Optical fiber sensors have been widely used in analytical chemistry, physiology, biology and other areas. With the development of sub-micron chemical fiber optical sensors with millisecond response times, it becomes possible to combine the capacity of biochemical sensing with well- established scanning probe microscopies, such as AFM, NSOM. Using near-field optical probe and microscopy, we can simultaneously obtain topographic and optical images of a variety of samples. By implementing a commercial NSOM system with the chemical sensor, we are developing an optical fiber-based scanning sensor microscope using evanescent wave excitation and chemically modified optical fiber probes. Further application of such microscope will be discussed. The major advantage of this microscopic imaging technique is its potential capability of topographic and biochemical imaging of a sample.

Scanning near-field optical microscopy and Raman scattering were combined to obtain subwavelength molecular resolution. Near-field microscopy allows to overcome the diffraction limit valid for all lens or mirror based optical instruments and lateral resolutions well below 100 nm have been claimed. Raman spectroscopy yields information on the vibrational states of a molecule and therefore allows to distinguish between different chemical compounds easily. This is an advantage to the more widely used near-field fluorescence microscopy. To enhance the notoriously weak near-field Raman signal the sample was brought onto a Raman enhancing surface (silver coated Teflon nanospheres). Additionally brilliant cresyl blue acted as a Raman label for our DNA samples. On such samples Raman images with a resolution better than 100 nm have been obtained. A single near-field SERS spectrum was measured in approximately 60 seconds. The acquisition time currently depends critically on the transmission of the near-field probes. Nevertheless, it is possible to measure whole near-field Raman images in a reasonable time. From these Raman images a preliminary distinction of different constitutions of adsorbed molecules can be done.

The reflection-back-to-the-fiber SNOM under shear-force control with sharp and cold uncoated tips provides reliable submicroscopic resolution on rough surfaces without topographic artifacts. The high lateral resolution is the result of a sudden increase in reflectivity when the tip goes close to the surface at about 50% vibration damping. This technique has been tested for samples of piratical importance in the field of organic colorants and for Raman spectroscopy.

A new technique developed in this laboratory and based on near-field scanning optical microscopy (NSOM) is used to study the field-induced reorientation of molecules in local regions of thin film materials. A highly concentrated electric field is applied across the sample, between the metal-coated near-field probe and the sample substrate. Molecular motion induced in the sample by modulation of the electric field is observed using NSOM methods. Lock-in detection of the optical response to a sinusoidally- modulated field, recorded under cross-polarized, transmitted-light conditions allows for the recording of dynamics images. The local rate of reorientation is measured for individual locations in a sample by recording the response in either the time or frequency domains. Dynamics information is obtained with microsecond(s) ec time resolution and nanometer-scale spatial resolution. This method is applied in studies of polymer-dispersed liquid crystal films. In these materials, small droplets of nematic liquid crystal are dispersed in an otherwise uniform poly(vinyl alcohol) film. The liquid crystal droplets are birefringent, forming electrically-switchable light-scattering centers. A simple forced-oscillator model for the reorientation dynamics in the liquid crystal is presented. Variations in the time scale and extent of molecular reorientation are observed as a function of field strength, droplet size, droplet shape, and position probed. The data are interpreted based on knowledge of the important intermolecular forces active in these materials.

A novel pipette SNOM/AFM probe has been developed for its simple fabrication and applicability to wide wavelength range. The pipette probe is simply fabricated by a successive process of pulling, bending with a CO₂ laser, making a hole and coating with a metal layer. The hole is made on the tube at the back side of the tip for applying a light. The pipette probe is mounted on the SNOM/AFM system which includes a confocal microscope. The light is introduced to the hole directly by focusing from a confocal microscope or through an optical fiber. The probe provided clear topographic and optical images for a sample of a patterned chromium layer on a glass substrate and fluorescence beads. A confocal images was also obtained in a wide area of the same samples.

A scanning near-field optical/atomic-force microscope (SNOAM) system was applied to simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic-force microscopy cantilever and a scanning near-field optical microscopy probe. The SNOAM system used 458 or 488 nm from Ar ion laser multiline of excitation of green fluorescent protein (GFP), since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E.coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope.

Single adeno- and tobacco mosaic viruses have been studied by correlated fluorescence spectroscopy and tapping-mode atomic force microscopy. The size and shape of spatially isolated, fluorescently tagged viruses are measured on the nanometer scale, and the fluorescent labels in each virus are determined by wavelength-resolved spectroscopy. This work extends ultrasensitive measurement to the single-virus level and is expected to have applications in studying gene therapy vectors and virus-cell interactions.

Advancements in near-field scanning optical microscopy (NSOM) tip design as well as an interferometric feedback mechanism are presented for the common goal of imaging living biological samples under physiological conditions. The ability of a cantilevered tip to track the subtle topography changes of a fragile lipid film in an aqueous environment is demonstrated. In order to further the imaging capabilities, the probes have been chemically etched to reduce the spring constants of the tips, thereby lowering the forces imparted on the sample. An optical feedback mechanism used as an alternative to the conventional force feedback is also described. Utilizing this optical feedback mechanism, images have been obtained of fixed cells underwater. Finally, progress towards modifying the NSOM tip for chemical sensor applications is discussed in the context of eventually measuring ion fluxes through single protein channels. Together these advancements demonstrate the potential of NSOM for studying live cells.

Fluorescence microscopy on single light harvesting 2 complexes from photosynthetic bacterium Rhodopseudomonas Acidophila strain 10050 has been carried out. The polarization of the fluorescence emitted by individual complexes immobilized on a surface was detected as a function of time. Abrupt changes in the polarization of the emitted light of single chromophores were observed. These abrupt changes are attributed to a localization of the exciton on a small fraction of the chromophores on the closely coupled ring shaped aggregate of the complex.

The physical adsorption and subsequent drying of biomolecules on surfaces is a common manufacturing step throughout the bio-diagnostics industry. To address the lack of knowledge concerning the molecular details of such commercially important processes, we have studied the effect of drying on absorbed layers of protein using the AFM. AFM images of adsorbed layers of monoclonal antibody on polystyrene microtitre plates after drying exhibited non- homogeneous distributions of protein reminiscent of the de- wetted structures often seen with polymer thin-films. Variation of the drying rate resulted in notably different protein distributions at micrometer and sub-micrometer scales. No obvious effect on the polystyrene microtitre plate nano-topography on the de-wetted distributions of antibody was apparent. To further explore the possible effects of nano-topography on the de-wetted distributions of antibodies and other proteins during drying, highly oriented pyrolytic graphite, with a surface topography consisting of hydrophobic basal plane areas disrupted by edge plane defects, has been studied. For this surface, de-wetting during drying of adsorbed antibody and bovine serum albumin layers appears to be significantly influenced by the edge plane defects including specific parameters such as the edge plane defect density.

The identification of specific binding molecules is of increasing interest in the context of drug development based on combinatorial libraries. Scanning Probe Microscopy (SPM) is the method of choice to image and probe individual biomolecules on a surface. Functional identification of biomolecules is a first step towards screening on a single molecule level. As a model system we use recombinant single- chain Fv fragment (scFv) antibody molecules directed against the antigen fluorescein. The scFv's are covalently immobilized on a flat gold surface via the C-terminal cysteine, resulting in a high accessibility of the binding site. The antigen is immobilized covalently via a long hydrophilic spacer to the silicon nitride SPM-tip. This arrangement allows a direct measurement of binding forces. Thus, closely related antibody molecules differing in only one amino acid at their binding site could be distinguished. A novel SPM-software has been developed which combines imaging, force spectroscopic modes, and online analysis. This is a major prerequisite for future screening methods.

Surface potential measurement on organic thin films and isolated biological samples have been performed using a scanning Maxwell-stress microscopy (SMM). The SMM is a type of scanning probe microscopy designed to image microscopic electrical properties, such as the local surface potential distribution. The present microscope afforded information of the lateral distribution of molecular dipole moments in phase separated monolayer, and the topographic image of purple membrane fragments as well as dielectric properties of samples.

Scanning near-field optical/atomic-force microscopy was first applied to detect fluorescence hybridization of DNA immobilized on nano-particle media. Hybridization can also be used to determine the sequence of unknown DNA. 100-nm in diameter of polystyrene sphere carboxylate was used as the nano-particle media. Template DNA including target sequence was chemically modified with animo group at the 5'-end of single-stranded DNA. Amine-coupling reaction made covalent bond between template DNA and carboxyl group on the surface of the media. Single-stranded DNA of specific base sequence labeled either fluorescent dye, that is used to detect the complementary base sequence by hybridization. Simultaneous imaging the colloidal particles showed us topography and near-field fluorescence images of them. All particles were observed in the topographic image, however, some particles were realized in the fluorescence image. This result indicated that fluorescent hybridized DNAs on the surface of the media were visualized specifically. High density arrays or integration of media is a fast and effective means of accessing the gene variation. However, in this study, detection of hybridized fluorescent DNA conjugated with particle is a major purpose rather than arrangement technique.

We have studied, using AFM, the structural basis of the outer membrane permeability for the bacterium E. col. The surface of the bacteria is visualized with an unprecedented details. Our AFM images clearly reveal that the outer membrane exhibits protrusions, which correspond to patches of LPS containing hundreds to thousands of LPS molecules. The packing of the nearest neighbor patches is tight, and as such the LPS layer provides an effective permeability barrier for the Gram-negative bacteria. We have also studied the mechanism of their permeability increase upon metal depletion. Our AFM images reveal that LPS molecules are released from the boundaries of some patches during the initial EDTA treatment. Further metal depletion produces a very distinct structure at the outer membrane: appearance of irregularly shaped pits. The pits are likely formed as a result of liberation of LPS patches and lipoproteins, exposing areas of peptidoglacan surface. Our study has proven AFM to be a very useful technique in providing structural basis for the functions of organisms.

A fine structure of bacteria flagella is an important problem of molecular cell biology. Bacteria flagella are the self-assembled structures that allow to use the flagellum protein in a number of biotechnological applications. However, at present, there is a little information about high resolution scanning probe microscopy study of flagellum structure, in particular, about investigation of Vibrio cholerae flagella. In our lab have been carried out the high resolution comparative investigation of V. cholerae flagella by means of various microscopes: tunneling (STM), scanning force (SFM) and electron transmission. As a scanning probe microscope is used designed in our lab versatile SPM with replaceable measuring heads. Bacteria were grown, fixed and treated according to the conventional techniques. For STM investigations samples were covered with Pt/Ir thin films by rotated vacuum evaporation, in SFM investigations were used uncovered samples. Electron microscopy of the negatively stained bacteria was used as a test procedure.

The supramolecular formation of tetracarboxyphenylporphyrin (TCPP) and anti-TCPP antibody was studied by a biosensor (BIAcore) and atomic force microscope (AFM). Results of BIAcore for the complexes of antibodies with TCPP suggested the supramolecular formation with the binding of two antibodies and a porphyrin molecule. AFM images of cyclic trimer, pentamer and linear oligomers constituted by antibodies and porphyrin dimers were obtained.

We applied atomic force microscopy (AFM) to the analysis and classification of metaphase chromosomes. Human chromosomes were isolated from blood and spread over a glass substrate. We found that air-dried and Giemsa stained chromosomes had a granular surface and the height of approximately 250 nm; however unstained chromosomes had a smooth surface and the height was approximately 100 nm. Giemsa staining caused swelling of the chromosome structure. For the structural analysis, chromosomes were treated with hyaluronidase or a citric acid buffer. The effects of the treatments on chromosomal components, spiral structure and 30-nm solenoid fiber were observed. Each step of G-banding treatments of chromosomes was also visualized by AFM. The trypsin treatment collapsed the chromosomes and subsequent Giemsa staining caused dramatically reswelling of the chromosomes. The height of the G-positive region was approximately 200 nm but the unstained region was approximately 50 nm. The difference in thickness observed was produced by binding of the dye. The AFM image of the banding patterns of treated chromosomes was clearer than the image obtained with an optical microscope. These images made it possible to visualize the karyotyping of chromosomes using AFM. Detection of in situ hybridization using AFM and microdissection of chromosomes using AFM were also investigated.

Recently we have derived the angular spectrum transfer function (ASTF) for photon scanning tunneling microscope and collection mode scanning near field optical microscope (collection-SNOM) under dark illumination. The paper present discussed image restoration using the ASTF. It was found that the key to achieve better restoration is to find a ASTF as close to that of the system as possible, which then depends on the determination of tip size, tip sample distance, incident angle of illuminating laser beam, etc. When low values are used for tip diameter and tip sample distance improvement of image quality is obvious after restoration. But when the value adopted for tip sample distance is 0.05 (lambda) higher than real one, high frequency oscillation in the restored image become intolerable, which we call over restoration. Slight over restoration was also observed when the value employed for tip diameter is 0.02 (lambda) larger than real one. The appearance of over restoration on the other hand can be utilized for the estimation of tip diameter and tip sample distance of the system. Generally the image seems more sensitive to tip sample distance than to tip size.

We describe a near-field apertureless fluorescence microscope, capable of imaging fluorescent latex beads with subwavelength precision. The instrument is based on a home- built tapping-mode atomic-force microscope, to which an inverted optical microscope was added. The fact that the wavelength of the fluorescence that we observe is different from the wavelength of the illumination allows for a relatively straightforward detection mechanism. Sample images are presented, along with evidence that the observe effect is of optical origin.