The atomic force microscope (AFM) is capable of measuring the interaction between tip and sample with high sensitivity and unparalleled spatial resolution. The chemical functionalization of the AFM tips has expanded the versatility of the AFM to experiments where specific molecular interactions are measured. We present here measurements of the interaction between complementary strands of DNA. A necessary prerequisite for the quantitative analysis of the interaction force is knowledge of the spring constant of the cantilevers. We report a method that allows for the in situ measurement of the absolute value of the spring constant of cantilevers based on spectral analysis of the thermal excitations of the cantilever.

Scanning force microscopy has been used to study the structure of chromatin fibers at low salt concentrations. Chicken erythrocyte chromatin fibers in low ionic strength buffer solutions were deposited on mica and imaged in ambient conditions with a tapping mode scanning force microscope. Individual nucleosomes can be clearly discerned in the images of the fibers. Native chromatin fibers show an asymmetrical, 3D structure of sinuous fiber trajectory with irregularly positioned nucleosomes. Fibers depleted of linker histones H1 and H5 have a completely extended 'beads-on-a-string' structure, with linker DNA visible between single nucleosomes. Molecular modeling of the fiber architecture and computer simulation of the imaging process provided more evidence on the observed organization of chromatin at low salt conditions. These results have implications on mechanisms of transcription control and chromatin compaction.

Chromatin fibers have been hydrolyzed by trypsin and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and scanning force microscopy (SFM). At early points of hydrolysis, when mostly only the tails of the linker histones have been cleaved, nucleosomes appear to pile upon each other within a fiber. Later, once significant hydrolysis of the N-terminal tail of histone H3 has occurred, fibers exhibit an open, three-dimensional arrangement of nucleosomes. Linker DNA between adjacent nucleosomes is observed for the first time. Adjacent nucleosomes appear to have a 'zig-zag' arrangement. Finally, when all the tails of the linker histones and the N-terminal tails of H3 have been cleaved, then the fibers exhibit (i) a flat two- dimensional arrangement of nucleosomes, (ii) linker DNA between nearly all nucleosomes, and (iii) a zig-zag arrangement among some nucleosomes. We suggest that (i) the linker histone globular domains help fix the angle of the DNA entering and exiting the nucleosome, (ii) the angle, however, is not sufficient to maintain the three-dimensionality of the fiber, and (iii) the N-terminal tails of histone H3 ares necessary for the three-dimensional conformation of the fiber at low ionic strength.

Unfixed chicken erythrocyte fibers in very low salt have been imaged using the scanning force microscope (SFM) operating in the tapping mode in air at ambient humidity. These images reveal a 3D organization of the fibers. The planar 'zig-zag' conformation is rare, and extended 'beads- on-a-string' fibers are seen only in chromatin depleted of H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies indicate that chromatin fibers may exist as irregular 3D arrays of nucleosomes even at low ionic strength. The basic subunit of chromatin, the nucleosome, is composed of a core particle of 146 bp of DNA wrapping 1.75 left-handed superhelical turns around an octamer of core histones and of DNA connecting consecutive core particles. The linker of lysine-rich histones (H1 family) bind the DNA entering and exiting the nucleosome core particle. We suggest that by binding the entry/exit DNA, histone H1 may fix the entry/exit DNA angle. The fixed entry/exit angle, the rigidity of the linker DNA at low ionic strength, and the natural variability of the linker DNA length determine an irregular 3D fiber of chromatin. Our results emphasize the role of H1 in determining the entry/exit DNA angle, which further helps determine the mutual disposition of adjacent nucleosomes an the packing of the chromatin fiber.

The sperm of bambooleaf wrasse, a marine teleost, are immotile when they are diluted in a solution isotonic to the seminal plasma, but they begin to swim when they are suspended in sea water. What changes arise in morphology of the sperm cell after the motility initiation? The semen collected from the abdomen of a matured wrasse was mixed with either thinned sea water or sea water. A drop of the same specimen was placed on a cleaned silicon wafer, respectively. After fixed chemically, they were rinsed with distilled water and dried naturally in room temperature. These samples were examined carefully with use of an atomic force microscopy. Although the axonemes of intact sperms were found to be crushed as if the axonemes were cut open along doublet microtubules. The motility initiated sperm was strong enough to resist the force caused by surface tension of water in the drying process and could maintain the structure of the axoneme. These experimental facts suggest that the binding characteristics in the structure of the axoneme after the initiation of the motility were clearly changed stronger that before.

In this presentation, we describe about the high resolution imaging of flagellar filaments of a salmonella with a diameter of 25 nm by a photon scanning tunneling microscope (PSTM). In our PSTM system, we have used a metal coated fiber probe with a nanometric dielectric protrusion fabricated by indigenous technique developed by us. By using this probe, flagellar filaments could be imaged as having a full width at half maximum of 50 nm. Obtained images strongly depended on the separation of the sample and to the probe, and the diameter of the aperture polarization of the irradiated light.

F-actin filaments have been found to adopt conformations with both right handed and left handed topographies, as revealed by AFM imaging in air. The left handed conformation has not been observed previously by either AFM or electron microscopy. Either handedness can be produced by controlling the conditions under which the molecules are deposited for imaging by Scanning Force Microscopy or Transmission Electron Microscopy. Low salt conditions favor the left handed form, and high salt conditions favor the right handed form. The ability of F-actin to undergo larger changes may still reflect smaller changes that take place under physiological conditions.

We report on two new techniques for obtaining fast time resolution in scanning tunneling microscopy. These techniques allow dynamical studies of processes that lie outside the ordinary bandwidth of the STM. The first of these methods uses a magnetostrictive tip to directly gate the tunnel current. The second method uses a nonlinear characteristic of the tunneling interaction to detect optically induced ultrafast signals. In both techniques, demodulation of the high-speed signal is performed at the tunnel junction itself, which should ultimately lead to the greatest possible time resolution. These stroboscopic techniques may be used whenever repetitive signals can be arranged, as is the case for many electronic, magnetic, and optical phenomena at surfaces.

We propose a Fluorescent Scanning Near-Field Optical Microscope (FSNOM) based upon the measurement of the decay time of a fluorescent particle adsorbed on a SNOM tip. The purpose of the experiment is to measure the decay time variations of the fluorescent particle when the tip is scanned at a few nanometers from the surface of a sample. We describe the experimental set-up and present a theoretical model: it calculates, by a perturbation method, the variations of the life-time on a rough surface. The formalism is rapidly described and first theoretical life- time signals are presented.

We demonstrate the ability of near-field scanning optical microscopy (NSOM) technique to detect inhomogeneities of the dynamics of excess carriers in oxidized silicon wafers. NSOM is used to improve the spatial resolution of a standard IR-scattering optical technique, which is carried out in a noncontact fashion. Continuous wave infrared light is used as a detector of the time dependent carrier population produced by a pulsed visible laser. We will show high resolution images of carrier lifetime, and discuss some aspects of the NSOM measurement that differentiate it from its far field counterpart.

Recent application of tapered fiber tips in near field optical microscopy has demonstrated a resolution far exceed the diffraction limit. We investigate the propagation and tunneling of monochromatic light through a tapered fiber tip near a dielectric surface. The transmission rate through the tip is given for various circumstances and found to increase significantly with the dielectric constant of the sample.

Specific photophysical phenomena appearing at a surface under superlocal action of light radiation are considered theoretically under diffusion approximation. Stationary spatial distribution of electron temperature is found and estimations of light energy flux density needed for various values of surface overheat are done.

The work includes theoretical analysis of heat-up of an optical near- field probe with laser radiation passing through it. Stationary solution is found regarding the probe heating due to partial absorption of the radiation by the metal film and linear heat exchange between the probe and its environment. Temperature space distribution down the tapered part of the probe is found. The threshold power of continuous radiation leading to melting of the probe is estimated.

We investigated the adhesion forces between single protein molecules and the silicon-nitride tip of an atomic force microscope. Force curves were taken on a sample with single adsorbed proteins while the tip was raster scanned laterally. Out of these force maps we can calculate several images showing for instance the topography or the adhesion force as a function of lateral position. Two systems were investigated here: actin adsorbed on mica and tubulin adsorbed on positively charged silanized surfaces, the adhesion force of the tip on the protein was smaller by about a factor of three to five compared to the force measured on the substrate. This is in agreement with previous studies of lysozyme and DNA adsorbed on mica. The data were analyzed by estimating the van der Waals force between the tip and a single protein and between the tip and a flat substrate. The measured adhesion force between the tip and the substrate can be understood by van der Waals. However in the case of the proteins the observed adhesion is larger than expected by only van der Waals forces. So we conclude that there are additional interactions determining the adhesion between the tip and the protein.

Most scanning tunneling microscopy (STM) work dealing with ordered organic films have primarily relied on highly ordered pyrolytic graphite as a substrate (HOPG). A second substrate, molybdenum disulfide (MoS₂), is appearing more often as a substrate. In this paper, we introduce a new substrate for the imaging of ordered systems. Tungsten diselenide (WSe₂) is a layered material similar in structure to MoS₂. The change in surface periodicity for HOPG to MoS₂ caused significant changes in the ordering of liquid crystals. This is not completely unexpected given the two different types of surfaces. In this contribution two n-alkyl-cyanobiphenyls (mCBs; m equals 6,8) were imaged on WSe₂. The differences between MoS₂ and WSe₂ are less drastic when compared to HOPG, yet the ordered structure that was found for 8CB was a new monolayer type structure while the 6CB structure is very similar to what has been found for this molecule on MoS₂.

Taxol stabilized microtubules were imaged in their native state in buffer solution without any fixation by an atomic force microscope (AFM) operated in tapping mode in liquids. Glass cover slips were derivatized with a positively charged silane to adsorb the filaments. The adsorbed microtubules could be imaged stably without any visible damage for hours. In cases where a microtubule crossed above another one, it often broke to follow the curvature of the underlying one. Longitudinal structures with a spacing of about 8 nm could be resolved suggesting they are protofilaments.

We have used the near-field scanning optical microscope (NSOM) to study the inhomogeneity as well as initiate photochemical processes in conjugated polymer films. A simple transmission-mode NSOM is constructed for these studies. A low noise, large area Si photo-detector is mounted directly between the PZT scanning stage and the sample. This method provides a simple way to covert the commercial AFM/STM scanning stage to a near-field optical microscope.

Near field scanning optical microscopy of thin birefringent samples is described. The system utilized is the linear polarizing near field microscope, resulting in a pure birefringence image of the sample. The sign of the birefringence is also preserved. Two specific classes of sample are studied. These include thin sections of Kevlar fibers, and polymer dispersed liquid crystals. Results are correlated with simultaneously obtained topographic images. Based on experimental observations, the relative strength of the optical indices of the structures is determined

We present a new method to improve the brightness of tapered fiber probes for near-field scanning optical microscope. The new probes are fabricated by adding high refractive index materials onto the pulled tapered fiber tips before coated with metal. With a tip size of 100 nm, the far-field optical power of the new tapered probe which has 25 nm thickness of zinc sulfide on tip end is about 5 times larger than the same sized traditional fiber probe.

Photon tunneling in the near field of a surface illuminates under Kretschmann's conditions has long been recognized. This has led to a specific scanning probe technique referred to as Photon Scanning Tunneling Microscopy (PSTM). More recently it was shown that AFM tips can be used as convenient optical converters owing to their transparency in the visible of near infrared domain. However the capture mechanism involved in very small silicon tips shows a strong scattering contribution leading to long range resonances. In the present work a series of experiments is presented which rely on infrared (1.06 micrometers ) evanescent wave capture by a Si tip at the surface of a semi-insulating InP prism. From our experimental results it is deduced that the conversion is mainly controlled by the classical dielectric model even though if a strong scattering is induced at the tip end. It is shown that the transmitted light is restricted to a limited solid angle which roughly corresponds to the tip cone angle. An important emission of scattered light is also observed from the outside which follows an exponential proximity dependence. The angular variation of the collected intensity is also studied as a function of the incidence angle; this observation could indicate that an important depth of the tip is involved in the collection mechanism.

Apertured photon scanning tunneling microscopy (APSTM) was used to image the internal structure of an etched optical fiber with an intracore Bragg grating. Direct evidence of the subwavelength periodic variation of the index of refraction along the fiber is obtained by probing evanescent field intensity modulated by the etched fiber grating structure. The images we observed suggests local non-uniformity in the grating structure. Local anomalies appear in the values of the grating spacing. The modulation of the evanescent field intensity originates from variations in the index of refraction as well as from surface structure induced by etching of the fiber.

The new born near field microscopy has already been used in many different topographic and spectroscopic investigations such as topography and spectroscopy. In this communication, we propose to exploit the capacity of a particular optical near field configuration, the so called Scanning Tunneling Optical Microscope (STOM), to scrutinize the field inside a Fabry-Perot cavity. This original analysis is possible due to the detection of evanescent fields by optical tunneling microscopes. In the proposed setup, we insert a STOM tip between the two mirrors of the resonant cavity. The tip is assumed to probe the field inside the resonant cavity. Beside the interest of mapping the standing waves in a cavity with a minimal perturbation, such a configuration allows one to imagine a new near field optical microscope working in dark field regime. Moreover, by setting the cavity in antiresonant mode, the evanescent field on the prism surface can be locally canceled. We will explain theoretically that the light density inside the prism is strongly attenuated since, in this case, the entrance face of the prism plays the role of a highly reflecting mirror. In such conditions, if the tip is brought sufficiently close to the object surface, the resulting coupling with the object will break the antiresonant regime allowing the light to enter the prism. We will show that this effect enhances the sensibility of optical tunneling microscopes. In this communication, we will describe the experimental setup and show the standing waves inside the Fabry-Perot and the first dark field images. These results will be compared with theoretical modelings based on a very precise self-consistent numerical method.

We present a method to measure the actual amplitude of the tip vibration in shear force microscope and near-field scanning optical microscope (NSOM). The method is based on the measurement of the laser light focused on and scattered by the tip. Different from the method already used in NSOM, the equilibrium point of the tip vibration is set to move and both dc and ac part of the scattering light is measured. The dc and ac data can be analyzed to give the actual vibrational amplitude.

A combined scanning shear-force and near-field optical microscope was used to detect the liquid-vapor interface and the smectic layer structure through the thickness of a film of the alkyl cyranobiphenyl liquid crystal 8CB on a HOPG substrate. With noncontact imaging of a thin precursor layer, it was possible to follow the movement of a monolayer of the 8CB. Noncontact imaging combined with polarization SNOM was used to study the birefringent nature of a thin film of a liquid crystal polymer.