SS-NEX
HS-AFM
SS-NEX_head110323
PS-NEX_僗僉儍僫僿僢僪170831
PS-NEX_寀岝尠旝嬀晹170823

超高速视频级原子力显微镜—HS-AFM

超高速视频级原子力显微镜—HS-AFM

 

原子力显微镜(Atomic Force Microscope,AFM),一种可用来研究包括绝缘体在内的固体材料表面结构的分析仪器。它通过检测待测样品表面和一个微型力敏感元件之间的极微弱的原子间相互作用力来研究物质的表面结构及性质。将一对微弱力极端敏感的微悬臂一端固定,另一端的微小针尖接近样品,这时它将与其相互作用,作用力将使得微悬臂发生形变或运动状态发生变化。扫描样品时,利用传感器检测这些变化,就可获得作用力分布信息,从而以纳米级分辨率获得表面形貌结构信息及表面粗糙度信息。原子力显微镜可以测量材料物理性质、力学性能、磁学性能、热学性能、电学性能等方面的一些特征信息,但在扫描成像速度上一直存在局限性,太慢的扫描速度导致原子力显微镜无法捕捉到分子间的相互作用过程和一些快速的分子动态变化。

 

超高速视频级原子力显微镜(High-Speed Atomic Force Microscope,HS-AFM)由日本 Kanazawa 大学 Prof. Ando 教授团队研发,日本RIBM公司(生体分子计测研究所株式会社,Research Institute of Biomolecule Metrology Co., Ltd)商业化的产品,可以达到视频级成像的商业化原子力显微镜。HS-AFM突破了传统原子力显微镜“扫描成像速慢”的限制,能够在液体环境下超快速动态成像,分辨率为纳米水平。样品无需特殊固定,不影响生物分子的活性,尤其适用于生物大分子互作动态观测。超高速视频级原子力显微镜HS-AFM主要有两种型号,SS-NEX样品扫描(Sample-Scanning HS-AFM)以及PS-NEX探针扫描(Probe-Scanning HS-AFM)。推出至今,全球已有100多位用户,发表 SCI 文章 300 余篇,包括Science, Nature, Cell 等顶级杂志。

 

相较于目前市场上的原子力显微镜成像设备,HS-AFM突破了 “扫描成像速慢”的限制,扫描速度高可达 20 frame/s,并且有 4 种扫描台可供选择。样品无需特殊固定染色,不影响生物分子的活性,尤其适用于生物大分子互作动态观测。液体环境下直接检测,超快速动态成像,分辨率为纳米水平。探针小,适用于生物样品;悬臂探针共振频率高,弹簧系数小,避免了对生物样品等的损伤。悬臂探针可自动漂移校准,适用于长时间观测。采用动态PID控制,高速扫描时仍可获得清晰的图像。XY轴分辨率2nm;Z轴分辨率0.5nm。

HS-AFM不仅拥有超高扫描速率与原子级别分辨率,而且具有操作的简易性,使得对单分子动态过程的捕捉变得十分方便,为科研工作者研所和理解生物物理、生物化学、分子生物学、病毒学以及生物医学等领域的单分子动态过程提供了一款强大的工具。

全新的HS-AFM采用了新的高频微悬臂架构,更低噪音、更高稳定性的2控制器,高速扫描器,缓冲防震设计,主动阻尼,动态PID,驱动算法优化,多种前沿技术,可以实现在超高速下获取高分辨的生物样品信息。新系统整合了基于工作流程的操作软件,直观的用户界面与流程化、自动化的设置使得研究人员可以专注于实验设计,不需要复杂的操作和条件设置,快速获取数据,加速研究的产出。

 


日本RIBM公司的超高速视频级原子力显微镜HS-AFM的创新点:

★   高频微悬臂

弹性系数:   0.1 N/m   

曲率半径:   <10 nm 

共振频率:   400-600kHz in liquid

★   高速扫描台

20 frames/s.   (Standard scanner)

★   缓冲防震+主动阻尼+动态PID+算法优化

缓冲防震

主动阻尼

动态PID控制:可自动改变反馈增益,保证了HS-AFM在高速扫描条件下仍可获得清晰的图像

探针自动漂移校准,适用于长时间样品观测

 

     

 

日本RIBM公司的超高速视频级原子力显微镜HS-AFM的的应用领域:

 

从单分子到单细胞,都可直接观测

 

 

1、肌动蛋白

 

2、CRISPR-Cas9

 

 

3、膜联蛋白

 

 

4、IgG

 

 

5、活细胞

 

 

6、细菌视紫红质

 

 

7、DNA纳米结构

 

 

8、仿生聚合物

 

 

日本RIBM公司的超高速视频级原子力显微镜HS-AFM的视频案例

 

1:IgG

在溶液中观察到抗体(IgG)。

IgG呈"Y"形,两个Fab区区分清晰。

由于锚定能力较弱,IgG保持其抗原结合能力。

 

 

 

2:Plasmid DNA

传统AFM在没有强锚定的情况下,DNA分子图像出现摆动。

然而,强锚定可能会削弱真实的结构和行为。

HS-AFM能清晰显示质粒的结构和运动,无强锚定。

 

 

 

3:DNA内切酶的消化:DNase I

DNA酶I是一种随机消化DNA的核酸内切酶。视频中的箭头表示DNase I消化DNA的部分。

请参考从DNA末端消化的核酸外切酶Bal31的视频。

 

 

 

4:DNA外切酶消化:Bal31

Bal31是一种从DNA链末端消化DNA的核酸外切酶。

视频显示Bal31的活性沿着DNA移动,并逐渐从DNA链的末端消化。

最后,DNA分子被完全消化,但环状DNA未被消化。高光点是Bal31分子,它们与DNA的不同位置结合。
 

 

 

5:DNA聚合酶的DNA延伸:Phi29

双链DNA(黄色)随着时间的推移而拉长。单链λDNA作为模具固定在基板上。

由于从随机六聚体引物(Red)结合到λDNA模体,phi29聚合酶(Black)以dNTP为底物合成互补DNA。
 

 

 

6:链亲和素2D晶体中的点缺陷

成功地观察到点缺陷在晶体中的扩散。

从图像上看,两个单空位缺陷的轨迹跟踪相对于晶格的两个轴是明显的各向异性的。

 

 

 

 

日本RIBM公司的超高速视频级原子力显微镜HS-AFM的文献列表

 

Title Journal
Biophysical reviews top five: atomic force microscopy in biophysics Biophysical Reviews
Reconstruction of Three-Dimensional Conformations of Bacterial
ClpB from High-Speed Atomic-Force-Microscopy Images
Frontiers in Molecular
Biosciences
A facile combinatorial approach to construct a ratiometric fluorescent
sensor: application for the real-time sensing of cellular pH changes
Chemical Science
DNA Nanotechnology to Disclose Molecular Events at the Nanoscale
and Mesoscale Levels
Springer Nature
Quantitative description of a contractile macromolecular machine Science Advances
Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ
Visualized by High-Speed Atomic Force Microscopy
International Journal of
Molecular Sciences 2021, Vol. 22, Page 1697
Localization atomic force microscopy Nature 2021 594:7863
Movements of mycoplasma mobile gliding machinery detected by
high-speed atomic force microscopy
mBio
An ultra-wide scanner for large-area high-speed atomic force
microscopy with megapixel resolution
Scientific Reports 2021 11:1
A molecularly engineered, broad-spectrum anti-coronavirus lectin
inhibits SARS-CoV-2 and MERS-CoV infection in vivo
Research Square
Influenza virus ribonucleoprotein complex formation occurs in the
nucleolus
bioRxiv
Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible
β-Barrel Structure in Solution and Keeps This Structure in
Dehydration
Journal of Physical Chemistry B
Ultrastructure of influenza virus ribonucleoprotein complexes during
viral RNA synthesis
Communications Biology on
A facile combinatorial approach to construct a ratiometric fluorescent
sensor: application for the real-time sensing of cellular pH changes
Chemical Science
Deformation of microtubules regulates translocation dynamics of
kinesin
Science Advances
Unraveling the host-selective toxic interaction of cassiicolin with lipid
membranes and its cytotoxicity
bioRxiv
Nanostructure and thermoresponsiveness of poly( N -isopropyl
methacrylamide)-based hydrogel microspheres prepared via aqueous free radical precipitation polymerization
RSC Advances
JRAB/MICAL-L2 undergoes liquid–liquid phase separation to form
tubular recycling endosomes
Communications Biology 2021
4:1
Correlation of membrane protein conformational and functional
dynamics
Nature Communications 2021
12:1
Non-close-packed arrangement of soft elastomer microspheres on
solid substrates
RSC Advances
Folding RNA–Protein Complex into Designed Nanostructures Methods in Molecular Biology
A glutamine sensor that directly activates TORC1 Communications Biology 2021
4:1
Architecture of zero-latency ultrafast amplitude detector for high-
speed atomic force microscopy
Applied Physics Letters
Correlative AFM and fluorescence imaging demonstrate nanoscale
membrane remodeling and ring-like and tubular structure formation by septins
Nanoscale
Desiccation-induced fibrous condensation of CAHS protein from an
anhydrobiotic tardigrade
bioRxiv
Construction of ferritin hydrogels utilizing subunit–subunit
interactions
PLOS ONE
Monomeric α-synuclein (αS) inhibits amyloidogenesis of human
prion protein (hPrP) by forming a stable αS-hPrP hetero-dimer.
Prion
Influence of protein adsorption on aggregation in prefilled syringes Journal of Pharmaceutical
Sciences
Dynamic mechanisms of CRISPR interference by Escherichia coli
CRISPR-Cas3
bioRxiv
An RNA Triangle with Six Ribozyme Units Can Promote a Trans-
Splicing Reaction through Trimerization of Unit Ribozyme Dimers
Applied Sciences
Faster high-speed atomic force microscopy for imaging of
biomolecular processes Review of Scientific Instruments ARTICLE scitation.org/journal/rsi Faster high-speed atomic force microscopy for imaging of biomolecular processes
Rev. Sci. Instrum
Title: Identification of lectin receptors for conserved SARS-CoV-2
glycosylation sites
bioRxiv
Structural and dynamics analysis of intrinsically disordered proteins
by high-speed atomic force microscopy
Nature Nanotechnology
Millisecond Conformational Dynamics of Skeletal Myosin II Power
Stroke Studied by High-Speed Atomic Force Microscopy
ACS Nano
High-Speed Atomic Force Microscopy Reveals Spatiotemporal
Dynamics of Histone Protein H2A Involution by DNA Inchworming
The Journal of Physical
Chemistry Letters
Nanostructures, Thermoresponsiveness, and Assembly Mechanism
of Hydrogel Microspheres during Aqueous Free-Radical Precipitation Polymerization
Langmuir
Chained structure of dimeric F 1-like ATPase in Mycoplasma mobile
gliding machinery 4
bioRxiv
Lipid Membrane Interaction of Peptide/DNA Complexes Designed for
Gene Delivery
Langmuir
High-Speed Atomic Force Microscopy to Study Myosin Motility Advances in Experimental
Medicine and Biology
Single-molecule level dynamic observation of disassembly of the
apo-ferritin cage in solution
Physical Chemistry Chemical
Physics
Atomic Force Microscopy of Biomembranes : A Tool for Studying the
Dynamic Behavior of Membrane Proteins
New Techniques for Studying
Biomembranes
Adenosine leakage from perforin-burst extracellular vesicles inhibits
perforin secretion by cytotoxic T-lymphocytes
PLOS ONE
High-Speed Atomic Force Microscopy Reveals the Structural
Dynamics of the Amyloid-β and Amylin Aggregation Pathways
International Journal of
Molecular Sciences 2020, Vol. 21, Page 4287
Molecular mechanism of the recognition of bacterially cleaved
immunoglobulin by the immune regulatory receptor LILRA2
Journal of Biological Chemistry
Biological physics by high-speed atomic force microscopy Philosophical Transactions of
the Royal Society A: Mathematical, Physical and Engineering Sciences
Viral RNA recognition by LGP2 and MDA5, and activation of signaling
through step-by-step conformational changes
Nucleic Acids Research
Key Nucleation Stages and Associated Molecular Determinants and
Processes in pH-Induced Formation of Amyloid Beta Oligomers as Revealed by High-Speed AFM
bioRxiv
Novel Babesia bovis exported proteins that modify properties of
infected red blood cells
PLoS Pathogens
DNA origami demonstrate the unique stimulatory power of single
pMHCs as T-cell antigens
Proceedings of the National
Academy of Sciences
Structural insights into the mechanism of rhodopsin
phosphodiesterase
Nature Communications
Structural insights into the mechanism of rhodopsin
phosphodiesterase
Nature Communications
High-Speed AFM Reveals Molecular Dynamics of Human Influenza A
Hemagglutinin and Its Interaction with Exosomes
Nano Letters
DNA Ring Motif with Flexible Joints Micromachines
Nanopores: a versatile tool to study protein dynamics Essays in Biochemistry
Geometrical Characterization of Glass Nanopipettes with Sub-10 nm
Pore Diameter by Transmission Electron Microscopy
Analytical Chemistry
Carbon nanotube porin diffusion in mixed composition supported
lipid bilayers
Scientific Reports
One-Step Calibration of AFM in Liquid Frontiers in Physics
Nanoscale interaction of RecG with mobile fork DNA Nanoscale Advances
Convergent evolution of processivity in bacterial and fungal
cellulases
Proceedings of the National
Academy of Sciences
Convergent evolution of processivity in bacterial and fungal
cellulases
Proceedings of the National
Academy of Sciences of the United States of America
Interaction of the motor protein SecA and the bacterial protein
translocation channel SecYEG in the absence of ATP
Nanoscale Advances
Nanoreporter of an Enzymatic Suicide Inactivation Pathway Nano Letters
High-speed atomic force microscopy highlights new molecular
mechanism of daptomycin action
Nature Communications
Direct visualization of the conformational change of FUS/TLS upon
binding to promoter-associated non-coding RNA
Chemical Communications
Thermoresponsive Micellar Assembly Constructed from a Hexameric
Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System
Journal of the American
Chemical Society
Schizorhodopsins: A family of rhodopsins from Asgard archaea that
function as light-driven inward H + pumps
Science Advances
Two-State Exchange Dynamics in Membrane-Embedded
Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM
Journal of Molecular Biology
Thermoresponsive structural changes of single poly(N-isopropyl
acrylamide) hydrogel microspheres under densely packed conditions on a solid substrate
Polymer Journal
High-Speed Atomic Force Microscopy Reveals Factors Affecting the
Processivity of Chitinases during Interfacial Enzymatic Hydrolysis of Crystalline Chitin
ACS Catalysis
Rad50 zinc hook functions as a constitutive dimerization module
interchangeable with SMC hinge
Nature Communications
Assembly mechanism of a supramolecular MS-ring complex to
initiate bacterial flagellar biogenesis in vibrio species
Journal of Bacteriology
Structural Dynamics of a Protein Domain Relevant to the Water-
Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy
Journal of Physical Chemistry B
Recent advances in bioimaging with high-speed atomic force
microscopy
Biophysical Reviews
Dynamic behavior of an artificial protein needle contacting a
membrane observed by high-speed atomic force microscopy
Nanoscale
Enhanced enzymatic activity exerted by a packed assembly of a
single type of enzyme
Chemical Science
High Speed AFM and NanoInfrared Spectroscopy Investigation of A
β1–42 Peptide Variants and Their Interaction With
POPC/SM/Chol/GM1 Model Membranes
Frontiers in Molecular
Biosciences
The hierarchical assembly of septins revealed by high-speed AFM Nature Communications
Millisecond dynamics of an unlabeled amino acid transporter Nature Communications
Atg9 is a lipid scramblase that mediates autophagosomal membrane
expansion
Nature Structural & Molecular
Biology
A Simplified Cluster Analysis of Electron Track Structure for
Estimating Complex DNA Damage Yields
International Journal of
Molecular Sciences
Supramolecular tholos-like architecture constituted by archaeal
proteins without functional annotation
Scientific Reports
Studies on the impellers generating force in muscle Biophysical Reviews
Diversity of physical properties of bacterial extracellular membrane
vesicles revealed through atomic force microscopy phase imaging
Nanoscale
High-Speed AFM Reveals Molecular Dynamics of Human Influenza A
Hemagglutinin and Its Interaction with Exosomes
Nano Letters
DNA density-dependent uptake of DNA origami-based two-or three-
dimensional nanostructures by immune cells
Nanoscale
Spatiotemporally tracking of nano-biofilaments inside the nuclear
pore complex core
Biomaterials
High-speed atomic force microscopy directly visualizes
conformational dynamics of the HIV Vif protein in complex with three host proteins
Journal of Biological Chemistry
Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and
Structure Observed by High-Speed Atomic Force Microscopy
ACS Nano
Liquidity Is a Critical Determinant for Selective Autophagy of Protein
Condensates
Molecular Cell
Capturing transient antibody conformations with DNA origami
epitopes
Nature Communications
Biophysics in Kanazawa University Biophysical Reviews
Dynamics of oligomer and amyloid fibril formation by yeast prion
Sup35 observed by high-speed atomic force microscopy
Proceedings of the National
Academy of Sciences
Annexin-V stabilizes membrane defects by inducing lipid phase
transition
Nature Communications
Structure and mechanism of bactericidal mammalian perforin-2, an
ancient agent of innate immunity
Science Advances
Atomic Force Microscopy Visualizes Mobility of Photosynthetic
Proteins in Grana Thylakoid Membranes
Biophysical Journal
Construction of a Hexameric Hemoprotein Sheet and Direct
Observation of Dynamic Processes of Its Formation
Chemistry Letters
Single-molecule imaging analysis reveals the mechanism of a high-
catalytic-activity mutant of chitinase A from Serratia marcescens
Journal of Biological Chemistry
Direct observation and analysis of TET-mediated oxidation
processes in a DNA origami nanochip
Nucleic Acids Research
On-membrane dynamic interplay between anti-GM1 IgG antibodies
and complement component C1q
International Journal of
Molecular Sciences
Zwitterionic Polypeptides: Chemoenzymatic Synthesis and Loosening
Function for Cellulose Crystals
Biomacromolecules