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Journal Article•DOI•

Elucidation and Identification of Double-Tip Effects in Atomic Force Microscopy Studies of Biological Structures

Chen Yong
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.

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Citations
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Journal Article•DOI•
Effects of long-term serial cell passaging on cell spreading, migration, and cell-surface ultrastructures of cultured vascular endothelial cells

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Huanhuan Liao1, Hui He1, Yuan Chen1, Fangfa Zeng1, Jie Huang1, Li Wu1, Yong Chen1 •
Nanchang University1
01 Mar 2014-Cytotechnology
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....

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  • ...Therefore, we utilized AFM to image the three regions (Fig....

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  • ...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....

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  • ...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....

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  • ...Keywords Cell passaging Cell spreading Cell migration Cell-surface roughness Actin filaments Atomic force microscopy (AFM) Human umbilical vein endothelial cells (HUVECs)...

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Journal Article•DOI•
Double-Tip Artifact Removal From Atomic Force Microscopy Images

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Yun-feng Wang1, Jason I. Kilpatrick2, Suzanne P. Jarvis2, Francis M. Boland1, Anil Kokaram1, David Corrigan1 •
Trinity College, Dublin1, University College Dublin2
01 Jun 2016-IEEE Transactions on Image Processing
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

Journal Article•DOI•
End-to-end differentiable blind tip reconstruction for noisy atomic force microscopy images

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Yasuhiro Matsunaga, Sotaro Fuchigami, Tomonori Ogane, Shoji Takada
26 Sep 2022-Dental science reports
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

Journal Article•DOI•
Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action

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K. Umeda, Steven J. McArthur, Noriyuki Kodera
06 Feb 2023-Microscopy
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

Self-Assembly Mechanisms of Organosilanes and Porphyrins Investigated with Scanning Probe Microscopy

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Chambers, Phillip Charles
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....

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References
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Journal Article•DOI•
NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal Vγ2Vδ2 T-cell expansion

[...]

Yong Chen1, Lingyun Shao1, Lingyun Shao2, Zahida Ali1, Jiye Cai1, Jiye Cai3, Zheng W. Chen1 •
University of Illinois at Chicago1, Fudan University2, Jinan University3
15 Apr 2008-Blood
TL;DR: Interestingly, expanded Vgamma2Vdelta2 T cells bearing TCR nanoclusters or nanodomains were able to rerecognize phosphoantigen and to exert better effector function as nanoscale insight into the in vivo T-cell immune response is provided.

70 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]....

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Journal Article•DOI•
Oriented, Active Escherichia coli RNA Polymerase: An Atomic Force Microscope Study

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Neil H. Thomson1, Bettye L. Smith2, Bettye L. Smith1, Nils Almqvist1, Lutz Schmitt3, Mikhail Kashlev, Eric T. Kool1, Paul K. Hansma •
University of Rochester1, University of California, Santa Barbara2, Stanford University3
01 Feb 1999-Biophysical Journal
TL;DR: Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols, which resists protein adsorption and phase-segregate.

65 citations

Journal Article•DOI•
Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

[...]

Yong Chen1, Jie Qin1, Zheng W. Chen1•
University of Illinois at Chicago1
01 Oct 2008-Journal of Lipid Research
TL;DR: This study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations on the apical membrane of polarized cells.

54 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]....

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Journal Article•DOI•
Atomic force bio-analytics of polymerization and aggregation of phycoerythrin-conjugated immunoglobulin G molecules

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Yong Chen1, Jiye Cai1, Qingcai Xu1, Zheng W. Chen2, Zheng W. Chen3 •
Jinan University1, University of Illinois at Chicago2, Harvard University3
01 Nov 2004-Molecular Immunology
TL;DR: Findings may help to understand the nano-structures of bio-labeled IgG or antibodies, and facilitate the potential use of PE-conjugated antibodies as markers or immunosensers for AFM bio-analytics of biomolecules in cells and membranes.

51 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]....

    [...]

Journal Article•DOI•
The application of atomic force microscopy to the study of living vertebrate cells in culture

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James A. Dvorak1•
National Institutes of Health1
01 Jan 2003-Methods
TL;DR: This review was written to encourage researchers in the biological and biomedical sciences to consider AFM as a potential (and potent) tool for their cell biological research.

44 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]....

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