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An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution
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 molecula...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217563/ https://www.ncbi.nlm.nih.gov/pubmed/34155261 http://dx.doi.org/10.1038/s41598-021-92365-y |
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author | Marchesi, Arin Umeda, Kenichi Komekawa, Takumi Matsubara, Takeru Flechsig, Holger Ando, Toshio Watanabe, Shinji Kodera, Noriyuki Franz, Clemens M. |
author_facet | Marchesi, Arin Umeda, Kenichi Komekawa, Takumi Matsubara, Takeru Flechsig, Holger Ando, Toshio Watanabe, Shinji Kodera, Noriyuki Franz, Clemens M. |
author_sort | Marchesi, Arin |
collection | PubMed |
description | 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 µm(2)) 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. |
format | Online Article Text |
id | pubmed-8217563 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-82175632021-06-22 An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution Marchesi, Arin Umeda, Kenichi Komekawa, Takumi Matsubara, Takeru Flechsig, Holger Ando, Toshio Watanabe, Shinji Kodera, Noriyuki Franz, Clemens M. Sci Rep Article 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 µm(2)) 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. Nature Publishing Group UK 2021-06-21 /pmc/articles/PMC8217563/ /pubmed/34155261 http://dx.doi.org/10.1038/s41598-021-92365-y Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Marchesi, Arin Umeda, Kenichi Komekawa, Takumi Matsubara, Takeru Flechsig, Holger Ando, Toshio Watanabe, Shinji Kodera, Noriyuki Franz, Clemens M. An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title | An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title_full | An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title_fullStr | An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title_full_unstemmed | An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title_short | An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
title_sort | ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217563/ https://www.ncbi.nlm.nih.gov/pubmed/34155261 http://dx.doi.org/10.1038/s41598-021-92365-y |
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