An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution

Abstract 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...

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Autores principales:	Arin Marchesi, Kenichi Umeda, Takumi Komekawa, Takeru Matsubara, Holger Flechsig, Toshio Ando, Shinji Watanabe, Noriyuki Kodera, Clemens M. Franz
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/93d7225a0676483f9988c6a963df01f9
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spelling	oai:doaj.org-article:93d7225a0676483f9988c6a963df01f92021-12-02T18:02:54ZAn ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution10.1038/s41598-021-92365-y2045-2322https://doaj.org/article/93d7225a0676483f9988c6a963df01f92021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92365-yhttps://doaj.org/toc/2045-2322Abstract 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.Arin MarchesiKenichi UmedaTakumi KomekawaTakeru MatsubaraHolger FlechsigToshio AndoShinji WatanabeNoriyuki KoderaClemens M. FranzNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Arin Marchesi Kenichi Umeda Takumi Komekawa Takeru Matsubara Holger Flechsig Toshio Ando Shinji Watanabe Noriyuki Kodera Clemens M. Franz An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution
description	Abstract 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.
format	article
author	Arin Marchesi Kenichi Umeda Takumi Komekawa Takeru Matsubara Holger Flechsig Toshio Ando Shinji Watanabe Noriyuki Kodera Clemens M. Franz
author_facet	Arin Marchesi Kenichi Umeda Takumi Komekawa Takeru Matsubara Holger Flechsig Toshio Ando Shinji Watanabe Noriyuki Kodera Clemens M. Franz
author_sort	Arin Marchesi
title	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_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_sort	ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/93d7225a0676483f9988c6a963df01f9
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An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution

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