Elsevier

Micron

Volume 33, Issue 4, January 2002, Pages 385-397
Micron

Review
Imaging and manipulation of biological structures with the AFM

https://doi.org/10.1016/S0968-4328(01)00026-9Get rights and content

Abstract

Many biologists have dreamt of physically touching and manipulating the biomolecules they were investigating. With the invention of the atomic force microscope (AFM), this dream has come true. Here, recent applications of the AFM to image and to manipulate biological systems at the nanometer scale are reviewed. Macromolecular biological assemblies as well as individual biomolecules can be subjected to controlled nanomanipulation. Examples of AFM application in imaging and nanomanipulation include the extraction of chromosomal DNA for genetic analysis, the disruption of antibody–antigen bonds, the dissection of biological membranes, the nanodissection of protein complexes, and the controlled modulation of protein conformations. Also reviewed is the novel combination of single molecule imaging and force spectroscopy which allows biomolecules to be imaged, and inter- and intramolecular forces to be measured. Future application of these nanotechniques will reveal new information on the structure, function and assembly of biomolecules.

Introduction

Understanding how complexes of biomolecules are assembled is of fundamental importance to elucidate their structure and function. To gain new insights into these supramolecular assemblies, techniques are required that provide information at the level of single molecules (Science, 1999, Nature Structural Biology, 2000). Among these, the atomic force microscope (AFM) is unique since it not only allows individual molecules to be imaged (Shao and Yang, 1995, Czajkowsky and Shao, 1998, Engel and Müller, 2000) but also to be manipulated (Clausen-Schaumann and Seitz, 2000, Fisher et al., 2000, Rief, 2000).

Since the invention of the AFM (Binnig et al., 1986) amazing progress has been made in the imaging of biomolecules. This progress is due to the combined efforts of numerous laboratories to improve instrumentation (Hansma et al., 1994, Putman et al., 1994, Viani et al., 1999, Humphris, 2000), AFM probes (Walters et al., 1996, Cheung et al., 2000, Schmitt et al., 2000), sample preparation methods (Müller, 1997a, Wagner, 1998), and image acquisition conditions (Müller et al., 1999b). The AFM has several unique features that are very useful for biological studies: Firstly, it can be operated in solution allowing biological structures to be observed in their native environment (Drake et al., 1989). Secondly, the AFM provides such a high signal-to-noise ratio that single proteins can be observed at a resolution better than 1 nm (Mou, 1995, Müller and Schabert, 1995b, Walz et al., 1996, Czajkowsky, 1998, Reviakine, 1998, Scheuring et al., 1999, Fotiadis et al., 2000). Thirdly, conformational changes of single biomolecules can be directly visualized (Müller, 1995a, Müller, 1996a, Müller and Engel, 1999). In fact single biomolecules can now be observed at work (Kasas et al., 1997, Grandbois, 1998, Viani et al., 2000) for a review see (Engel and Müller, 2000).

Furthermore, the past few years have demonstrated that the combination of AFM imaging and manipulation, allows the precise and controlled modification of biological systems from the level of the cell down to the scale of individual molecules (Schoenenberger and Hoh, 1994, Thalhammer et al., 1997, Fotiadis et al., 1998). In this review the unique possibilities provided by the AFM are presented and the novel information thereby obtained is discussed.

Section snippets

Principles of AFM imaging

The principle of the AFM is relatively simple: A sharp probe (tip) mounted at the end of a flexible cantilever raster-scans over the sample surface in a series of horizontal sweeps (Binnig et al., 1986). Deflections of the cantilever caused by the probe-sample interaction are detected by an optical detector (Meyer and Amer, 1988, Alexander et al., 1989). This signal is used to minimize the force applied to the sample by moving the sample up and down via a servo-system. The surface topography is

Imaging and dissection of isolated DNA and human metaphase chromosomes

The first controlled nanomanipulation of biomolecules using the AFM was performed on genetic material (Hansma et al., 1992). Isolated DNA adsorbed to a mica surface was dissected, both in air (Henderson, 1992, Vesenka et al., 1992) and in propanol (Hansma et al., 1992) by increasing the force applied to the AFM probe to about 5 nN. This demonstrated for the first time the feasibility of dissections in the nanometer range.

In the nucleus of eukaryotic cells, DNA is packaged with proteins to form

Disruption of antibody and antigen bonds

Immunolabelling and other labelling techniques are nowadays well established and used routinely in light and electron microscopy. They provide biologists with a wealth of information at both the cellular and the molecular levels. Recently, labelling techniques have found an application in combined light and atomic force microscopy (Putman et al., 1993, Schabert et al., 1994, Hillner et al., 1995). The use of these two complementary microscopy techniques in parallel, for example to examine

Nanodissection of membranes

Cell-to-cell interactions involve the adhesion of two cell membranes to one another. This is mediated by the extracellular regions of specific membrane proteins. When isolated and reconstituted, such membrane proteins also interact via their extracellular surfaces giving rise to ‘sandwiched’ structures. Examples are the extracellular surfaces of gap junction connexins (Hoh et al., 1993), porin OmpF from Escherichia coli (Schabert et al., 1995) and the major intrinsic protein (MIP) from sheep

Nanodissection of the photosystem I reaction center

The 340 kDa pigment-containing reaction center photosystem I (PSI) from the thermophilic cyanobacterium Synechococcus sp. consists of 11 protein subunits (Schubert et al., 1997). Three of these 11 subunits, PsaC, -D and -E, are extrinsic and adhere to the complex via electrostatic interactions (Schubert et al., 1997). Isolated PSI complexes assemble in an up-and-down manner to form 2D crystals in which their lumenal and stromal surfaces are alternately exposed (Karrasch et al., 1996, Fotiadis et

Modulating polypeptide loops of individual bacteriorhodopsin molecules

On AFM topographs of the cytoplasmic surface, BR exhibits trimeric structures arranged in a trigonal lattice of 6.2±0.2 nm side length (Müller et al., 1995b). At imaging forces of 100 pN, each subunit in the trimer features a pronounced protrusion extending 0.8±0.2 nm above the lipid surface (Fig. 5A, top and Fig. 5B). This protrusion is thought to arise from the loop connecting the α-helices E and F (Müller et al., 1995a). Increasing the applied force to about 200 pN changes the AFM topographs

Single molecule imaging and assessment of intermolecular forces

Structural, chemical and morphological studies have shown that the S-layers of bacteria are one of the most simple membrane structures developed during evolution (Baumeister et al., 1988). They completely cover the cell surface and are usually composed of a single protein that is endowed with the ability to assemble into highly ordered monomolecular arrays by an entropy driven process (Sleytr et al., 1993). Experimental results indicate that the integrity of the S-layer lattice is maintained by

Single molecule imaging and detection of inter- and intramolecular forces

Membrane proteins acquire their function through specific folding of their polypeptide chain and through specific interactions with the lipid bilayer and adjacent proteins. The stability and resistance of membrane proteins to unfolding is predominantly investigated by studying their thermal or chemical denaturation (Haltia and Freire, 1995, White and Wimley, 1999). Membrane proteins are designed to remain stable when embedded in lipid leaflets. Thus, their overall stability and resistance to

Summary and outlook

The dissection of chromosomes and the controlled extraction of specific DNA segments are selected examples from the rapidly increasing number of experiments, where the AFM has been used for not only imaging biomolecules under native conditions, but also as a mechanical ‘scalpel’ and ‘shovel’. A refinement and extension of these applications will make the AFM a valuable tool for cytogenetic studies.

Imaging and manipulation of double-layered 2D crystals of membrane proteins has allowed the

Acknowledgements

This work was supported by the M.E. Müller Foundation of Switzerland, by the Swiss National Foundation for Scientific research (Grant 4036-44062 to A.E.) and by the European Union-Biotech Program (Grant BIO4-CT98-0024 to A.E.). We thank Dr Stefan Thalhammer for kindly providing Fig. 1 and Dr Cora-Ann Schönenberger for critical reading of the manuscript.

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