Faster high-speed atomic force microscopy for imaging of biomolecular processes

Abstract

High-speed atomic force microscopy (HS-AFM) has enabled observing protein molecules during their functional activity at rates of 1–12.5 frames per second (fps), depending on the imaging conditions, sample height, and fragility. To meet the increasing demand for the great expansion of observable dynamic molecular processes, faster HS-AFM with less disturbance is imperatively needed. However, even a 50% improvement in the speed performance imposes tremendous challenges, as the optimization of major rate-limiting components for their fast response is nearly matured. This paper proposes an alternative method that can lower the feedback control error and thereby enhance the imaging rate. This method can be implemented in any HS-AFM system by minor modifications of the software and hardware. The resulting faster and less-disturbing imaging capabilities are demonstrated by the imaging of relatively fragile actin filaments and microtubules near the video rate, and of actin polymerization that occurs through weak intermolecular interactions, at ∼8 fps.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy

“…HS-AFM possesses the nanometer resolution suitable for visualizing single molecules and sufficient temporal resolution for meaningful analyses of dynamics in biological processes without molecular labeling − ,,,,, (i.e., scanning speed up to 20 ms per frame). In previous investigations, HS-AFM enabled the real-time visualization of the DNA cleavage mediated by SpCas9 .…”

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

“…A related raster scan profile that is used in some laboratory-built AFM devices is the revolving back and forth raster pattern in which, after completion of one positive scan profile, the tip is translated back to the beginning of the image frame [ 36 ]. Another type of related scan profile is the uniformly same direction pattern recently introduced by Fukuda and Ando [ 9 ] in which both the line and vertical scanning both occur in the same direction throughout the scan. This latter approach has been shown to have the added benefits of simplifying feedback control of the HS-AFM tip and also greatly maintain sample integrity in biological HS-AFM experiments.…”

“…Although this question has been extensively considered in the field of single particle tracking conducted using optical microscope procedures [ 3 – 5 ], the same type of problem has been less-well investigated in atomic force microscopy (AFM) measurements [ 6 – 8 ]. As continual scientific and technological developments produce ever faster AFM scan rates [ 9 , 10 ] the potential use of AFM devices to track the motion of slowly moving diffusible components in the cell membrane, such as protein receptors and membrane channels [ 11 ], is becoming tantalizingly extant. In preparation for this potentially game-changing technological improvement in the high-speed atomic force microscopy (HS-AFM) technique, here we focus on the question of what biological and instrumental factors would affect the feature assignment of mobile cell membrane components observed using HS-AFM [ 12 , 13 ] in a manner that will lead to improved experimental design.…”

Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

“…The actin filaments at different Rng2CHD binding ratios were gently immobilized onto this lipid bilayer during sample approaching (∼5–7 min), before the HS-AFM imaging. An HS-AFM imaging process was performed as described in detail elsewhere ( Ando et al, 2013 ), except for an additional use of a recently developed OTI mode ( Fukuda & Ando, 2021 ). Half-helical pitches (HHPs) of actin filaments were analyzed by measuring the distance between the crossover points of two single-actin protofilaments along the filaments using the home-built software (UMEX Viewer for Drift Analysis), which allowed us to semi-automatically determine and measure the distance between highest points of two neighboring actin protomers (e.g., HHPs) by making a topographical line profile along actin filaments ( Fig S4 ).…”