Near-field Scanning Optical Microscopy Probe Research Articles

Contrast enhancement using polarization-modulation scanning near-field optical microscopy (PM-SNOM)

Polarization contrast scanning near-field optical microscopy (SNOM) provides information on the orientation of molecules and molecular aggregates on surfaces. Other than in transmission or fluorescence SNOM, the control of polarization demands for SNOM probes having extinction ratios better than 20 : 1 in at least two perpendicular polarization directions. Most common SNOM probes consist of tapered and metal-coated fiber tips. While the birefringence of fibers can be compensated, the tapering often exhibits depolarizing effects that make good qualitative or even quantitative measurements difficult. In an attempt to improve the quality of the apertures, we have modified metal-coated fiber probes with a focused ion beam (FIB). As an example of investigations where a very good polarization control is needed, we present and discuss polarization-modulation SNOM (PM-SNOM) measurements on dye crystals. The modulation of the direction of linearly polarized light and the use of lock-in techniques allows the simultaneous detection of absorption together with magnitude and orientation of optical anisotropy.

人工膜と生体膜の接点を探る 最近の膜研究の方法 原子間力制御型近視野顕微鏡による生体イメージング

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 (AFM) cantilever and a scanning near-field optical microscopy (NSOM) probe. Optical resolution of this system indicated about 50-100 nm in a fluorescence mode for fluorescent latex beads on a quartz glass plate. The SNO-AM 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 fluoresence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. Nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, individual E. coli cells expressed different fluorescent intensity. Fluorescence obtained by SNOAM indicated GFP oxidation possibly occured near cell surface. SNOAM also provided us with simultaneous topographical and optical images of human chromosomes. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. Topographic images clearly indicated duplicated structure on metaphase chromosome, while fluorescence images were a different shape probably because it depended on the combination of SYBRTM Green I and chromosome DNA. Atomic force images have some artefacts, however they can be corrected by comparison with the fluorescence image. Topography and fluorescence images of RBL-2H3 mast cells surface were determined with/without DNP-BSA stimulation by SNOAM system. Near-field fluorescence images were obtained from the granules stained with quinacrine. Fluorescence profiles and intensities were largely changed after allergen stimulation. Exocytotic events of granules were specially discussed based on SNOAM.

Simultaneous topographic and fluorescence imagings of recombinant bacterial cells containing a green fluorescent protein gene detected by a scanning near-field optical/atomic force microscope.

A scanning near-field optical/atomic force microscope (SNOAM) system was applied for 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 as a scanning near-field optical microscopy probe. Optical resolution of this system was about 50-100 nm in fluorescence mode for fluorescent latex beads on a quartz glass plate. Green fluorescent protein (GFP) is a convenient indicator of transformation and should allow cells to be separated by fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant Escherichia coli. The SNOAM system used 458- or 488-nm irradiation from a multiline Ar ion laser for excitation of 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. A nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, it can be seen that individual E. coli cells expressed different fluorescence intensities. Fluorescence obtained by SNOAM indicated that GFP oxidation possibly occurred near the cell surface. A SNOAM system also indicated the possibility of precise imaging of native cells in liquid.

Fabrication of multimode fiber tapers with a high reproducibility of the subwavelength extremity size: application to scanning near-field optical microscope probes

A new method for the fabrication of multimode fiber tapers is presented. The process is aimed at elaborating fiber tips with a subwavelength size extremity. After creating a Biconical Taper (BT) with a standard fusion splicer, the elongated part is immersed in an HF acid solution. The light transmitted through the BT is collected by a photodiode, and thus the chemical etching is monitored by analysing the transmitted power. After calibration, it gives rise to a high reproducibility of the tip extremity with a subwavelength size. It has been applied to the fabrication of UV-Scanning Near-Field Optical Microscope probes. Multimode fibers are only available for UV light.

Studying topography and sub-surface structure of 8CB liquid crystal films with shear-force microscopy

Scanning shear-force microscopy, which is usually employed to track scanning near-field optical microscopy probes across samples, was used to observe the sub-surface smectic layer structure through the thickness of a film of the alkyl cyanobiphenyl liquid crystal 8CB on a highly oriented pyrolytic graphite substrate from force-distance measurements. With the noncontact imaging shear-force microscopy supplies, it was possible to follow, in the topography, the movement of a monolayer of the 8CB molecules. This has enabled the diffusion of molecules in a precursor film of 8CB to be observed without significantly disrupting the free liquid surface.

Investigations of liquid crystals and liquid ambients using near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) has been utilized to study the behavior of liquid crystals at room temperature. The material, 4′-octyl-4-biphenylcarbonitrile (8cb) was studied at the air-liquid interface. The NSOM probe is shown to take an active role in modifying, and subsequently reading, the liquid crystal with ∼ 65 nm resolution. The optical modification is proven to be independent of topographic modification. Also, NSOM operation using shear force feedback in water is demonstrated with better than 50 nm lateral spatial resolution.

Thermal/optical effects in NSOM probes

The coupling of thermal and optical effects in near-field scanning optical microscope (NSOM) fitted with a coated tapered fiber probe is considered. Heating of the probe occurs due to the absorption of light in the metal coating in the tapered region of the probe at relatively large (tens of microns) distances from the probe apex. Conduction of heat that occurs with a time constant of tens of milliseconds results in an elevated probe temperature over a rather large region of the taper. This in turn, affects the optical throughput of the NSOM. Experimental measurement of thermal/optical coupling and a discussion of coupling mechanisms is included.

Modification of the optical properties of liquid crystals using near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) has been utilized to study the behavior of liquid crystals at room temperature. The material, 4′-octyl-4-biphenylcarbonitrile (8cb) was studied at the air–liquid interface. The NSOM probe is shown to take an active role in modifying, and subsequently reading, the liquid crystal with about 65 nm resolution. The optical modification is proven to be independent of topographic modification. Results indicate potential technological uses in data storage and displays.