The Atomic Force Microscope Biology Essay

The atomic force microscope, or AFM, is an imaging tool that is used to generate high resolution images of nonconductive hard or soft surfaces. The capability of the AFM to provide high-resolution images of nonconductive soft surfaces makes it a great tool for imaging a range of biological samples [1, 2]. Biomolecules ranging in size from DNA to blood cells can be imaged with the AFM because of this capability.

Because it can operate in liquid environments and there is no special coating of the sample necessary, biological studies under physiological conditions are possible. This is very unlike samples and working environments for other methods such as the scanning electron microscope or the tunneling electron microscope, even though they provide images of similar resolution as the AFM [2]. Similarly, in contrast to other imaging methods, biological samples can be prepared in such a way that their molecular dynamics can be viewed with the AFM for long periods of time while still in a natural state without damage [3]. And, the AFM has extremely high spatial resolution that allows users to study individual biological molecules such as proteins and the relationships of their molecular dynamics and functions relative to their structures and functionalities. While the AFMs have the capability of distinguishing single atoms in hard materials, it can still be used to image these soft biological specimens providing the applied force of the stylus is very low so as to prevent structural damage to the softer biological samples [3].

The AFM has been modified to operate under cryogenic temperatures and under very low vacuum for viewing DNA plasmids under contact mode [1]. However, when viewing high-resolution images of wet biological molecules, it must be kept in mind that it is easy to deform these molecules from the force applied to the AFM tip. Heat also induces motion in the samples causing difficulties in obtaining a sharp image, which can be counteracted by operating the AFM at low temperatures.

Cryogenic AFM operation and subsequent sample temperature lowering increases mechanical stability in DNA macromolecules and virtually eliminates any thermally induced effects. The cantilever tip is also less subject to thermal effects and so less noise is seen from the instrument itself. However, this means that the sample is no longer in a physiological state, much like with electron microscopes.

Operating the AFM at liquid nitrogen temperature is suitable for DNA and most other proteins because they freeze and become hard and rigid when cooled to below 180 K [1].

The imaging speed of the AFM has been of concern in much of the recent research when imaging biological specimens [2,3,4]. Short time ranges are required to capture clear topographs of moving specimens [3].

Typically when trying to image a specific biological sample or molecule such as chromosomes, AFMs must make multiple scans of large surfaces because these objects are randomly distributed on the substrate [2]. And, so, most of the time spent imaging is devoted to locating the object and not to producing an image of the object itself. To rectify this, an AFM can be integrated with EPI illumination from an inverted optical microscope. This modified AFM can image objects 1pm or larger. Specimens of interest can be quickly selected by moving the entire sample with an XY stage. An image is then taken with the optical microscope and then is either reflected or fluoresced. This image can then be compared to the image taken by the AFM based on the amount of overlap in the fields of view [2].

Faster imaging times are necessary to obtain real-time images of dynamic biological processes, including mineral dissolution and growth and polymer crystallization, and work is being done in probe sensitivity enhancement and faster in and out of plane speeds of the scanner [4].

Cells must be kept alive when they are viewed interacting with stimuli [5]. Cells are typically just a few micrometers in size, and proteins, in tens of angstroms, are even smaller. The study of living cells and their interactions with their external environment is crucial when developing medications. Other essential factors include cell adhesion and growth characteristics. Nanoparticles and nanostructures can be used for cell or molecule modification and drug delivery without causing great damage to the cells or molecules themselves because of their relative sizes.

Traditionally, the optical microscope has been used to view cells, but since live cells have very low contrast and their internal structures are virtually colorless, they must be stained and fixated to a slide or subjected to other processes that kill them before they can be viewed. And the optical microscope, like any other imaging method, has its limitations. The image must be dark or highly refractive, resolution is limited to about 0.2 microns by diffraction, and unfocused light from surrounding sources distort the image. Electron microscopes overcome most of the limits of the optical microscope, but still the cells lives are compromised in the sample preparation. Biological cells are soft and do not adhere well to substrate surfaces unlike hard material samples. This makes imagining of such materials much more difficult with the AFM but the AFM has the advantage over the other imaging methods in that it is capable of imaging micron sized objects like cells in a wet environment, and thus cell life and integrity is not so easily compromised [5].

Pilli, flagella, polysaccharides, proteins and most other nanostructures found in biofilms are in the extracellular matrix and range from 50-150 nm in width and 1-10 nm in thickness [6]. These nanostructures have a direct influence on bacterial biofilm stability based on their spatial arrangement. For example, this arrangement and biofilm stability is important in dealing with wastewater treatment, biofiltration, and biocide enhancement. This presents a need for enhanced imaging methods with resolution on the nanoscale.

Standard analysis of biofilms and biopolymers is done using confocal laser scanning microscopy, or CLSM. Like other traditional methods for viewing biological molecules, CLSM requires staining and spatial resolution is limited. CLSM alone cannot determine the arrangement and distribution of nanostructures in the matrix of biofilms and biopolymers. However, the CLSM can be combined with the AFM. Imaging of such small molecules is possible with the AFM and the images show the heterogeneous composition of biofilms in a nanometer scale. So, combining these two provides excellent nanoscale resolution of CLS micrographs and it is therefore easy to identify and quantify nanostructures and their interactions within the biofilms.

Further combination of the CLSM-AFM system with Raman spectroscopy provides even further enhancement. If a metal or metal coated tip is used with the AFM, the laser focus improves the signals from the Raman spectroscope and a fingerprint of spectra can be obtained allowing better quantification of sample composition [6].

Many applications require the need for highly advanced sensitive biosensors [7]. The major problem with their design is that of the interaction of the bio-interface with the transducer surface. Nanopatterned surfaces offer a solution to this disadvantage because they can be used as an interface between the incompatible biological sample and the transducer surface.

Diagnosis of diseases can be time consuming and early stage detection is not always possible. Especially when dealing with critical diseases, high performance and high sensitivity biosensors could satisfy the need for early disease screening, detection, and diagnosis. Their development and ability to define diseases in their early stages would be an asset to the current healthcare system and would reduce the need for therapy and cost of care while simultaneously giving a more positive outlook to the patient.

Other applications for biosensors are the food industry and rapid detection of biological pathogens and bioterrorism agents [7].

Hard material implants in biological systems suffer from host tissue detachment, bioincompatibility, low adhesion, and can result in cell integration [8]. Recent efforts towards tissue engineering to solve these problems have gained favor with current medical research. Tissue engineering in this area involves finding suitable alternatives for replacing the non-compatible hard material implants by using more compatible biological materials. Reduced need for repeated operations to replace implants, rejection of implants by body tissues, disease transmission, and even donated organ rejection is feasible. This research starts with the need for biodegradable tissue scaffolds on which cells can be adhered, or seeded. Requirements for the scaffolds are that they must be biocompatible, biodegradable, and provide a reliable structure for the cells to seed. The AFM provides an excellent method to study the surface morphology of these scaffolds and biocompatible implants as it can image in wet environments. And so, the biological environment that these scaffolds and tissues are found in naturally can be imitated in the AFM [8].

Since the AFM is an excellent tool for interatomic force studies, and lends itself well to topographical imaging of both hard and soft samples, both in ambient conditions and under liquids, it lends itself well to imaging of biological species. It is able examine a greater variety of surface interactions and develop mathematical models for similar inorganic, organic, and biological systems than other conventional techniques, especially the traditional optical light microscope.

While a robust tool for generating high resolution images of smooth surfaces on a nanometer level, the atomic force microscope, or AFM, is limited. The AFM tip range of measurement and the tip-sample interaction result in strong nonlinearities in tip motion, inaccuracies in measurements, and detrimental wear of the tip occur. Its simplicity and especially resolution does allow it to be used for viewing unmodified biological samples in a natural environment and in special cases of modified environments.