Integrated Confocal and Scanning Probe Microscopy for Biomedical Research

Mini-Review Article TheScientificWorldJOURNAL (2006) 6, 1609–1618 ISSN 1537-744X; DOI 10.1100/tsw.2006.269 Integrated Confocal and Scanning Probe Microscopy for Biomedical Research B.J. Haupt, A.E. Pelling, and M.A. Horton* London Centre for Nanotechnology, The Bone and Mineral Centre, The Rayne Institute, Department of Medicine, University College London, London WC1E 6JJ E-mail: ; ; Received October 24 2006; Accepted November 22, 2006; Published December 15, 2006 Atomic force microscopy (AFM) continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM) and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease. KEYWORDS: atomic force microscopy (AFM), confocal microscopy, fluorescence microscopy, cell biology, nanomechanics, mechanotransduction, mechanobiology INTRODUCTION In recent years, atomic force microscopy (AFM) has been increasingly used to address problems of biomedical relevance. Originally developed by Binnig et al. in 1986[1] to study the material and topographic features of nonconductive surfaces, it has now become a popular tool in the study of biological samples[2]. It is capable of imaging cells at high resolution[3,4,5,6,7,8], measuring the forces involved in intermolecular bonds[3,9,10,11], and investigating the mechanical properties of biological materials[5,6,12,13,14,15,16,17,18,19,20,21,22,23,24]. AFM has the advantage of being able to operate in air and fluid under physiological conditions, which has allowed biologically relevant, force spectroscopy studies of single biomolecule binding events[10,13] and a wide range of applications in cell biology, such as studying cell-surface morphology[6,7,8,25,26], the cytoskeleton[5,6,17,18,19,20,22,23, 24,27,28,29] and organelles[6,29,30,31], cell movement[16,31,32,33], and cell-matrix or cell-cell interaction forces[3,9,34,35,36] resulting from plasma membrane receptor binding[3,9,10,11,37]. In biomedical studies, AFM can be used as a micromanipulator, applying precise pico to nano Newton forces[3,14,35,38]. As an imaging tool of biological samples, AFM cannot compete with the speed at which traditional microscopy tools, such as confocal laser scanning microscopy (CLSM) and electron microscopy (EM), capture an image. However, its main advantage in biomedicine is the ability to probe and alter the mechanobiological characteristics of living cells and tissues in order to examine *Corresponding author. ©2006 with author. Published by TheScientificWorld, Ltd.; www.thescientificworld.com 1609 Haupt et al.: SPM in Biomedical Research TheScientificWorldJOURNAL (2006) 6, 1609–1618 specific, receptor-ligand interactions, material properties, and cell behavior. The new focus of using AFM to study biological samples is to combine it with optical imaging methods, such as CLSM and fluorescent imaging[5,6,7,8,9,12,13,14,17,18,19,20,24,26,29,30,37,38,39,40,41,42,43]. This would then allow the investigation of mechanotransduction/sensation pathways[12,14,18] as well as traditional imaging applications, such as the comparison to topographical features to the cell architecture[5,6,7,8,12,13, 14,17,19,26,29,39,40]. In this review, we show how the AFM has currently undergone a transformation from a surface science tool to a biological tool by integration with optical techniques in combination with its ability to act as a nanoscale force transducer/actuator and imaging device. We will discuss the advantages of such combinations, current research [7, 8, 10-14, 18, 24, 26, 37, 38, 40-43], the potential medical applications of AFM in diagnosis and detection of disease or ageing [44, 45], development of new and novel devices [38, 41-43], and highly specific drug studies [5, 6, 12, 19, 22-24]. PRINCIPLES OF AFM AND INTEGRATION WITH OTHER OPTICAL TECHNIQUES Imaging of a surface with AFM involves a microfabricated cantilever with a very small tip (with a contact area of only a few square nanometers) that is raster scanned above the surface of a sample (see Fig. 1). The movement of the cantilever is controlled by a x,y,z-piezoelectric ceramic tube that moves the cantilever, and a laser beam that is reflected off the back of the cantilever onto a quadrant photodiode that measures the cantilever deflection. A feedback loop linking the current applies the piezo and the detector enables precise control of the positioning of the cantilever and the force applied to the sample[39,46]. Several different modes of operation have been developed for AFM and have been reviewed by Hansma et al.[46]. Contact mode imaging involves the cantilever scanning a sample at a constant applied force, where the tip remains in constant contact with the sample surface. In this mode, the cantilever is moved up and down in the z-direction to remain in contact and, from this, a topographical image is obtained. Noncontact mode is another imaging mode that involves rapidly oscillating the cantilever in the z-direction, either mechanically (Tapping mode) or magnetically (MAC mode) above the surface while scanning. As the oscillating cantilever approaches the surface, its amplitude is decreased and it is this that is used as the feedback for imaging. This imaging mode is far less damaging to soft samples such as cells and tissues[4]. The last mode we shall describe is force mode from which force-distance measurements are made. In this mode, the cantilever is moved only in the z-direction towards and away from the surface with the deflection (z) being constantly measured (see Fig. 2). With knowledge of the spring constant (k) of the cantilever, it is possible to calculate the force (F) at any given deflection through Hookes Law (F = -kz). This mode can be used to study single ligand-receptor binding forces[3,9] or determine the material properties of biological samples[5,6,12,13,14,15,16,17,18,19,20,21,22,23,24]. Measuring Receptor-Ligand Binding Events With AFM, it has been possible to quantify receptor-ligand binding forces, typically in the range of 15–250 pN for protein interactions and up to 220 nN for cellular binding forces[9,10,11,34,35,36,37, 47,48,49,50,51,52]. To be able to study single receptor-ligand events requires that the tip of the AFM be “functionalized” by attaching a specific interactive molecule (either the “receptor” or the “ligand”) that will act as a probe to detect bind (...truncated)