Constant A.J. Putman

A detailed analysis of the optical beam deflection technique for use in atomic force microscopy

A Michelson interferometer and an optical beam deflection configuration (both shot noise and diffraction limited) are compared for application in an atomic force microscope. The comparison shows that the optical beam deflection method and the interferometer have essentially the same sensitivity. This remarkable result is explained by indicating the physical equivalence of both methods. Furthermore, various configurations using optical beam deflection are discussed. All the setups are capable of detecting the cantilever displacements with atomic resolution in a 10 kHz bandwidth.

Adhesion force imaging in air and liquid by adhesion mode atomic force microscopy

A new imaging mode for the atomic force microscope(AFM), yielding images mapping the adhesion force between tip and sample, is introduced. The adhesion mode AFM takes a force curve at each pixel by ramping a piezoactuator, moving the silicon‐nitride tip up and down towards the sample. During the retrace the tip leaves the sample with an adhesion dip showing up in the force curve. Adhesion force images mapping parameters describing this adhesion dip, such as peak value, width, and area, are acquired on‐line together with the sample topography. Imaging in air gives information on the differences in hydrophobicity of sample features. While imaging a mercaptopentadecane‐gold layer on glass in demineralized water, the adhesion force could be modulated by adding phosphate buffered saline.

A new imaging mode in Atomic Force Microscopy based on the error signal

A new imaging mode, the error signal mode, is introduced to atomic force microscopy. In this mode, the error signal is displayed while imaging in the height mode. The feedback loop serves as a high-pass filter that filters out the low spatial frequency components of the surface, leaving only the high spatial frequency components of the surface to contribute to the error signal and to be displayed. At a scan rate of typically 10 lines per second, images taken in this mode show very fine detail. Since the applied force stays nearly constant, the error signal mode is especially suitable for imaging soft biological samples with a high level of detail without damaging the surface.

High-resolution imaging of chromosome-related structures by atomic force microscopy.

An atomic force microscope (AFM) was combined with a conventional optical microscope. The optical microscope proved to be very convenient for locating objects of interest. In addition, the high‐resolution AFM image can be compared directly with the traditional optical image. The instrument was used to study chromosome structures. High‐resolution chromosome images revealed details of the 30‐nm chromatide structure, confirming earlier electron microscopic observations. Chromosomes treated with trypsin revealed a banding pattern in height which is very similar to the optical image observed after staining with Giemsa. Furthermore, it is shown that the AFM can be used to locate DNA probes on in situ hybridized chromosomes. Images of the synaptonemal complex isolated from rat spermatocytes revealed details that improve the understanding of the three‐dimensional structure of this protein.

Single‐asperity friction in friction force microscopy: The composite‐tip model

Friction measurements on muscovite mica and glass have been performed with a friction force microscope using Si3N4 tips. The environment was changed from ambient conditions to N2‐ or Ar‐gas conditions. In ambient conditions, the friction‐versus‐load curves showed a nonlinear behavior closely following the Hertzian contact mechanics of a single‐asperity contact: friction force ∼load2/3. For the same tip under gaseous conditions the friction force increased linearly with the load, indicating multi‐asperity contact. A new model is proposed to explain this change in the nature of the friction behavior. In this composite‐tip model, the tip is formed by the actual Si3N4 tip and ‘‘solidlike’’ contaminants present in enclosed cavities between tip and sample surface.

Atomic force microscope with integrated optical microscope for biological applications

Since atomic force microscopy (AFM) is capable of imaging nonconducting surfaces, the technique holds great promises for high‐resolution imaging of biological specimens. A disadvantage of most AFMs is the fact that the relatively large sample surface has to be scanned multiple times to pinpoint a specific biological object of interest. Here an AFM is presented which has an incorporated inverted optical microscope. The optical image from the optical microscope is not obscured by the cantilever. Using a XY stage to move the sample, an object is selected with the optical microscope and an AFM image of the selected object can be obtained. AFM images of chromosomes and K562 cells show the potential of the microscope. The microscope further enables a direct comparison between optically observed features and topological information obtained from AFM images.

A theoretical comparison between interferometric and optical beam deflection technique for the measurement of cantilever displacement in AFM

A shot-noise- and diffraction-limited Michelson interferometer and two optical beam deflection configurations are compared for application in an atomic force microscope. The results show that under optimal conditions the optical beam deflection method is just as sensitive as the interferometer. This remarkable result is explained by indicating the physical equivalence of both methods.

Atomic force microscopy combined with confocal laser scanning microscopy: a new look at cells

A stand-alone atomic force microscope (AFM) has been developed, which features a large scan area and which allows operation under liquid. This system was combined with a confocal laser scanning microscope (CLSM). Information about cell structures, obtained by CLSM, can be complemented with images of the cell surface obtained with the AFM. This is illustrated by studying the pseudopodia of cells from a human cell line (K562-cells, predecessor of erythroblasts) and the cytoskeleton of monkey kidney cells (in air and under liquid), both stained with F-actin-specific fluorescent probes. Images of the cytoskeleton during the cytotoxic interaction between a natural killer and a K562 target cell are presented. Our results show that combination of these techniques can provide new information about cells and cellular structures.

ATOMIC FORCE MICROSCOPY OF POLLEN GRAINS, CELLULOSE MICROFIBRILS, AND PROTOPLASTS

SummaryAtomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.

Experimental observation of single-asperity friction at the atomic scale

Abstract Friction forces between a sharp silicon nitride tip (radius of curvature, 10 nm) and mica were measured using a friction force microscope. Under low load conditions (