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 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.

Practical considerations for feature assignment in high-speed AFM of live cell membranes

“…In the simulations that follow, the scanned image area containing the mobile component is subjected to raster scanning at either a slow, medium or fast scanning speed such that t SCAN ∈ (1,10,100) s. Interpreted through the lens of Eqn. 5 this implies that for a square image scan area of L=10 μm, and a typical AFM device setup with a tip size ϕ ∈ (10, 100) nm, the required AFM cantilever tip oscillation frequencies and tip lateral scan velocities will be in the respective ranges, f ∈ (100, 1×10 6 ) and υ x ∈ (0.01, 10) mm/s—values which are compatible with current state of the art HS-AFM devices [ 36 , 64 ]. In a corresponding fashion we bracket a range of diffusion constants relevant to the motion of lipids, proteins and protein complexes within a typical phospholipid cell membrane, sampling from D q ∈ [0.01,0.1,1] μm 2 s –1 .…”

“…Non-penetrative measurements of a cell’s behavior principally involve the high spatial density recording of relatively soft interactions between the AFM tip and the outer cell membrane [ 28 , 29 ]. However, due to its non-equilibrium nature the cell membrane is a dynamic structure that changes over time [ 30 – 36 ]. In terms of how this change occurs, we make a distinction between (i) relatively large-scale (~100nm) gross morphological changes in membrane shape that occur slowly with a time constant, τ 1 (~minutes) [e.g.…”

Simulation atomic force microscopy for atomic reconstruction of biomolecular structures from resolution-limited experimental images

“…Application of simulation AFM can reach much beyond just the interpretation of what is observed in an AFM image. We have previously shown that new structural models with atomistic resolution can be retrieved for higher oligomeric arrangements of the Annexin V protein seen under HS-AFM [ 20 ]. Simulation AFM was recently applied in a demonstration study of a membrane transporter protein, showing that based on topographic imaging of only one membrane side, the functionally coupled conformational state at the remote opposite site can be predicted [ 22 ].…”

“…The simulated scanning method computationally emulates scanning of the molecular structure to produce pseudo AFM images. It has been employed in several studies to interpret experimental AFM images of proteins [8,[17][18][19][20][21][22], and in molecular simulations of flexible fitting biomolecular structures to experimental images [23] or to deduce information on the AFM tip shape [24].…”

Atomic reconstruction of biomolecular structures from AFM images and quantitative validation of experimental data using simulated AFM scanning

“…15 The simulated scanning method computationally emulates scanning of the molecular structure to produce pseudo AFM images. It has been employed in several studies to interpret experimental AFM images of proteins, 8,16,17 and in molecular simulations to perform flexible fitting of biomolecular structures to experimental images 18 or to deduce information on the AFM tip shape. 19 The strengths of the stand-alone BioAFMviewer software are its user-friendly versatile interface and rich functionality.…”