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2022 | Book

High-Speed Atomic Force Microscopy in Biology

Directly Watching Dynamics of Biomolecules in Action

Author: Prof. Dr. Toshio Ando

Publisher: Springer Berlin Heidelberg

Book Series : NanoScience and Technology

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About this book

This first book on high-speed atomic force microscopy (HS-AFM) is intended for students and biologists who want to use HS-AFM in their research. It provides straightforward explanations of the principle and techniques of AFM and HS-AFM. Numerous examples of HS-AFM studies on proteins demonstrate how to apply this new form of microscopy to specific biological problems. Several precautions for successful imaging and the preparation of cantilever tips and substrate surfaces will greatly benefit first-time users of HS-AFM. In turn, the instrumentation techniques detailed in Chapter 4 can be skipped, but will be useful for engineers and scientists who want to develop the next generation of high-speed scanning probe microscopes for biology.
The book is intended to facilitate the first-time use of this new technique, and to inspire students and researchers to tackle their own specific biological problems by directly observing dynamic events occurring in the nanoscopic world. Microscopy in biology has recently entered a new era with the advent of high-speed atomic force microscopy (HS-AFM). Unlike optical microscopy, electron microscopy, and conventional slow AFM, it allows us to directly observe biological molecules in physiological environments. Molecular “movies” created using HS-AFM can directly reveal how molecules behave and operate, without the need for subsequent complex analyses and roundabout interpretations. It also allows us to directly monitor morphological change in live cells, and dynamic molecular events occurring on the surfaces of living bacteria and intracellular organelles. As HS-AFM instruments were recently commercialized, in the near future HS-AFM is expected to become a common tool in biology, and will enhance and accelerate our understanding of biological phenomena.

Table of Contents

Frontmatter

Principle and Techniques of HS-AFM

Frontmatter
Chapter 1. Principle of AFM
Abstract
AFM is a member of the superfamily of scanning probe microscopy (SPM) techniques. SPM detects changes in a certain physical quantity (e.g., electric current, electric capacitance, light intensity, magnetic force or mechanical force).
Toshio Ando
Chapter 2. Cantilever Mechanics
Abstract
In Sect. 1.​3, some mechanical properties of a cantilever beam are briefly described to explain AFM imaging modes. Since AFM relies on the mechanics of a cantilever beam as a sensor, this chapter focuses on its fundamental mechanics.
Toshio Ando
Chapter 3. Feedback Control and Imaging Rate
Abstract
Achieving feedback control to maintain the tip–sample contact strength is essential in the AFM imaging of biological samples. However, feedback control with a chasing-after nature cannot be carried out in real time; there is always a time delay and hence a control error. Faster imaging requires faster feedback control with a shorter time delay.
Toshio Ando
Chapter 4. HS-AFM System and Optimized Instrumental Components
Abstract
This chapter describes in detail almost all instrumental components and new techniques developed to establish HS-AFM, including small cantilevers, the OBD detector, the amplitude detector, the phase detector, scanners, the feedback controller, the drift compensator, and vibration damping methods.
Toshio Ando
Chapter 5. Tip-Scanning HS-AFM
Abstract
Sample stage-scanning (stationary tip) AFM restricts the sample mounting area to the Z-scanner, which excludes large samples from AFM observations. This restriction is more rigorous in HS-AFM. In contrast, tip-scanning (stationary sample) AFM has no such restriction. There is an additional advantage: a tip-scanning AFM system can be easily combined with other systems such as optical microscopes.
Toshio Ando
Chapter 6. Interactive HS-AFM (iHS-AFM)
Abstract
The disruption of the higher-order structure of protein assemblies and the disturbance of protein–protein interactions with the AFM tip can provide information on these objects that cannot be attained by imaging alone. Sample manipulation techniques have been introduced to conventional AFM systems, ranging from a simple system to a sophisticated system containing a haptic device. Thus far, only simple techniques have been incorporated into HS-AFM that can be installed by software modifications of the operating system. Although interactive HS-AFM (iHS-AFM) has been used only in a few cases, interesting results have been provided.
Toshio Ando
Chapter 7. Influence of Tip–Sample Interactions on Specimens
Abstract
HS-AFM imaging is often performed to visualize dynamic processes of protein molecules during their functional activity. Therefore, we must be careful of the possible influences of tip–sample contact on the structure and the functional activity of the protein under study. Although the degree of this influence depends on the sample fragility, it is always good to know how large influences can occur quantitatively, depending on imaging conditions.
Toshio Ando
Chapter 8. Toward the Next Generation of HS-AFM
Abstract
Further advanced HS-AFM systems are currently under development, which will be established and followed by more technical developments over the next 5–10 years. These efforts will further improve both the basic performances and functionality of HS-AFM. It is also plausible that other types of HS-SPM for biological studies will emerge. In fact, high-speed scanning ion conductance microscopy (HS-SICM) has already been developed (Leitao et al. in ACS Nano 15:17613–17622, 2021; Watanabe et al. in Rev. Sci. Instrum. 90:123704, 2019;), although its spatiotemporal resolution is still far lower than that of HS-AFM. This chapter describes very recent progress in the basic performances of HS-AFM (i.e., higher imaging rate and lower disturbance), followed by future prospects of HS-AFM performances.
Toshio Ando

Biological Applications of HS-AFM

Frontmatter
Chapter 9. Overview of Bioimaging with HS-AFM
Abstract
This chapter gives an overview of bioimaging studies with HS-AFM performed in recent years, efficiently illustrating how this new field has evolved and what biomolecular phenomena have been preferentially studied with HS-AFM. One can notice that many interesting biomolecular systems and phenomena remain to be examined with HS-AFM. Moreover, this chapter summarizes several issues to be considered in HS-AFM imaging; what limitations exist and what we should bear in mind for successful studies with HS-AFM.
Toshio Ando
Chapter 10. Substrate Surfaces
Abstract
The substrate surfaces onto which samples are placed play an essential role in successful dynamic imaging by HS-AFM. In contrast to single-molecule fluorescence microscopy, for HS-AFM, the surface roughness should be small enough to identify molecules of interest, which typically have single-nanometer dimensions. Moreover, because AFM can visualize the sample only from one direction perpendicular to the substrate surface, sample molecules sometimes have to be attached to the surface in a specific orientation so that their characteristic structural features and portions of interest of the molecules can be visualized. These requirements are common in both static and dynamic AFM imaging. However, in dynamic AFM imaging, sample molecules should not strongly interact with a surface, which could interfere with their function. Nevertheless, when the interaction is too weak, the molecules move too fast on the surface to be clearly imaged even with HS-AFM.
Toshio Ando
Chapter 11. Canonical Motor Proteins
Abstract
Motor proteins, not only actin- and microtubule-based motors but also many other mechanoenzymes, are the most fascinating targets for HS-AFM imaging because their structure (topography), dynamics and function can be directly and simultaneously displayed in filmed images. This makes a striking contrast to, for example, active transporters that pump or transport ions and small molecules across the membrane; these transport activities hardly appear on HS-AFM images. Nevertheless, few motor proteins have yet been imaged with HS-AFM. This chapter focuses on HS-AFM studies on canonical motor proteins, myosin V and F1-ATPase. HS-AFM studies on noncanonical motor proteins are described in other chapters.
Toshio Ando
Chapter 12. Membrane-Remodeling Proteins
Abstract
Many cellular processes depend on membrane shape changes. Cytokinesis, exocytosis and endocytosis, phagocytosis, T-tubule formation in muscle, crista formation in mitochondria, fission and fusion of organelles, and fission and fusion of vesicular compartments as transport carriers all proceed via membrane rearrangements. Their initial process proceeds from membrane bulging and budding either toward or away from the cytoplasm.
Toshio Ando
Chapter 13. Intrinsically Disordered Proteins (IDPs)
Abstract
IDPs are partially or entirely disordered. Their intrinsically disordered regions (IDRs) are highly flexible and dynamic, and therefore, in IDRs, the binding residues for targets and even residues responsible for weak intramolecular interactions are widely distributed in space and time.
Toshio Ando
Chapter 14. Self-assembly
Abstract
Self-assembly is ubiquitous in biological systems and underlies the formation of a wide variety of ordered biological architectures. Although molecular self-assembly is an excellent strategy for the automated and energy-efficient construction of large ordered structures in most cases, it sometimes works incorrectly to produce substances detrimental to health, exemplified by amyloid/prion-related aggregates that self-replicate to grow by interacting with native soluble proteins.
Toshio Ando
Chapter 15. Structural Changes of Membrane Proteins
Abstract
Transmembrane proteins (here referred simply to as membrane proteins) are exposed to different environments on the two sides of the membrane. Membrane proteins function mainly for communication between the two sides of the membrane, for example, as receptors, ion channels, and transporters.
Toshio Ando
Chapter 16. Peripheral Membrane Proteins (PMPs)
Abstract
PMPs play important roles in various biological processes, including cell signaling, membrane trafficking, metabolism of membrane components, transport of hydrophobic molecules, and the formation of membrane pores.
Toshio Ando
Chapter 17. Molecular Chaperones
Abstract
After being synthesized on the ribosome, peptide chains must fold into unique 3D structures to become functional, except for IDPs lacking folded domains. The amino acid sequence determines the folded structure. However, during the folding process, polypeptide chains often misfold either spontaneously or under cellular stress, resulting in the formation of nonfunctional proteins and aggregates. To avoid this adverse result, life has evolved molecular chaperones that promote proper folding of nascent polypeptides by facilitating folding and preventing misfolding and aggreagation. However, properly folded proteins are often subjected to several types of stress, resulting in misfolding and aggregation.
Toshio Ando
Chapter 18. Other Topics
Abstract
This chapter describes HS-AFM imaging studies on topics that are not categorized into the above chapters: structural fluctuations, Brownian motion, and ion-dependent structure of proteins. HS-AFM analysis of structural fluctuations and Brownian motion can provide information on energy landscapes.
Toshio Ando
Backmatter
Metadata
Title
High-Speed Atomic Force Microscopy in Biology
Author
Prof. Dr. Toshio Ando
Copyright Year
2022
Publisher
Springer Berlin Heidelberg
Electronic ISBN
978-3-662-64785-1
Print ISBN
978-3-662-64783-7
DOI
https://doi.org/10.1007/978-3-662-64785-1