Fluorescence lifetime imaging with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.     

Observation of the dynamics of live cardiomyocytes through a free-running scanning near-field optical microscopy setup

We report the observation of live-cell dynamics by noncontact scanning near-field optical microscopy (SNOM) modified to work with living biological samples that are fully immersed in liquid. We did not use the SNOM setup in strictly near-field conditions (we used 1-mum constant-height mode); however, we could examine the dynamics of rhythmically beating cardiac myocytes in culture with extremely high vertical sensitivity below the nanometric range. We could halt scans at any point to record localized contraction profiles of the cell membrane. We show that the contractions of the organisms changed shape dramatically within adjacent areas. We believe that the spatial dependency of the contractions arises because of the measurement system's ability to resolve the behavior of individual submembrane actin bundles. Our results, combining imaging and real-time recording in localized areas, reveal a new, to our knowledge, noninvasive method for using SNOM setups for studying the dynamics of live biological samples.     

Direct near-field optical imaging of higher order plasmonic resonances

We map in real space and by purely optical means near-field optical information of localized surface plasmon polariton (LSPP) resonances excited in nanoscopic particles. We demonstrate that careful polarization control enables apertureless scanning near-field optical microscopy (aSNOM) to image dipolar and quadrupolar LSPPs of the bare sample with high fidelity in both amplitude and phase. This establishes a routine method for in situ optical microscopy of plasmonic and other resonant structures under ambient conditions.     

High spatial resolution imaging with near-field scanning optical microscopy in liquids.

The mechanism of tuning fork-based shear-force near-field scanning optical microscopy is investigated to determine optimal experimental conditions for imaging soft samples immersed in liquid. High feedback sensitivity and stability are obtained when only the fiber probe, i.e., excluding the tuning fork prongs, is immersed in solution, which also avoids electrical shorting in conductive (i.e., buffer) solutions. Images of MEH-PPV were obtained with comparable spatial resolution in both air and water. High optical resolution (approximately160 nm fwhm) was observed.     

Absorption coefficient imaging by near-field scanning optical microscopy in bacteria

We present a method for obtaining a position-dependent absorption coefficient from near-field scanning optical transmission microscopy. We show that the optical transmission intensity can be combined with the topography, resulting into an absorption coefficient that simplifies the analysis of different materials within a sample. The method is tested with the dye rhodamine 6G, and we show some analysis in biological samples such as bacteria KIebsiella pneumoniae and Pseudomonas aeruginosa. The calculated absorption coefficient images show important details of the bacteria, in particular for P. aeruginosa, in which membrane vesicles are clearly seen.     

Scanning near-field optical data contrast measurement: a tomographylike near-field reconstruction

A method for the reconstruction of near-field optical signals is proposed to produce a tomographylike map of the field above the nanostructures being studied. The data obtained through lock-in detection are processed employing Chebyshev's polynomial function of the distance between the probe and the sample. The method is first applied to numerically generated near-field evanescent data, with three different decreasing lengths, and then applied to an experimental signal. Therefore the contrast of the signal above nanostructures is discussed to underline the discrepancy between the scanning near-field optical microscopy data and the reconstructions.     

Heating mechanisms in a near-field optical system

A finite-difference-time-domain and two finite-difference-thermal models are used to study various heating mechanisms in a near-field optical system. It is shown that the dominant mechanism of sample heating occurs from optical power that is transferred from the probe to a metallic thin-film sample. The optical power is absorbed in the sample and converted to heat. The effects of thermal radiation from the probe 's coating and thermal conduction between the probe and the sample are found to be negligible. In a two-dimensional waveguide with TE polarization, most of the optical power is transferred directly from the aperture to the sample. In a two-dimensional waveguide with TM polarization, there is significant optical power transfer between the probe 's aluminum coating and the sample. The power transfer results in a wider thermal distribution with TM polarization than with TE polarization. Using computed temperature distributions in a Co -Pt film, we predict the relative size of thermally written marks in a three-dimensional geometry. The predicted mark size shows a 30 % asymmetry that is due to polarization effects.     

Design and optimization of a near-field scanning optical microscope for imaging biological samples in liquid

We describe a near-field scanning optical microscope capable of imaging biological samples in liquid. The microscope uses a straight optical fiber near-field probe and optical shear-force feedback for tip-sample distance regulation. Physical aspects of the design are discussed, and phenomena related to operation in liquid are revealed. Careful calibration of the instrument in air and in liquid is shown, and for the first time to our knowledge, near-field fluorescence images of a biological cell in liquid are presented.     

Optical near-field Raman imaging with subdiffraction resolution

We report optical near-field Raman imaging with subdiffraction resolution (approximately 120 nm) without field enhancement effects. Chemical discrimination on tetracyanoquinodimethane organic thin films showing localized salt complexes is accomplished by detailed Raman maps. Acquisition times that are much shorter than previously reported are due to the high Raman efficiency of the materials and to careful collection and detection of the optical signals in our near-field Raman spectrometer.     

Theoretical study of near-field optical storage with a solid immersion lens

Both the reflection inside a hemisphere solid immersion lens (SIL) and the reflection inside the gap between the SIL and the optical recording medium are considered. The near-field SIL imaging theory for high numerical aperture is developed by using the vector diffraction and thin-film optics. Numerical results show that the spot size, Strehl ratio, and sidelobe intensity have an oscillatory behavior with the change of thickness of the air gap, which results from the interference effect of the transmitted field. We find that for smaller spot size, the Strehl ratio is smaller but the sidelobe intensity is larger. A certain thickness of air gap is useful for optical storage, which is less than 63 nm for the system in the simulated examples.     

Nanoscopic imaging of human chromosomes via a scanning near-field optical/atomic-force microscopy (SNOAM)

Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographic and fluorescence images of human chromosomes. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever. Optical resolution was approximately 50–100 nm in fluorescence mode. Conventional karyotyping information was linked with SNOAM topographic analyses such as location of centromere and length of individual chromosomes. The height profile clearly indicated higher teromere regions. The SNOAM fluorescence images were different shapes from topographic images probably due to results from the combination of fluorescence dye and chromosome DNA.     

Fabrication and characterization of fluorescent rare-earth-doped glass-particle-based tips for near-field optical imaging applications

Fluorescent rare-earth-doped glass particles glued to the end of an atomic force microscope tip have been used to perform scanning near-field optical measurements on nanostructured samples. The fixation procedure of the fluorescent fragment at the end of the tip is described in detail. The procedure consists of depositing a thin adhesive layer on the tip. Then a tip approach is performed on a fragment that remains stuck near the tip extremity. To displace the particle and position it at the very end of the tip, a nanomanipulation is achieved by use of a second tip mounted on piezoelectric scanners. Afterward, the particle size is reduced by focused ion beam milling. These particles exhibit a strong green luminescence where excited in the near infrared by an upconversion mechanism. Images obtained near a metallic edge show a lateral resolution in the 180-200-nm range. Images we obtained by measuring the light scattered by 250-nm holes show a resolution well below 100 nm. This phenomenon can be explained by a local excitation of the particle and by the nonlinear nature of the excitation.     

Dual-optical-mode near-field scanning optical microscopy

The simultaneous operation of near-field scanning optical microscopy (NSOM) in reflection and transmission modes is demonstrated. In the transmission mode, a low-noise, large-area silicon photodetector was mounted between the piezoelectric transducer scanning stage and the sample. In the reflection mode, either a photomultiplier tube or two large-area silicon detectors was used for signal collection. The reflection-mode setup consisting of two silicon detectors provides a large numerical aperture of 0.9 as well as symmetrical detection of emitting photons. The dielectric thin films and the light-emitting polymers were used to demonstrate the capability of these two modes of NSOM. A comparison between these two different setups is also presented.     

Differential near-field scanning optical microscopy

We theoretically and experimentally illustrate a new apertured near-field scanning optical microscopy (NSOM) technique, termed differential NSOM (DNSOM). It involves scanning a relatively large (e.g., 0.3-2 mum wide) rectangular aperture (or a detector) in the near-field of an object and recording detected power as a function of the scanning position. The image reconstruction is achieved by taking a two-dimensional derivative of the recorded power map. Unlike conventional apertured NSOM, the size of the rectangular aperture/detector does not determine the resolution in DNSOM; instead, the resolution is practically determined by the sharpness of the corners of the rectangular aperture/detector. Principles of DNSOM can also be extended to other aperture/detector geometries such as triangles and parallelograms.     

Characterization of near-field optical probes

Radiation and collection characteristics of four different near-field optical-fiber probes, namely, three uncoated probes and an aluminum-coated small-aperture probe, are investigated and compared. Their radiation properties are characterized by observation of light-induced topography changes in a photosensitive film illuminated with the probes, and it is confirmed that the radiated optical field is unambiguously confined only for the coated probe. Near-field optical imaging of a standing evanescent-wave pattern is used to compare the detection characteristics of the probes, and it is concluded that, for the imaging of optical-field intensity distributions containing predominantly evanescent-wave components, a sharp uncoated tip is the probe of choice. Complementary results obtained with optical phase-conjugation experiments with the uncoated probes are discussed in relation to the probe characterization.     

Optical field study of near-field optical recording with a solid immersion lens

The use of a solid immersion lens (SIL) is an important technique for increasing areal density in optical recording. Here an approximate method is presented for analyzing the optical fields in a SIL above a half-space and a SIL above a multilayer recording medium. Both propagating and evanescent components are included in the distribution of fields below the SIL. An approximate closed-form expression is given for the decay of the intensity away from the SIL surface above a half-space. In the case of a SIL above a recording medium the model describes the strong oscillations that are observed in the reflected Kerr rotation and ellipticity as the medium spacing is varied. These oscillations are attributed to standing waves in the propagating field component.     

Theory of near-field magneto-optical imaging

Scanning near-field optical microscopy has been recently applied to the imaging of magnetic samples. It was shown experimentally that an apertureless microscope suffers a substantial loss of resolution when used for magneto-optical imaging compared with that for conventional imaging. No such change is observed for aperture microscopes. We explain this observation by developing a model for the imaging process that incorporates the response of the probe. We calculate real observable properties such as the rotation of polarization at the detector or the circular dichroism signal and thus simulate magneto-optical images of a domain structure in cobalt for both aperture and apertureless microscopes.     

Phase separation in polyaniline with near-field scanning optical microscopy

We report the studies of conjugated polymers, polyaniline thin films, with a near-field scanning optical microscope. Because of the absorption variation in different oxidation states, transmission-mode near-field scanning optical microscope images were employed to map out the distribution of the oxidation states on a submicrometer scale. When the near-field wavelength is varied (between 632.8 and 543.5 nm), the phase separation between the oxidized and the reduced repeated units, with domain sizes on a nanometer-length scale, is observed.     

Compact stand-alone near-field optical microscope combined with force detection

We present a compact stand-alone near-field optical microscope combined with force detection in which manufactured atomic force microscope (AFM) microcantilevers are used for both optical and force detection. Because of the stand-alone design, the combination allows a great variety of operation modes, including the scanning tunneling optical microscope (STOM), and possibly the reflection scanning near-field optical microscope modes. The first images obtained in the AFM and the STOM mode are presented. A polarization study is carried out to confirm the optical nature of the detected signal and to discuss possible artifacts.