Fluorescence imaging with a laser trapping scanning near-field optical microscope

We investigated fluorescence imaging using a near-field scanning optical microscope which uses a laser-stabilized gold nanoparticle as a near-field probe. This microscope is suitable for observations of biological specimens in aqueous solutions because the probe particle is held by a noncontact force exerted by a laser beam. Theoretical calculations based on Mie scattering theory are presented to evaluate the near-field enhancement by a gold particle of 40 nm diameter. We also present fluorescence images of a single fluorescent bead and discuss the near-field contribution to the fluorescence image in this type of microscope.     

Fluorescence lifetime imaging using a confocal laser scanning microscope

The implementation of a fast fluorescence life-time imaging method in a confocal laser scanning microscope is described. The set up utilizes a low-power continuous wave (CW) argon ion laser equipped with an electro-optic chopper producing nanosecond pulses with a repetition rate up to 25 MHz. A time-gated detection technique enables the measurement of the lifetime of a pixel in 40 μs. The first confocal fluorescence lifetime contrast images are presented. Application of fluorescence lifetime imaging in multilabelling experiments for discrimination between different labels with overlapping emission bands, for probing the local environment of a fluorescent molecule, and for quantitative fluorescence are discussed.     

Imaging 'intact' myofibrils with a near-field scanning optical microscope

Fluorescently labelled myofibrils were imaged in physiological salt solution by near-field scanning optical microscopy and shear-force microscopy. These myofibrils were imaged in vitro , naturally adhering to glass while retaining their ability to contract. The Z-line protein structure of the myofibrils was antibody labelled and easily identified in the near-field fluorescence images. The distinctive protein banding structure of the myofibril was also seen clearly in the shear-force images without any labelling requirement. With the microscope in the transmission mode, resolution of the fluorescence images was degraded significantly by excessive specimen thickness (>1 μm), whereas the shear-force images were less affected by specimen thickness and more affected by poor adherence to the substrate. Although the exact mechanism generating contrast in the shear-force images is still unknown, shear-force imaging appears to be a promising new imaging modality.     

Magnetic domain imaging with a scanning near-field optical microscope using a modified Sagnac interferometer

We report on the combination of a scanning near-field optical microscope and a modified Sagnac interferometer for magnetic-domain imaging in the reflection mode. The Sagnac interferometer is used for detection of the magneto-optical Kerr effect. Since the interferometer is inherently insensitive to polarization changes caused by topography effects, magnetic-domain imaging is not limited to samples with flat surfaces. In this way, it is possible to image magnetic bits written on the tracks of a magneto-optical disc that has a rather pronounced surface profile.     

Magnetic domain imaging with a scanning near-field optical microscope using a modified Sagnac interferometer

We report on the combination of a scanning near-field optical microscope and a modified Sagnac interferometer for magnetic-domain imaging in the reflection mode. The Sagnac interferometer is used for detection of the magneto-optical Kerr effect. Since the interferometer is inherently insensitive to polarization changes caused by topography effects, magnetic-domain imaging is not limited to samples with flat surfaces. In this way, it is possible to image magnetic bits written on the tracks of a magneto-optical disc that has a rather pronounced surface profile.     

A storage Dewar near-field scanning optical microscope

A near-field scanning optical microscope for operation within a storage Dewar is described. It was designed for studies of opaque samples and operates in the collection mode. Illumination can be either through the tip or from the side via a separate fiber. Scans can be begun within 2 h after start of cooldown. Its rigid design allows high resolution and long scans with no additional vibration isolation. To illustrate its performance, measurements of photoluminescence in GaAs/AlGaAs heterostructures are presented. The signal and noise levels for the two illumination modes are examined.     

Shear force imaging of DNA in a near-field scanning optical microscope (NSOM)

A near-field scanning optical module has been constructed as an accessory for a Nanoscope IIIa commercial scanning probe microscope. Distance feedback and topographic registration are accomplished with an uncoated optical fibre scanning tip by implementation of the shear force technique. The tip is driven by a piezoelectric actuator at a resonance frequency of 8–80 kHz. A laser diode beam is scattered by the tip and detected by a split photodiode, with lock-in detection of the difference signal. The amplitude ( r ) and phase (τ) responses were characterized as a function of the calibrated tip–sample separation. Using an r cos τ feedback signal, imaging of pUC18 relaxed circular plasmid DNA spread on mica precoated with cetylpyridinium chloride was achieved. The apparent width (28 ± 5 nm) was approximately four times that achieved by scanning force measurements with the same instrument; the apparent height of the DNA (0.6 ± 0.3 nm) was similar with the two techniques. These results demonstrate the applicability of the shear force signal for imaging biological macromolecules according to topography and in conjunction with the optical signals of a near-field scanning optical microscope (NSOM).     

Mid-infrared scanning near-field optical microscope resolves 30 nm

We explore the performance of a scanning near-field infrared microscope, which works by scattering tightly focused CO2 laser radiation (lambda = 10 microm) from the apex of a metallized atomic force microscope tip. The infrared images of test samples prove a spatial resolution of 30 nm and are free of topographical and inertial artefacts, thus they should be of great interest for practical applications. We also observe that the infrared contrast vanishes when the input beam polarization is orthogonal to the tip axis, in accordance with theoretical expectations for a mechanism of longitudinal field interaction.     

Optical-fibre scanning near-field optical microscope for cryogenic operation

We have developed a new type of scanning near-field optical microscope (SNOM) utilizing optical fibres. The probe tip is controlled by shear force feedback with a fibre interferometer and signal light is collected directly by a multimode fibre. These features make the SNOM head more compact and less sensitive to vibration. Further advantages of this new type of SNOM are that it obviates the need for optical windows in the cryostat and offers easy optical alignment.     

Optical-fibre scanning near-field optical microscope for cryogenic operation

We have developed a new type of scanning near-field optical microscope (SNOM) utilizing optical fibres. The probe tip is controlled by shear force feedback with a fibre interferometer and signal light is collected directly by a multimode fibre. These features make the SNOM head more compact and less sensitive to vibration. Further advantages of this new type of SNOM are that it obviates the need for optical windows in the cryostat and offers easy optical alignment.     

The scanning near-field optical microscope as a tool for proteomics

The identification of the entire genetic code of human DNA is more or less completed. With this knowledge, research in identifying the real information lying in the genes, will begin. This information is contained in the proteins, which are the main biological actors in the cell. For this reason proteins will be targeted in biological investigations in the future. The structure, affinity and reactivity of each identified protein has to be determined, which is a primary goal in the field of proteomics. This will require new and better strategies to identify protein-protein interaction. Our approach, based on the detection and visualization of single proteins by scanning near-field optical microscopy (SNOM), has allowed us to visualize various fixed and fluorochrome-labelled proteins at the nanometer scale. Subsequently SNOM may then be developed to efficiently detect the specific behavior of a certain protein in response to other biomolecules.     

Vibration sensitivity of the scanning near-field optical microscope with a tapered optical fiber probe

In this paper the Rayleigh-Ritz method was used to study the scanning near-field optical microscope (SNOM) with a tapered optical fiber probe's flexural and axial sensitivity to vibration. Not only the contact stiffness but also the geometric parameters of the probe can influence the flexural and axial sensitivity to vibration. According to the analysis, the lateral and axial contact stiffness had a significant effect on the sensitivity of vibration of the SNOM's probe, each mode had a different level of sensitivity and in the first mode the tapered optical fiber probe was the most acceptive to higher levels of flexural and axial vibration. Generally, when the contact stiffness was lower, the tapered probe was more sensitive to higher levels of both axial and flexural vibration than the uniform probe. However, the situation was reversed when the contact stiffness was larger. Furthermore, the effect that the probe's length and its tapered angle had on the SNOM's probe axial and flexural vibration were significant and these two conditions should be incorporated into the design of new SNOM probes.     

Inducing superconductivity at a nanoscale: photodoping with a near-field scanning optical microscope

The local modification of an insulating GdBa2Cu3O6.5 thin film, made superconducting by illumination with a near-field scanning optical microscope (NSOM), is reported. A 100-nm aperture NSOM probe acts as a sub-wavelength light source of wavelength lambda(exc) = 480-650 nm, locally generating photocarriers in an otherwise insulating GdBa2-Cu3O6.5 thin film. Of the photogenerated electron-hole pairs, electrons are trapped in the crystallographic lattice, defining an electrostatic confining potential to enable the holes to move. Reflectance measurements at lambda = 1.55 microm at room temperature show that photocarriers can be induced and constrained to move on a approximately 200 nm scale for all investigated lambda(exc). Photogenerated wires present a superconducting critical temperature Tc= 12 K with a critical current density Jc = 10(4) A cm(-2). Exploiting the flexibility provided by photodoping through a NSOM probe, a junction was written by photodoping a wire with a narrow (approximately 50 nm) under-illuminated gap. The strong magnetic field modulation of the critical current provides a clear signature of the existence of a Josephson effect in the junction.     

Inducing superconductivity at a nanoscale: photodoping with a near-field scanning optical microscope

The local modification of an insulating GdBa₂Cu₃O_6.5 thin film, made superconducting by illumination with a near-field scanning optical microscope (NSOM), is reported. A 100-nm aperture NSOM probe acts as a sub-wavelength light source of wavelength λ_exc = 480–650 nm, locally generating photocarriers in an otherwise insulating GdBa₂–Cu₃O_6.5 thin film. Of the photogenerated electron–hole pairs, electrons are trapped in the crystallographic lattice, defining an electrostatic confining potential to enable the holes to move. Reflectance measurements at λ = 1.55 μm at room temperature show that photocarriers can be induced and constrained to move on a ≈200 nm scale for all investigated λ_exc. Photogenerated wires present a superconducting critical temperature T _c = 12 K with a critical current density J _c = 10⁴ A cm⁻². Exploiting the flexibility provided by photodoping through a NSOM probe, a junction was written by photodoping a wire with a narrow (≈ 50 nm) under-illuminated gap. The strong magnetic field modulation of the critical current provides a clear signature of the existence of a Josephson effect in the junction.     

Fluorescence scanning near-field optical microscopy of polyfluorene composites

Fluorescence scanning near-field optical microscopy (SNOM) is used to investigate binary polyfluorene-based composites of varying composition. The samples investigated contain blends of the polymer poly(9,9'-dioctylfluorene-cobenzothiadiazole), F8BT, with similar polyfluorenes of wider band gap. Images acquired from a film containing 50% by weight F8BT exhibit a high degree of correlation between the topography and fluorescence, with an F8BT-rich phase which protrudes from the surface of the film forming isolated regions with sizes from hundreds of nanometres to several micrometres. A film containing 10% by weight F8BT also has micrometre-size F8BT-rich regions, but also present are small and locally varying proportions of F8BT in the other polyfluorene component phase, indicating a hierarchy of phases within this sample. The fluorescence and topographic images of a third sample studied, containing 90% by weight F8BT, display no correlation, demonstrating that it is not always appropriate to use topographic information to determine the phase structure within polymer blends. The fluorescence SNOM images acquired from these samples are able to assist our understanding of the photovoltaic efficiency of devices fabricated from these films, which are governed by the extent of the interfacial area between these two constituent polymers.     

Fluorescence imaging and investigations of directly labelled chromosomes using scanning near-field optical microscopy

Scanning near-field optical microscopy (SNOM) has been successfully employed to generate high resolution (<100nm) fluorescence images of directly tagged human chromosomes. Direct tagging, fluorescence in-situ hybridisation processes (with and without amplification) are investigated and their fluorescence response to near-field excitation are compared. Using the simultaneous topography mode of SNOM, chromosome morphology was seen to differ as a result of the two processes; with chromatin collapse more extensive when the amplified direct tagging procedure was used. The results are discussed in the context of developing locus specific direct tags together with high resolution SNOM imaging for the observation of chromosome aberrations.     

The height regulation of a near-field scanning optical microscope probe tip

A nonoptical detection of the optical fibre tip has been developed. By detecting the output signal from a tiny piezoelectric detector attached to the vibrating fibre tip, the distance between the fibre tip and the sample has been successfully controlled. The frequency responses of the system composed of tip, the dither and the detector have been studied. The difference between the shear-force detection and the tapping-mode detection is discussed. It is found that the shear force exerted on the tip reduces the vibration amplitude with an unvaried resonance frequency. However, in the tapping mode, the resonance frequency varies with the tip–sample distance as the force is exerted on the fibre tip only within a half period. This requires better adjustments for the tapping-mode detection.     

Implementation of a short-tip tapping-mode tuning fork near-field scanning optical microscope

We present the implementation of a short‐tip tapping‐mode tuning fork near‐field scanning optical microscope. Tapping frequency dependences of the piezoelectric signal amplitudes for a bare tuning fork fixed on the ceramic plate, a short‐tip tapping‐mode tuning fork scheme and an ordinary tapping‐mode tuning fork configuration with an 80‐cm optical fibre attached are demonstrated and compared. Our experimental results show that this new short‐tip tapping‐mode tuning fork scheme provides a stable and high Q factor at the tapping frequency of the tuning fork and will be very helpful when long optical fibre probes have to be used in an experiment. Both collection and excitation modes of short‐tip tapping‐mode tuning fork near‐field scanning optical microscope are applied to study the near‐field optical properties of a single‐mode telecommunication optical fibre and a green InGaN/GaN multiquantum well light‐emitting diode.     

Probe design optimization for a high-resolution scattering-type scanning near-field optical microscope

The scattering-type scanning near-field optical microscope (SNOM) has a probe with a sharp tip for use in high resolution imaging. As sharp a tip as possible is generally considered ideal for the observations, but actually, a sharp tip does not always provide a high resolution SNOM image. We numerically examined the scattering property of the SNOM probe by the three dimensional finite difference time domain method. In this paper, we show the criterion for the ideal scattering probe which satisfies the simple relation between radius and taper angle of the tip.     

The height regulation of a near-field scanning optical microscope probe tip

A nonoptical detection of the optical fibre tip has been developed. By detecting the output signal from a tiny piezoelectric detector attached to the vibrating fibre tip, the distance between the fibre tip and the sample has been successfully controlled. The frequency responses of the system composed of tip, the dither and the detector have been studied. The difference between the shear-force detection and the tapping-mode detection is discussed. It is found that the shear force exerted on the tip reduces the vibration amplitude with an unvaried resonance frequency. However, in the tapping mode, the resonance frequency varies with the tip-sample distance as the force is exerted on the fibre tip only within a half period. This requires better adjustments for the tapping-mode detection.