Atomic Force Microscopy: Imaging AFM
1-Introduction  2-Optical Beam Deflection 3-Feedback Operation 4-Contact Mode (Static Mode)
5-VIbration Mode (Dynamic Mode)
1 - Introduction
The atomic force microscope (AFM) was originally designed as an imaging tool. It was modified from the design of the scanning tunneling microscope (STM). The AFM acquires topographic images by methodically scanning a sample with a flexible probe, called a cantilever, which bends according to the contours of the sample's surface. The bending of the cantilever is translated into an image map, which reveals the height differences in the surface being scanned. It is possible to image biological samples under physiological conditions as imaging can be done in both air and liquid. The resulting resolution of such maps is at the atomic level.
The imaging AFM has been used to image many biological samples ranging from genetic material to cells to bone. We will highlight a few of these studies. One of the earliest biological materials to be imaged was DNA, which has been imaged in many forms to date, including double and single-stranded forms as well as more complex structures. The AFM has also been used for many applications including DNA sizing, previously only achieved using gel electrophoresis, DNA mapping, hybridization studies and examinations of protein- DNA interactions. AFM studies of RNA were also conducted. Unlike DNA, which mainly forms a double- helical structure, RNA has the ability to form more advanced structures that do not rely solely upon Watson-Crick base-pairing. One example are the so called "kissing-loop" structures imaged by Hansma, et al . Not only was the AFM used in imaging of such structures, many of them three dimensional, but also played an important role in designing them. Unlike other imaging techniques, AFM sudies can be done under physiological conditions allowing for the imaging of biological processes. Images of transcription complexes have been obtained, for example E.coli RNA polymerase in complex with DNA. These studies are the only of their kind that can answer certain specific questions as to how the RNA transcription process takes place. One is able to visualize how the DNA does not get entangled in the nascent RNA strands. Such studies detailing the structure- function relationship of the transcription process are key in furthering our understanding of gene expression.
Also imaging of cells was conducted to examine the structure of the cellular cortex in detail. The cell cytoskeleton is known to be involved in affecting cell shape as well as movement and other cellular responses to biochemical and biophysical signals. At present, relatively little is known about the mechanical organization of cells at a subcellular level. Pesen, et al studied the cell cortex of bovine pulmonary artery endothelial cells (BPAECs) using atomic force microscopy (AFM) and confocal fluorescence microscopy (CFM). They were able to identify a coarse and fine mesh which make up the cortical cytoskeleton . These two types of mesh appear to be intertwined. Such details are not distinguished in imaging studies using fixed cells.
Other imaging studies have looked at tendon and bone, both of which are composed of type I tropo-collagen. This was done by acquisition of high resolution AFM images of type I collagen in conjunction with force spectroscopy studies, namely protein unfolding which is described in the following section. In these studies the AFM was used to investigate the mechanical properties of this collagenous tissue, which are altered in diseases such as osteoporosis. Being familiar with such properties is important for gaining further understanding as well as preventing and curing bone diseases [9].
2-Optical Beam Deflection
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