This Title All WIREs
How to cite this WIREs title:
WIREs Nanomed Nanobiotechnol
Impact Factor: 6.14

Atomic force microscopy of biological samples

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

Abstract The ability to evaluate structural–functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini‐review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high‐resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non‐contact imaging modes. These non‐contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion‐conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH). WIREs Nanomed Nanobiotechnol 2010 2 618–634 This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

The scanning tunneling microscope (STM) and the atomic force microscope (AFM) differ primarily in the manner in which they sense proximity to the surface. The STM senses changes in surface topography electronically by monitoring a tunneling current, between a conductive tip and a conductive sample, as the surface is scanned. The AFM senses the surface by contacting or near contacting a surface with a sharp tip on the end of a microcantilever. With AFM, height information is gained by reflecting a laser beam off the back surface of the cantilever onto a photodiode. As the sample is scanned, feedback electronics raises or lower the tip, in response to changes in surface topography, to maintain a constant position where the laser strikes the photodiode. The voltages applied to raise or lower the tip serve as the height input for the image. Both instruments use the same control electronics so that only the surface sensing device is different. This allows for the instrument to be operated as either an STM or AFM.

[ Normal View | Magnified View ]

Schematic cross‐section of the experimental setup including the reaction scheme of the tip generation/substrate collection mode experiment for imaging glucose with an AFM‐tip integrated biosensor (substrate: periodically micropatterned silicon nitride/gold substrate with 1 µm gold electrodes (Quantifoil, Germany). Simultaneously recorded height (a, c) and current (b, d) current image of glucose conversion with an AFM‐tip integrated glucose sensor recorded in AFM contact mode and SECM generator/collector mode. (b) current image in absence of glucose; (d) current image in presence of 3 mM glucose in phosphate buffer pH 7.2. (Courtesy of A. Kueng, B. Mizaikoff, C. Kranz: unpublished results School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA.)

[ Normal View | Magnified View ]

Live cell imaging with scanning ion conductance microscopy (SICM). The image in (A) is an untreated human embryonic cell line NCL‐1 imaged in L‐15 at room temperature. This unpublished image is provided by Dr Julia Gorelik. (Untreated amphibian kidney epithelial A6 cell line (B) imaged in L‐15 at room temperature by Dr. Yuri Korchev.)

[ Normal View | Magnified View ]

In this study ATP‐dependent human Swi‐Snf remodeling complex was incubated with mouse mammary tumor virus (MMTV) promoter nucleosomal arrays and deposited on mica surfaces pretreated with aminopropyltriethoxysilane (APTES) activated with glutaraldehyde.131 In the three AFM images taken before ATP and after ATP was added changes (arrowheads) in the nucleosome complexes are clearly seen. (Images courtesy of Dr. Stuart Lindsay, BioDesign Institute, Arizona State University, Tempe, Arizona.)

[ Normal View | Magnified View ]

Simultaneous recorded atomic force microscope (AFM) images (A) topography and (B) recognition (TREC) on gently fixed MyEnd cells acquired with a fibrinogen‐coated AFM tip. In both images the outlined areas identify places where fibrinogen attached to the cantilever interacts with vascular endothelial (VE)‐cadherin. The topographic image is not affected by interaction of the fibrinogen on the AFM tip while the recognition image shows dark spots where the interaction between fibrinogen and (VE)‐cadherin occurs. This AFM capability, developed by the Hinterdorfer group, separates the minimum and maximum peaks of an oscillating cantilever as it interacts with a sample surface thereby allowing simultaneous acquisition of topographic and recognition images. The minima of the oscillating cantilever wave contributes the topographic information while the maxima is the source of the recognition image. Scale bars on both images are 200 nm. (Images courtesy of Dr. Peter Hinterdorfer, Johannes Kepler University of Linz, Linz, Austria.)

[ Normal View | Magnified View ]

AFM images of African green monkey kidney cells (CV‐1) taken in contact mode using a Nanoscope III atomic force microscope (AFM) (Veeco Instruments, Santa Barbara, CA). In Image (a) the scale bar is 10 µm with a Z range of 8 µm. By adjusting the scanning force to 20–50 pN high‐resolution images (b) of the cell surface reveal globular structures (black arrows) and elongated particles (white arrow) with lateral diameters of ∼20 nm with heights of ∼10 nm. The CV‐1 images were kindly provided by Dr. Christian LeGrimellec, INSERM/UNIV‐MONTP 1/CNRS, Montpellier, France. The (c) image taken in contact mode, on a PicoPlus AFM (Molecular Imaging, Tempe, AZ), shows several E. coli bacteria mounted on a gelatin surface and imaged in water.67

[ Normal View | Magnified View ]

The first atomic force microscope (AFM) image shows an isolated cell membrane from Deinococcus radiodurans with pores structures. As the cantilever tip is brought in contact with the surface and retracted the force‐extension curve shows six force peaks, of about 300 pN each, required to extract all six protomers of a single bacterial pore from the surface. The distance between each of the protomer disruption events is 7.3 nm. The second image of the same surface shows that an entire bacterial pore was extracted from the surface. (Reprinted with permission from Ref 109. Copyright 1999 National Academy of Sciences, USA).

[ Normal View | Magnified View ]

Sensing forces with the atomic force microscope (AFM) cantilever is a non‐imaging application of AFM. As shown in (a), the number 1 position on the figure shows that the cantilever is not yet on the surface but moving toward the surface (red arrow). The red line on the force curve is the approach while the blue line is the retraction of the cantilever. As the cantilever touches the surface it begins to deflect, as indicated by position 2. This serves as the signal for the cantilever to retract, position number 3 (blue arrow). When force curves are done in air there is a water bridge that can form between the cantilever tip and the surface that causes an adhesion event requiring force to break this contact, as the cantilever tip abruptly jumps off the surface (position number 4). By knowing the spring constant of the cantilever and determining the slope of the cantilever deflection, one can calculate the spring constant of the surface. In (b), the same process was repeated in water, notice that the water bridge between the cantilever tip and surface does not form and therefore no adhesion event. In (c), a specific probe (biotin) is attached to the cantilever tip, by a polyethylene glycol tether, while the surface is covered with avidin. In a liquid environment the tip approaches the surface makes contract, is retracted, moves off the surface, and an adhesion event occurs due to the biotin/avidin interaction. The force required to break this interaction can also be calculated and since the biotin is tethered to the cantilever the adhesion event will occur at approximately the length of the tether.

[ Normal View | Magnified View ]

Browse by Topic

Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Nanotechnology Approaches to Biology > Cells at the Nanoscale

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts