Biological Applications of the AFM: From Single Molecules to Organs S. Kasas, N. H. Thomson, B. L. Smith, P. K. Hansma, J. Miklossy, H. G. Hansma Department of Physics, University of California Santa Barbara, Santa Barbara, CA 93106 Received 13 August 1996; revised 18 October 1996 ABSTRACT: The application domains of the atomic force microscope between single molecules, measurement of the micromechanical have increased dramatically in recent years. We present a short review properties of the sample, and detection of protein motion. Finally, of the contributions of this microscope to biology. These are illustrated we will discuss the future of AFM in biology. through the study of different samples, starting with the imaging of single molecules all the way up through the length scales, and ending with imaging of tissues. So that nonbiologists can appreciate the II. WORKING PRINCIPLE significance of these studies, special attention has been paid to a The principle of the AFM is simple: a sharp tip fixed at the end description of the samples and to point out the motivation of these of a flexible cantilever is raster-scanned over the surface of a studies and their implications for the field of medicine.  1997 John sample. As the tip interacts with the surface, the cantilever de- Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 151–161, 1997 flects and its deflections are monitored and used to reconstruct the topography of the sample [122,145]. Surfaces can be imaged nondestructively because the interatomic spring constant of the I. INTRODUCTION sample is on the order of 10 N/m, in comparison with typical The atomic force microscope ( AFM ) , also known as the scanning contact mode AFM cantilevers, which have spring constants in force microscope (SFM), was developed 10 years ago [12] and the range of 0.01–1 N/m. The cantilever is a macroscopic spring is an instrument which probes the interaction forces between a that has a lower spring constant, meaning that the applied force sharp tip and the surface of a sample. The ability of this micro- can be kept well below the force which would disturb the atoms scope to achieve high resolution (subnanometer) in liquids and from their sites, while still achieving measureable cantilever de- to probe the mechanical properties of the sample at a nanometric flections [122]. If the microscope is operated in liquids or in scale make this instrument increasingly interesting for the study vacuum, high resolution is more readily achieved, since the strong of biological specimens. Consequently, the number of AFM arti- capillary forces, due to the thin liquid film present on all samples cles published per year in this field has grown exponentially in air, are absent. [48]. Every conceivable type of biological material has been explored using the AFM (for reference, see review articles [18,19,79,82,86,111,122,126 ] . This review presents selected bio- III. THE SCANNER logical applications to illustrate the microscope’s potential in the The precise positioning of the sample relative to the tip is one field of biology. of the practical reasons why AFM can achieve subnanometer In the first part, we outline the general working principle of resolution on hard samples. Scanning is accomplished using pi- the instrument and its different imaging modes. AFM applications ezoelectric translators, with a precision on the order of angstroms. can be classified in different ways. We have chosen to divide In most AFMs the sample is positioned on top of a four- them into imaging and nonimaging categories. One advantage of segment piezoelectric tube and is scanned under a fixed tip. The the AFM is the ease with which data can be acquired over a wide mechanical components of the microscope are designed to be range of length scales. When discussing imaging, we will present rigid and compact, insuring the high mechanical stability required some applications of the AFM at different magnifications, from for atomic resolution imaging. Figure 1 depicts the typical archi- single molecules to organs. After this, we will discuss the nonim- tecture of such an AFM. For biological applications it can be aging category, which includes measurement of forces involved useful to observe the sample simultaneously with an optical mi- croscope, hence, free-standing AFMs mounted on the top of an inverted optical microscope have been developed [113]. In this Correspondence to: S. Kasas, University de Fribourg Suisse, Institut d’Histolo- architecture, the sample is fixed, and the tip, which is mounted gie Perolles, CH-1705 Fribourg, Switzerland Contract grant sponsor: NATO on a piezo tube, moves about the sample [ 51,138 ] . The disadvan- Contract grant sponsor: NSF; Contract grant numbers: DMR-9622169, DMR- tage of this architecture is poorer mechanical stability, limiting 9123048, MCB 9317466 Contract grant sponsor: Digital Instruments the resolution of the instrument. However, since the sample is  1997 John Wiley & Sons, Inc. CCC 0899–9457/97/020151-11 4433 / 8402$$4433 05-22-97 10:29:27 ista W: IST