Elucidation and Identification of Double-Tip Effects in Atomic Force Microscopy Studies of Biological Structures
27 Jul 2012-Journal of Surface Engineered Materials and Advanced Technology (Scientific Research Publishing)-Vol. 2012, Iss: 3, pp 238-247
TL;DR: The results can serve as a foundation to design computer-based automatic detection of double-tip AFM images during nanoscale measuring and imaging of biomolecules and even non-biological materials or structures, and then personal experience is not needed any longer to evaluate artifactual images induced by the double- Tip/probe effect.
Abstract: While atomic force microscopy (AFM) has been increasingly applied to life science, artifactual measurements or images can occur during nanoscale analyses of cell components and biomolecules. Tip-sample convolution effect is the most common mechanism responsible for causing artifacts. Some deconvolution-based methods or algorithms have been developed to reconstruct the specimen surface or the tip geometry. Double-tip or double-probe effect can also induce artifactual images by a different mechanism from that of convolution effect. However, an objective method for identifying the double-tip/probe-induced artifactual images is still absent. To fill this important gap, we made use of our expertise of AFM to analyze artifactual double-tip images of cell structures and biomolecules, such as linear DNA, during AFM scanning and imaging. Mathematical models were then generated to elucidate the artifactual double-tip effects and images develop during AFM imaging of cell structures and biomolecules. Based on these models, computational formulas were created to measure and identify potential double-tip AFM images. Such formulas proved to be useful for identification of double-tip images of cell structures and DNA molecules. The present studies provide a useful methodology to evaluate double-tip effects and images. Our results can serve as a foundation to design computer-based automatic detection of double-tip AFM images during nanoscale measuring and imaging of biomolecules and even non-biological materials or structures, and then personal experience is not needed any longer to evaluate artifactual images induced by the double-tip/probe effect.
Citations
More filters
TL;DR: It is found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments, which implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.
Abstract: The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies.
34 citations
Cites methods from "Elucidation and Identification of D..."
...An Agilent AFM series 5500 (Agilent Technologies, Santa Clara, CA, USA) was recruited to image individual whole cells and cell-surface ultrastructures in tapping mode at room temperature with a lateral scan rate of *0.5 Hz....
[...]
...Therefore, we utilized AFM to image the three regions (Fig....
[...]
...Next, we made use of our expertise of atomic force microscopy (AFM) (Chen 2012; Chen et al. 2011; Jin et al. 2011; Jin et al. 2012) to investigate the effects of cell passaging on cell-surface ultrastructures of HUVECs....
[...]
...Atomic force microscopy (AFM) Cells were plated on sterilized coverslips in the wells of a 6-well plate and cultured in a CO2 incubator at 37 C for 24 h....
[...]
...Keywords Cell passaging Cell spreading Cell migration Cell-surface roughness Actin filaments Atomic force microscopy (AFM) Human umbilical vein endothelial cells (HUVECs)...
[...]
TL;DR: This work applies a novel deblurring technique, using a Bayesian framework, to yield a reliable estimation of the real surface topography without any prior knowledge of the tip geometry (blind reconstruction), and focuses specifically on the double-tip effect.
Abstract: The atomic force microscopy (AFM) allows the measurement of interactions at interfaces with nanoscale resolution. Imperfections in the shape of the tip often lead to the presence of imaging artifacts, such as the blurring and repetition of objects within images. In general, these artifacts can only be avoided by discarding data and replacing the probe. Under certain circumstances (e.g., rare, high-value samples, or extensive chemical/physical tip modification), such an approach is not feasible. Here, we apply a novel deblurring technique, using a Bayesian framework, to yield a reliable estimation of the real surface topography without any prior knowledge of the tip geometry (blind reconstruction). A key contribution is to leverage the significant recently successful body of work in natural image deblurring to solve this problem. We focus specifically on the double-tip effect, where two asperities1 are present on the tip, each contributing to the image formation mechanism. Finally, we demonstrate that the proposed technique successfully removes the double-tip effect from high-resolution AFM images, which demonstrate this artifact while preserving feature resolution.1
An asperity is a localized sharp peak in the surface of an object.
7 citations
TL;DR: Differentiable blind tip reconstruction (DTR) as mentioned in this paper is an alternative to the BTR algorithm that estimates tip shape only from high-speed atomic force microscopy images using mathematical morphology operators.
Abstract: Abstract Observing the structural dynamics of biomolecules is vital to deepening our understanding of biomolecular functions. High-speed (HS) atomic force microscopy (AFM) is a powerful method to measure biomolecular behavior at near physiological conditions. In the AFM, measured image profiles on a molecular surface are distorted by the tip shape through the interactions between the tip and molecule. Once the tip shape is known, AFM images can be approximately deconvolved to reconstruct the surface geometry of the sample molecule. Thus, knowing the correct tip shape is an important issue in the AFM image analysis. The blind tip reconstruction (BTR) method developed by Villarrubia (J Res Natl Inst Stand Technol 102:425, 1997) is an algorithm that estimates tip shape only from AFM images using mathematical morphology operators. While the BTR works perfectly for noise-free AFM images, the algorithm is susceptible to noise. To overcome this issue, we here propose an alternative BTR method, called end-to-end differentiable BTR, based on a modern machine learning approach. In the method, we introduce a loss function including a regularization term to prevent overfitting to noise, and the tip shape is optimized with automatic differentiation and backpropagations developed in deep learning frameworks. Using noisy pseudo-AFM images of myosin V motor domain as test cases, we show that our end-to-end differentiable BTR is robust against noise in AFM images. The method can also detect a double-tip shape and deconvolve doubled molecular images. Finally, application to real HS-AFM data of myosin V walking on an actin filament shows that the method can reconstruct the accurate surface geometry of actomyosin consistent with the structural model. Our method serves as a general post-processing for reconstructing hidden molecular surfaces from any AFM images. Codes are available at https://github.com/matsunagalab/differentiable_BTR .
5 citations
TL;DR: In this paper , the authors explain the principle of high-speed atomic force microscopy (HS-AFM) and describe how the resolution is determined, and discuss recent attempts to improve the resolution of HS-AFMs to further extend the observable range of biological phenomena.
Abstract: Abstract High-speed atomic force microscopy (HS-AFM) is a unique approach that allows direct real-time visualization of biological macromolecules in action under near-physiological conditions, without any chemical labeling. Typically, the temporal resolution is sub-100 ms, and the spatial resolution is 2–3 nm in the lateral direction and ∼0.1 nm in the vertical direction. A wide range of biomolecular systems and their dynamic processes have been studied by HS-AFM, providing deep mechanistic insights into how biomolecules function. However, the level of mechanistic detail gleaned from an HS-AFM experiment critically depends on the spatiotemporal resolution of the system. In this review article, we explain the principle of HS-AFM and describe how the resolution is determined. We also discuss recent attempts to improve the resolution of HS-AFM to further extend the observable range of biological phenomena.
1 citations
01 Jan 2017
1 citations
Cites background from "Elucidation and Identification of D..."
...These separate tip protrusions result in duplicates of the same feature: true image and the “ghost” image.(167,158) Double tips typically come from a damaged AFM tip that often results in the formation of additional spikes by dislodging particles during manufacturing, tip use, and through the attachment of surface debris, that often occurs while imaging....
[...]
References
More filters
TL;DR: Lee et al. as mentioned in this paper used a vibrating tip with a heterodyne-interferometer sensor similar to that introduced by Martin, Williams, and Wickramasinghe.
Abstract: This article describes the extension of atomic force microprobe (AFM) technology to two dimensions (2D) for accurate measurement of submicron critical dimensions (CDs). The system utilizes a vibrating tip with heterodyne‐interferometer sensor similar to that introduced by Martin, Williams, and Wickramasinghe. [Y. Martin, C. C. Williams, and H. K. Wickramasinghe, J. Appl. Phys. 61, 4723 (1987)]. However, the tip vibrates in 2D with dual heterodyne detection. The sample is moved relative to the tip by means of coarse‐ and fine‐motion stages whose position is monitored with 3D interferometry. The system does not scan the sample, but operates like a nanorobot sensing the approach of the tip to the surface by means of the vibration damping. A special three‐point tip has been fabricated by Lee [K. L. Lee, D. W. Abraham, F. Serord, and L. Landstein, Proceedings of the 35th ISEIPB Conference, Seattle, WA, May 28–31, 1991 (unpublished), paper K1] with 0.1 μm shank diameter which allows measurement of submicrometer...
43 citations
"Elucidation and Identification of D..." refers background in this paper
...Artifactual or “ghost” images due to errors in STM and AFM probing have been described by some investigators since the invention of scanning probe techniques [22-25]....
[...]
TL;DR: By successively investigating erythrocytes exposed in air on mica for 5 days using tapping mode atomic force microscopy (TM-AFM), the efficacy of AFM is demonstrated as a potentially powerful analytical tool in forensic science.
38 citations
"Elucidation and Identification of D..." refers background in this paper
...One of the important research endeavors is to understand nanoscale structures and life events through the nano-measuring and imaging of cells [2-6] or thin sections of them [7], cellular organelles [8], proteins [9-13], polysaccharide [14], DNA [15] and others using the instruments such as scanning tunneling microscopy (STM), near field scanning optical microscope (NSOM) [16-18] and atomic force microscope (AFM) [19-21]....
[...]
TL;DR: In this paper, the results of atomic force microscopy (AFM) on carbon fibers from polyacrylonitrile and pitch are presented in comparison with Scanning Electron Microscopy (SEM) and Scanning Tunneling MicroScopy (STM) images.
Abstract: Results of Atomic Force Microscopy (AFM) on carbon fibers from polyacrylonitrile and pitch are presented in comparison with Scanning Electron Microscopy (SEM) and Scanning Tunneling Microscopy (STM) images Single fiber surfaces and their crosssections have been imaged on scales from microns to nanometers Morphological details beyond the resolution of SEM were revealed by AFM and STM Grain-type structure was verified on surface of numerous nanofibrils orlented along the main fiber direction Grains are bigger on pitch-based fibers generally, and on fibers of both types after treatment at higher temperatures In the atomic scale AFM images traces of graphitic structure were recorded AFM artefacts on rough surfaces are demonstrated ac19920414
32 citations
"Elucidation and Identification of D..." refers background in this paper
...Such artifactual images appear to be associated with steep corrugations or sharp structural features on a sample surface [26], and with the occurrence of an “extra-tip” on the scanning tipsample interface (double-tip or multiple-tip effect and tip asymmetry)....
[...]
TL;DR: The results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures.
31 citations
"Elucidation and Identification of D..." refers background in this paper
...One of the important research endeavors is to understand nanoscale structures and life events through the nano-measuring and imaging of cells [2-6] or thin sections of them [7], cellular organelles [8], proteins [9-13], polysaccharide [14], DNA [15] and others using the instruments such as scanning tunneling microscopy (STM), near field scanning optical microscope (NSOM) [16-18] and atomic force microscope (AFM) [19-21]....
[...]
TL;DR: The cold-induced re-distribution of lipid raft markers under a nearly-natural condition provide clues for their alternations, and help to propose a model in which raft lipids associate themselves or interact with protein components to generate functional membrane heterogeneity in response to stimulus.
Abstract: Whether and how cold causes changes in cell-membrane or lipid rafts remain poorly characterized. Using the NSOM/QD and confocal imaging systems, we found that cold caused microscale redistribution of lipid raft markers, GM1 for lipid and CD59 for protein, from the peripheral part of microdomains to the central part on Jurkat T cells, and that cold also induced the nanoscale size-enlargement (1/3- to 2/3-fold) of the nanoclusters of lipid raft markers and even the colocalization of GM1 and CD59 nanoclusters. These findings indicate cold-induced lateral rearrangement/coalescence of raft-related membrane heterogeneity. The cold-induced re-distribution of lipid raft markers under a nearly-natural condition provide clues for their alternations, and help to propose a model in which raft lipids associate themselves or interact with protein components to generate functional membrane heterogeneity in response to stimulus. The data also underscore the possible cold-induced artifacts in early-described cold-related experiments and the detergent-resistance-based analyses of lipid rafts at 4 degrees C, and provide a biophysical explanation for recently-reported cold-induced activation of signaling pathways in T cells. Importantly, our fluorescence-topographic NSOM imaging demonstrated that GM1/CD59 raft markers distributed and re-distributed at mounds but not depressions of T-cell membrane fluctuations. Such mound-top distribution of lipid raft markers or lipid rafts provides spatial advantage for lipid rafts or contact molecules interacting readily with neighboring cells or free molecules.
25 citations
"Elucidation and Identification of D..." refers background in this paper
...One of the important research endeavors is to understand nanoscale structures and life events through the nano-measuring and imaging of cells [2-6] or thin sections of them [7], cellular organelles [8], proteins [9-13], polysaccharide [14], DNA [15] and others using the instruments such as scanning tunneling microscopy (STM), near field scanning optical microscope (NSOM) [16-18] and atomic force microscope (AFM) [19-21]....
[...]