An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution.
An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution.
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DOI：
10.1038/s41598-021-92365-y
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发表时间：
2021-06-21
影响因子：
4.6
通讯作者：
Franz CM
中科院分区：
文献类型：
--
作者：
Marchesi A;Umeda K;Komekawa T;Matsubara T;Flechsig H;Ando T;Watanabe S;Kodera N;Franz CM
高速原子力显微镜（HS-AFM）是一种强大的工具，用于可视化单个生物分子的动力学。然而，在单分子HS-AFM成像应用中，x，y扫描仪的范围通常被限制在几百纳米，防止更大的分子组装，如二维蛋白质晶体生长或纤维状聚集的概览观察。使用压电驱动的x，y定位系统的机械放大的扫描仪设计的先前进展已经将HS-AFM图像帧的尺寸扩展到几十微米，但是这些大型扫描仪在高扫描速度下可能遭受机械不稳定性，并且仅记录具有有限像素数和相对低的横向分辨率（> 20-100 nm/像素）的图像，使单分子分析复杂化。因此，AFM系统能够在高速和纳米分辨率的图像大样本区域仍然是失踪。在这里，我们描述了一个HS-AFM样品扫描仪系统，能够记录包含高达1600万像素的大型地形图像（≤ 36 × 36 µm2），在整个图像帧中提供分子分辨率。尽管尺寸很大，但这款基于柔性的扫描仪具有高谐振频率（> 2 kHz），即使在高达7.2 mm/s的高扫描速度下也能稳定运行，从而最大限度地减少记录百万像素扫描所需的时间。我们还证明，在延时模式下操作这种高速扫描仪可以同时识别自发的二维膜联蛋白A5晶体生长的区域，解决大结晶域的角取向，甚至检测罕见的晶格缺陷，所有这些都不改变扫描帧大小或分辨率。首先从概览扫描中识别的动态过程可以在切换到常规HS-AFM扫描后以增加的帧速率在减少的扫描区域中进一步成像。在生物相关的时间范围内收集复杂样品的大面积，高分辨率图像的能力将HS-AFM的能力从单分子成像扩展到大型动态分子阵列的研究。此外，大面积HS-AFM扫描可以从单次扫描中生成详细的结构数据集，有助于对结构异质性样品（包括细胞表面）进行定量分析。
High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20–100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm2) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.
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影响因子：
13.3
作者：
Cisneros, David A.;Friedrichs, Jens;Muller, Daniel J.
通讯作者：
Muller, Daniel J.
BioAfmViewer：用于模拟生物分子结构和动力学的AFM扫描的交互式界面。
影响因子：
4.3
作者：
Amyot R;Flechsig H
通讯作者：
Flechsig H
影响因子：
4.6
作者：
Shibata M;Uchihashi T;Ando T;Yasuda R
通讯作者：
Yasuda R
DOI：
10.1073/pnas.211400898
发表时间：
2001-10-23
影响因子：
11.1
作者：
Ando, T;Kodera, N;Toda, A
通讯作者：
Toda, A
影响因子：
3
作者：
Reviakine, I;Bergsma-Schutter, W;Brisson, A
通讯作者：
Brisson, A