the mechanism of fibrillization of amyloid proteins.

AFM is not only used for images and visualization of specimens, it can also be used to study mechanical properties. In this study, mechanical stiffness of endothelial cells increased by 50% when stimulated with TNF-α. Force displacement curves were made on control and TNF-α stimulated endothelial cells which aided in determining the changes in elastic moduli. The image below represents the AFM-related portions of this study.

In the last experiment, the AFM consisted of a small portion of the actual experiment. The AFM was used to measure the laser impulse that is later used to detach cells from each other. Below is the figure describing the set up used and graphs showing oscillations of the cantilever. These oscillations were then used to calculate the impulse of the laser.

From the above results, AFM has been compared to the TEM. Preparation of samples for the TEM are time consuming and cause irreversible damages to the specimen. Again, as mentioned before, preparations for the SEM include dehydrating and coating before a biological sample can be analyzed which also causes damages to the organisms and possible inaccuracies to the images. Another limitation of SEM and TEM is the damage while imaging as well, and not just the damages during preparation. These two methods both require electrons to be bombarded onto the sample, therefore causing irreparable damage over time. Like all other imaging tools, AFM does have its limitations. Traditionally, an AFM scan will take a few minutes while SEM works at real-time. Another limitation of AFM is that it can only image areas as large as 150x150um and maximum heights of about 10-20um. Research is still being done on how to improve scans which will be discussed later. Future Directions
Researchers constantly develop new applications of AFM. It is such a diverse imaging tool from the fundamentals to the cantilever itself. Each aspect of it can be used in a way that it wasn’t exactly designed to be used. For example, in the paper above where it was used to measure the impulse forces of a laser. Recently, there have been many developments as well as sustained research on the type of material used for the cantilevers and for the probes. The dimensions of the probes have also been studied in depth. By changing the material of the probe, one can improve the parameters depending on a specific experiment. Also, refining the tips of the cantilevers like making them as narrow as possible and by making them longer, images with higher resolutions can be taken [9].

The applications of AFM are only expanding. Although only its use with living organisms have been discussed, AFM is highly used with regards to materials. Because materials are been developed and refined on a microscopic level, the need for AFM is increasing [9]. AFM can be directed to study the forces between particles and bubbles which lends itself to improving mineral separation in purposes such as desalination.

Because AFMs use is do broad and diverse, it is difficult to see where the most important future application lies. Interfaces are all around us and AFM allows us to study those interfaces and the forces that produced. Again, future improvements would be developing a tips made of a range of materials. Making the tip out of polymer has been a recent occurrence to aid in studying biological samples [10]. We can continue to improve the technique by maybe using more than one probe at a time to speed up the process of analyzing a sample as well as improving the size of the scanning area.