Polymerized LB films imaged with a combined atomic force microscope-fluorescence microscope

The first results obtained with a new stand-alone atomic force microscope (AFM) integrated with a standard Zeiss optical fluorescence microscope are presented. The optical microscope allows location and selection of objects to be imaged with the high-resolution AFM. Furthermore, the combined microscope enables a direct comparison between features observed in the fluorescence microscope and those observed in the images obtained with the AFM, in air or under liquid. The cracks in polymerized Langmuir-Blodgett films of lO,l2-pentacosadiynoic acid as observed in the fluorescence microscope run parallel to one of the lattice directions of the crystal as revealed by molecular resolution images obtained with the AFM. The orientation of these cracks also coincides with the polarization direction of the fluorescent light, indicating that the cracks run along the polymer backbone. Ripple-like corrugations on a submicrometer scale have been observed, which may be due to mechanical stress created during the polymerization process

Atomic force microscope featuring an integrated optical microscope

The atomic force microscope (AFM) is used to image the surface of both conductors and nonconductors. Biological specimens constitute a large group of nonconductors. A disadvantage of most AFM's is the fact that relatively large areas of the sample surface have to be scanned to pinpoint a biological specimen (e.g. cell, chromosome) of interest. The AFM presented here features an incorporated optical microscope. Using an XY- stage to move the sample, an object is selected with the aid of the optical microscope and a high-resolution image of the object can be obtained using the AFM. Results o­n chromosomes and cells demonstrate the potential of this instrument. The microscope further enables a direct comparison between optically observed features and topological information obtained from AFM images

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

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

Immunogold labels: cell-surface markers in atomic force microscopy

The feasibility of using immunogold labels as cell-surface markers in atomic force microscopy is shown in this paper. The atomic force microscope (AFM) was used to image the surface of immunogold-labeled human lymphocytes. The lymphocytes were isolated from whole blood and labeled by an indirect immunolabeling method using the monoclonal antibody anti-CD3 and a secondary antibody (Goat-anti-Mouse) linked to 30 nm colloidal gold particles. Some of the samples were enhanced by silver deposition o­nto the gold particles. The AFM images reveal the colloidal gold particles o­n the cell surface, with and without silver enhancement. Individual immunogold (-silver) particles are clearly resolved from the cell surface thus determining the location of antigens. The 30 nm gold particles appear in the AFM images having an average size of about 80 nm due to convolution between gold particle and AFM tip

Tapping mode atomic force microscopy in liquid

We show that standard silicon nitride cantilevers can be used for tapping mode atomic force microscopy (AFM) in air, provided that the energy of the oscillating cantilever is sufficiently high to overcome the adhesion of the water layer. The same cantilevers are successfully used for tapping mode AFM in liquid. Acoustic modes in the liquid excite the cantilever. o­n soft samples, e.g., biological material, this tapping mode AFM is much more gentle than the regular contact mode AFM. Not o­nly is the destructive influence of the lateral forces minimized, but more important, the intrinsic viscoelastic properties of the sample itself are effectively used to ''harden'' the soft sample

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

Vacuum chamber for sample attachment in atomic force microscopy

A small ring-shaped vacuum chamber has been constructed and connected to the piezotube used for scanning samples in the atomic force microscope (AFM). Samples made up of any material, up to 50 mm in diameter, can be firmly attached o­nto the piezotube without causing damage to the sample. A 50-l beer container forms a buffer between vacuum pump and chamber. With this supply of vacuum, the AFM can be operated for a 4-8 h period without turning o­n the vacuum pump again. Samples can be changed within 30 s. The scan frequency when using microscope slides is limited to 40 Hz due to resonance effects of the microscope slides

Stand-alone atomic force microscope featuring large, scan friction measurement, atomic resolution, and capability of liquid operation

We have developed a stand-along atomic force microscope featuring large scan, friction measurement, atomic resolution and capability of in liquid operation. Cantilever displacements are detected with optical beam deflection. Cantilever and laser diode are both attached at the piezo tube and thus scanned simultaneously. As a direct consequence the maximum scan range, 25 X 25 micrometers 2, is solely determined by the characteristics of the piezo tube and not by the dimensions of the cantilever and/or the waist of the laser beam. The stand- along atomic force microscope is suitable to be combined with any inverted optical microscope (including the confocal laser scanning microscope), as is illustrated with fluorescence and height images of K562-cells. Results on thin films consisting of a mixture of polymers show the strength of measuring friction and height simultaneously. Images of mica show that atomic resolution can be obtained both in height and friction mode

Chromosome structure investigated with the atomic force microscope

We have developed an atomic force microscope (AFM) with an integrated optical microscope. The optical microscope consists of an inverted epi-illumination system that yields images in reflection or fluorescence of the sample. With this system it is possible to quickly locate an object of interest. A high-resolution image of the object thus selected can then be obtained with the AFM that is built on top of the optical microscope. In addition, the combined microscopes enable a direct comparison between the optical image and the topography of the same object. The microscope is used to study the structure of metaphase chromosomes of eukaryotic cells. The topography of metaphase chromosomes reveal grooved structures that might indicate spiral structure of the chromatin. High resolution images reveal structures that can be ascribed to the end loops of the chromatin. The resolution of the AFM images was improved by using sharper tips obtained by carbon deposition on the Si3N4 cantilevers using a scanning electron microscope. Chromosomes which are treated to reveal the G- banding pattern in the optical microscope display a similar pattern when viewed with the AFM, as is shown by a direct comparison