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WIREs Syst Biol Med
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Potential role of atomic force microscopy in systems biology

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Abstract Systems biology is a quantitative approach for understanding a biological system at its global level through systematic perturbation and integrated analysis of all its components. Simultaneous acquisition of information data sets pertaining to the system components (e.g., genome, proteome) is essential to implement this approach. There are limitations to such an approach in measuring gene expression levels and accounting for all proteins in the system. The success of genomic studies is critically dependent on polymerase chain reaction (PCR) for its amplification, but PCR is very uneven in amplifying the samples, ineffective in scarce samples and unreliable in low copy number transcripts. On the other hand, lack of amplifying techniques for proteins critically limits their identification to only a small fraction of high concentration proteins. Atomic force microscopy (AFM), AFM cantilever sensors, and AFM force spectroscopy in particular, could address these issues directly. In this article, we reviewed and assessed their potential role in systems biology. WIREs Syst Biol Med 2011 3 702–716 DOI: 10.1002/wsbm.154 This article is categorized under: Laboratory Methods and Technologies > Genetic/Genomic Methods Laboratory Methods and Technologies > Proteomics Methods

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A simplified framework of systems approach. The first step involves defining a system that represents a structure or function of a biological entity, by breaking it down into its components to propose an analytical model (Mx). This is followed by experimental evaluation (Ex) of the model by simultaneous interrogation of the information pathways. Validate the analytical model in the light of experimental results (Δx = ExMx) and redefine the model to maximize the goodness of fit between the experimental results and the model. These steps are repeated until the agreement between them is statistically significant and biologically relevant.

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High resolution AFM images of pores formed by amyloid β1‐42 (a)47 and amyloid β17‐42 (b)96 oligomers in lipid bilayers. Open (c) and close (d) conformations of connexin‐43 hemichannels in the absence and presence of 1.8 mM Ca++, respectively.16 Real time aggregation of VEGF receptors on the surface of a endothelial cell before (e) and after (f) the addition of VEGF‐antibody.29 Real time proteolysis of collagen molecules before (g) and after (h) the addition of collagenase.98 Shear stress induced reorganization of cytoskeleton in living endothelial cells imaged by AFM.99 Under static conditions (i), the cells topography is smooth and under unidirectional shear stress the topography is reorganized (j) to minimize the shear stress gradient to reduce resistance to flow.

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Distribution of human plasma proteins and their concentration. The dynamic range of proteins in the plasma ranges from millimolar (10−3) to yactomolar (10−24) concentrations. It also shows the concentration dependence of the number of protein species. Current mainstream proteomic methods account for few hundreds of proteins only leaving the large majority (∼80%) unaccounted. (Reprinted with permission from Ref 2. Copyright 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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(a) Optical components of an AFM combined with a light fluorescence microscope. The optical focusing of the laser light is integrated within the piezotube which provides the lateral movement of the cantilever. The scanning lens is located inside the lower segment, which provides the vertical movement of the cantilever. After the positions of the lenses are adjusted, the scanning focused spot accurately tracks the cantilever, and the zero deflection signal from the four‐segment photodiode is independent of position within the scan area. The AFM is placed on the stage of an inverted microscope (Reprinted with permission from Ref 48. Copyright 1997 John Wiley & Sons, Inc.; For details see Hansma et al.49). Bottom right Internalization of small liposomes: light microscopy (b), fluorescence (c), and tapping mode AFM (d–e) imaging of fixed cells at different time intervals after incubating with cisplatin‐encapsulated liposomes. Height mode image (d) and error mode image (e) of the same cells. Error mode image shows better contrast and ultrastructural details. The liposomal position with respect to the cell membrane was determined from the height mode images. For cells incubated with cisplatin‐encapsulated liposomes for only 1‐h incubation sample, many large liposomes are seen on the cell surface (red arrows in b–e). On the other hand, small cisplatin‐encapsulated liposomes appear to be internalized in the cell cytoplasm (black arrows in d, e). The inset in (e) shows a zoomed‐in portion of the encircled area: the internalized liposomes are clusters of small liposomes (white arrows) of ∼250 nm diameter. After 16 h of incubation, no defined liposomes were observed Scan size of AFM images is 25 µm (Reprinted with permission from Ref 76. Copyright © 2006 American Chemical Society). (f) Schematics of a combined Scanning Ion Conductance Microscope (SICM) and AFM setup able to obtain simultaneous current and topographical information. A laser beam reflecting off a mirror glued to the back of a pipette with a nanometer provides the deflection signal for the topographic image (Reprinted with permission from Ref 48. Copyright 1997 John Wiley & Sons, Inc.). Intrapipette and bath electrodes measure electrical currents. (g) Simultaneous AFM and conductivity images of a nanopore fabricated in a silicon chip. (Reprinted with permission from Ref 50. Copyright 2007 American Chemical Society)

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(a) Schematic diagram of an AFM with optical detection system of cantilever deflection. Only two quadrants of the photodetector are shown. A feedback system compares the amplified output of the photodetector with a setpoint value chosen by the user. When the difference between both values is different to zero, a voltage is sent to the piezoelectric scanner, which moves the sample to bring the difference value back to zero. The inset shows cantilever deflections as a result of attractive and repulsive forces. (b) The probe plays a fundamental role in the image formation mechanism of scanning probe microscopies. The resulting image is a convolution of the topography of the sample and the geometrical features of the probe being scanned. If the probe has multiple protrusions close to one another, the resulting image will be distorted, displaying multiple features of the same object. The measured width of an object is also broadened because of the size of probe radius (c). However, the measured height is independent of it. The figure shows images of gold nanoparticles with 30 nm diameter. The measured height is close to 30 nm, but the width is larger.

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A simplified representation of the hierarchical nature of biological systems.

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