Methods for imaging DNA in liquid with lateral molecular-force microscopy.

Shear force microscopy is not normally associated with the imaging of biomolecules in a liquid environment. Here we show that the recently developed scattered evanescent wave (SEW) detection system, combined with custom-designed vertically oriented cantilevers (VOCs), can reliably produce true non-contact images in liquid of DNA molecules. The range of cantilever spring constants for successful shear force imaging was experimentally identified between 0.05 and 0.09 N m(-1). Images of λ-DNA adsorbed on mica in distilled water were obtained at scan rates of 8000 pixels s(-1). A new constant-height force mapping mode for VOCs is also presented. This method is shown to control the vertical position of the tip in the sample plane with better than 1 nm accuracy. The force mode is demonstrated by mapping the shear force above λ-DNA molecules adsorbed on mica in a liquid environment at different tip-sample separations.

Processive behaviour of kinesin observed using micro-fabricated cantilevers.

The mechanical characterization of biomolecular motors requires force sensors with sub-piconewton resolution. The coupling of a nanoscale motor to this type of microscale sensors introduces structural deformations in the motor according to the thermally activated degrees of freedom of the sensor. At present, no simple solution is available to reduce these effects. Here, we exploit the advantages of micro-fabricated cantilevers to produce a force sensor with essentially one degree of freedom and a spring constant of 0.03 pN nm(-1) for the study of the molecular motor protein kinesin-1. During processive runs, the cantilever constrains the movement of the cargo binding domain of kinesin in a straight line, parallel to the microtubule track, and excludes specific reaction coordinates such as cargo rotation. In these conditions, we measured a step size of 8.0 ± 0.4 nm and a maximal unloaded velocity of 820 ± 80 nm s(-1) at saturated adenosine triphosphate (ATP) concentration. We concluded that the motor does not need to rotate its tail as it moves through consecutive stepping cycles.

Tailoring gold nanostructures for near-field optical applications.

A method of combined thin-film deposition, electron beam lithography, and ion milling is presented for the fabrication of gold and silver nanostructures. The flexibility of lithographical processes for the variation of geometric parameters is combined with three-dimensional control over the surface evolution. Depending on the etching angle, different shapes ranging from cones over rods to cups can be achieved. These size- and shape-tunable structures present a toolbox for nano-optical investigations. As an example, optical properties of systematically varying structures are examined in a parabolic mirror confocal microscope.

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy.

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 microm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

Micro-fabricated mechanical sensors for lateral molecular-force microscopy.

Atomic force microscopy (AFM) has been very successful in measuring forces perpendicular to the sample plane. Here, we present the advantages of turning the AFM cantilever 90° in order for it to be perpendicular to the sample. This rotation leads naturally to the detection of in-plane forces with some extra advantages with respect to the AFM orientation. In particular, the use of extremely small (1µm wide) and soft (k≅10(-5)N/m) micro-fabricated cantilevers is demonstrated by recording their thermal power spectral density in ambient conditions and in liquid. These measurements lead to the complete characterisation of the sensors in terms of their stiffness and resonant frequency. Future applications, which will benefit from the use of this force microscopy technique, are also described.

Scanning probe evolution in biology.

Twenty years ago the first scanning probe instrument, the scanning tunneling microscope, opened up new realms for our perception of the world. Atoms that had been abstract entities were now real objects, clearly seen as distinguishable individuals at particular positions in space. A whole family of scanning probe instruments has been developed, extending our sense of touching to the scale of atoms and molecules. Such instruments are especially useful for imaging of biomolecular structures because they can produce topographic images with submolecular resolution in aqueous environments. Instruments with increased imaging rates, lower probe-specimen force interactions, and probe configurations not constrained to planar surfaces are being developed, with the goal of imaging processes at the single-molecule level-not only at surfaces but also within three-dimensional volumes-in real time.

[Atomic force microscope (AFM). A nanomanipulator for biophysical studies of stereocilia of the cochlear hair cells]. / Das Rasterkraftmikroskop (AFM). Ein Nanomanipulator für biophysikalische Untersuchungen an Stereozilien der Sinneszellen der Kochlea.

OBJECTIVES: Studies of the mechanoelectrical sensor system of the hair cell bundle in the cochlea require a manipulation device that enables controlled force application and movement of individual stereocilia in the nanometer range. METHODS: In our atomic force microscope (AFM) setup, the scan is directly controlled in an upright differential interference contrast (DIC) infrared video microscope with a water immersion objective and in the measured AFM image. Here we present studies on hair cells of the mammalian cochlea. RESULTS AND CONCLUSIONS: The resulting images revealed the tips of individual stereocilia of living sensory cells of the organ of Corti and the typical shape of the ciliary bundle. Scanning electron-microscopic (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that up to four AFM manipulations on the same cell did not cause obvious damage to the surface morphology of the stereocilia.