Optimized temporal response in multichannel two-photon fluorescence lifetime microscopy using a photonic crystal fibre

The integration of fibre optics into an imaging system for the convenient delivery and collection of light has resulted in many hybrid forms of novel biomedical optical instrumentation. Although it is extremely robust and cost effective, fibre integration requires special consideration in a time‐domain fluorescence lifetime imaging schema where multipath propagation in the fibre causes significant spread in photon transit times. In this study, we investigated the effect of the length of a multimode collection fibre on the temporal performance of a multichannel fluorescence lifetime microscope and demonstrated the effectiveness of a photonic crystal fibre as a means of optimizing the collection and delivery of emitted fluorescence in terms of temporal resolution. The findings are pertinent to all studies that employ a multimode optical fibre to collect and deliver an emitted fluorescence signal from a sample to a remote detector for measurement of the characteristic fluorescence lifetime.     

A computerized microspectrophotometer using fibre optics for transmission and detection of light

A computerized microspectrophotometer was developed to provide rapid and accurate sequential measurements on cells, including nuclear DNA content, by fluorescence staining, photometric grain counting from autoradiographs, and nuclear size estimated by measurement aperture area. To alleviate bulk and vibrations caused by additional stepper motors, the excitation light and filters as well as the emission filters and photomultipliers were detached from the main microscope frame. The light was directed to and from the microscope through fibre optic bundles. This configuration provided an excellent light gathering faculty and made optical alignment easier. The machine provided highly accurate sequential measurements on a relatively large number of cells by static photometry.     

Near-field imaging of organic nanofibres

The optical near‐field of orientated nanofibres of para‐hexaphenyl was investigated by a combination of localized far‐field ultraviolet excitation and scanning near‐field fluorescence detection. Morphological inhomogeneities of the nanofibres together with selective incoupling into the near‐field probe resulted in a detection sensitivity was dependent on individual nanoaggregates. Strongest near‐field intensity and radiation into the far‐field were observed at the crossing points of nanofibres. The near‐field observation of a distance‐dependent damping of the waveguiding along the nanofibres allowed us to determine the imaginary part of the dielectric function of the nanofibres, in quantitative agreement with far‐field optical data.     

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.     

Well-shaped fibre tips by pulling with a foil heater

The length of the molten zone determines the length of pulled optical fibre tips. Tips produced by laser or filament heating are rather lengthy. By using a foil heater the taper length can be shortened and cone angles in the order of 30° can reproducibly be obtained. For varying the drawing force there is an optimum temperature range where the taper shape is monotonic for the whole tip. The tip end diameter is well below 100 nm for optimized pulling conditions.     

Dynamic etching method for fabricating a variety of tip shapes in the optical fibre probe of a scanning near-field optical microscope

A novel etching method for an optical fibre probe of a scanning near-field optical microscope (SNOM) was developed to fabricate a variety of tip shapes through dynamic movement during etching. By moving the fibre in two-phase fluids of HF solution and organic solvent, the taper length and angle can be varied according to the movement of the position of the meniscus on the optical fibre. This method produces both long (sharp angle) and short (wide angle) tapered tips compared to tips made with stationary etching processes. A bent-type probe for a SNOM/AFM was fabricated by applying this technique and its throughput efficiency was examined. A wide-angle probe with a 50° angle at the tip showed a throughput efficiency of 3.3 × 10⁻⁴ at a resolution of 100 nm.     

Automated segmentation of wood fibres in micro‐CT images of paper

A novel algorithm has been developed and validated to isolate individual papermaking fibres in micro‐computed tomographic images of paper handsheets as a first step to characterize the structure of the paper. The three‐step fibre segmentation algorithm segments the papermaking fibres by (i) tracking the hollow inside the fibres via a modified connected component methodology, (ii) extracting the fibre walls using a distance transform and (iii) labelling the fibres through collapsed sections by a final refinement step. Furthermore, postprocessing algorithms have been developed to calculate the length and coarseness of the segmented fibres. The fibre segmentation algorithm is the first ever reported method for the automated segmentation of the tortuous three‐dimensional morphology of papermaking fibres within microstructural images of paper handsheets. The method is not limited to papermaking fibres, but can be applied to any material consisting of tortuous and hollow fibres.     

A scanning near-field optical microscope having scanning electron tunnelling microscope capability using a single metallic probe tip

A new microscope system that has the combined capabilities of a scanning near-field optical microscope (SNOM) and a scanning tunnelling microscope (STM) is described. This is achieved with the use of a single metallic probe tip. The distance between the probe tip and the sample surface is regulated by keeping the tunnelling current constant. In this mode of operation, information about the optical properties of the sample, such as its refractive index distribution and absorption characteristics, can be disassociated from the information describing its surface structure. Details of the surface structure can be studied at resolutions smaller than the illumination wavelength. The performance of the microscope is evaluated by analysing a grating sample that was made by coating a glass substrate with gold. The results are then compared with the corresponding SNOM and STM images of the grating.     

Femtosecond near-field scanning optical microscopy study of molecular thin films

A near-field scanning optical microscope has been combined with a two-colour time-resolved pump-probe measurement system. It has a noise-equivalent transmittance change of 5.0 × 10⁻⁵ for a probe pulse with an intensity of 30 nW. The system has been used for evaluating molecular thin films that have a domain structure, particularly for observing a gate action of the single domains. The results include key features to understand an origin of the domains and suggest that the film composition is uniform over a distance of several micrometres.     

Current-sensing scanning near-field optical microscopy using a metal probe for nanometre-scale observation of electrochromic films

A novel technique for scanning near‐field optical microscopy capable of point‐contact current‐sensing was developed in order to investigate the nanometre‐scale optical and electrical properties of electrochromic materials. An apertureless bent‐metal probe was fabricated in order to detect optical and current signals at a local point on the electrochromic films. The near‐field optical properties could be observed using the local field enhancement effect generated at the edge of the metal probe under p‐polarized laser illumination. With regard to electrical properties, current signal could be detected with the metal probe connected to a high‐sensitive current amplifier. Using the current‐sensing scanning near‐field optical microscopy, the surface topography, optical and current images of coloured WO₃ thin films were observed simultaneously. Furthermore, nanometre‐scale electrochromic modification of local bleaching could be performed using the current‐sensing scanning near‐field optical microscopy. The current‐sensing scanning near‐field optical microscopy has potential use in various fields of nanometre‐scale optoelectronics.     

Imaging of various surface properties of fluorescently labelled phospholipid Langmuir–Blodgett films with a combined scanning probe microscope

We present results of phase separation of a single-component system of 1,2-dihexadecanoyl- sn -glycero-3-phospho-[ N -(4-nitrobenz)-2-oxa-1,3-diazolyl]ethanolamine in which a liquid-condensed (LC) phase co-exists with a liquid-expanded (LE) phase. Domain formation in the co-existence region was studied using a newly developed combined scanning near-field optical microscope–atomic force microscope (SNOM–AFM). We demonstrate for the first time that the topographic, friction, fluorescence and surface potential distributions for a phase-separated single-component Langmuir–Blodgett film between the LE and LC phases can be simultaneously observed using the SNOM–AFM with a thin-step etched optical fibre probe.     

Photon scanning tunneling microscope,now and in the future

The concept of photon tunneling and the principle of the photon scanning tunneling microscope (PSTM) are described. The history of the PSTM and its development in China are reviewed. The principal problem in the recent development of the PSTM, together with its solution, is discussed. The distinguishing features and the future of the PSTM are described.     

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s^?1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.     

Modified fabrication process for aperture probe cantilevers

We report the development of cantilever- and fibre-based probes for scanning near-field optical microscopy. Both probe concepts rely on the integration of a microfabricated aperture tip with reproducible optical and mechanical properties. Numerical calculations were carried out using a finite integration code to investigate the polarization-sensitive transmission behaviour of aperture tips. In order to establish technological guidelines for the optimization of the properties of the optical tip the distinct influence of the tip geometry on the intensity distribution in the vicinity of the aperture is studied in detail.     

Photonic nanopatterns of gold nanostructures indicate the excitation of surface plasmon modes of a wavelength of 50–100 nm by scanning near-field optical microscopy

Scanning near‐field optical microscopy images of metal nanostructures taken with the tetrahedral tip (T‐tip) show a distribution of dark and bright spots at distances in the order of 25–50 nm. The images are interpreted as photonic nanopatterns defined as calculated scanning near‐field optical microscopy images using a dipole serving as a light‐emitting scanning near‐field optical microscopy probe. Changing from a positive to a negative value of the dielectric function of a sample leads to the partition of one spot into several spots in the photonic nanopatterns, indicating the excitation of surface plasmons of a wavelength in the order of 50–100 nm in metal nanostructures.     

Polarization properties of the near-field intensity reflected by metallic and dielectric one-dimensional structures

We investigate the state of polarization and near-field intensity distribution in the vicinity of rectangular groove objects ruled on metallic and dielectric materials. The sample is illuminated from the vacuum side by a linear combination of p- and s-polarised waves. Two rigorous methods of solution are used and compared in calculations of the total intensity at constant height when the light is incident normally onto the surface. Some calculations of the total intensity in the 'follow-the-profile mode' are also presented. It is shown that in the constant height mode, the contrast in the image can be reversed as the plane of observation moves away from the mean plane of the sample. We also found that the state of polarization depends strongly on the material and the distance to the plane of detection.     

A versatile multipurpose scanning probe microscope

A combined scanning probe microscope has been developed that allows simultaneous operation as a non‐contact/tapping mode atomic force microscope, a scattering near‐field optical microscope, and a scanning tunnelling microscope on conductive samples. The instrument is based on a commercial optical microscope. It operates with etched tungsten tips and exploits a tuning fork detection system for tip/sample distance control. The system has been tested on a p‐doped silicon substrate with aluminium depositions, being able to discriminate the two materials by the electrical and optical images with a lateral resolution of 130 nm.     

Near- and far-field second-harmonic imaging of quasi-phase-matching crystals

Second-harmonic scanning near- and far-field optical microscopy of an electric-field poled KTiOPO₄ quasi-phase-matching crystal has been accomplished. This has been done in order to reveal the walls that form the intersections between inverted and non-inverted crystal domains. The domain walls are seen clearly only in images recorded by means of second-harmonic generation because of a large nonlinear contrast, and they appear as bright stripes when studied in a reflection geometry but they are dark when studied in transmission. The images show that the duty cycle of the quasi-phase-matching crystal differs from the ideal and that the walls are not completely smooth. These effects, in combination with the observed scattering from the domain walls, are expected to lower the output of the crystal when used for frequency doubling. We conclude that the wall thickness is no more than approximately 100 nm, which makes it a suitable test object for the resolution capabilities of scanning near-field optical microscopes that are used for nonlinear imaging.     

Near-field fluorescence imaging and simultaneous observation of surface potentials

We present a new detection method to measure simultaneously surface potential and fluorescence intensity distributions using a combined scanning near-field optical microscope-atomic force microscope (SNOM-AFM). A surface potential image of phospholipid monolayers was obtained in non-contact mode using the SNOM-AFM with a thin-step etched optical fibre probe. For applying this technique, a phospholipid of dipalmitoylphosphatidylethanolamine labelled at the head with a nitrobenzoxadiazole group was used as a fluorescent and single component Langmuir–Blodgett film. It is well known that aggregation of the lipid molecules and their fluorescence intensities are very sensitive to its environmental conditions such as humidity and temperature. We demonstrated for the first time the near-field optical imaging and simultaneous observation of surface potentials with Maxwell stress microscopy.