Fluorescence Scanning Near-field Optical Microscopy Research Articles

Fluorescence enhancement near single TiO2 nanodisks

We present a near-field optical study of TiO2 nanodisks by fluorescence scanning near-field optical microscopy. The localization of light and the fluorescence enhancement near the dielectric structures are visualized with a lateral resolution of ∼λ/5 using an Er/Yb-codoped fluorescent nanocrystal glued at the end of a sharp scanning tip. We observed that the intensity patterns strongly depend on the disk size, forming lobes for a diameter close to the wavelength and a single bright spot for smaller structures. Although the experiments were performed out of resonance, a maximum fluorescence enhancement of 2.3 was observed near 700 nm-wide disks. The evolution of the fluorescence pattern as a function of the disk size is in good agreement with the near-field maps calculated by the finite-difference time-domain method, in both two and three dimensions above the structures.

Enhanced Emission from Single Isolated Gold Quantum Dots Investigated Using Two-Photon-Excited Fluorescence Near-Field Scanning Optical Microscopy.

New approaches in molecular nanoscopy are greatly desired for interrogation of biological, organic, and inorganic objects with sizes below the diffraction limit. Our current work investigates emergent monolayer-protected gold quantum dots (nanoclusters, NCs) composed of 25 Au atoms by utilizing two-photon-excited fluorescence (TPEF) near-field scanning optical microscopy (NSOM) at single NC concentrations. Here, we demonstrate an approach to synthesize and isolate single NCs on solid glass substrates. Subsequent investigation of the NCs using TPEF NSOM reveals that, even when they are separated by distances of several tens of nanometers, we can excite and interrogate single NCs individually. Interestingly, we observe an enhanced two-photon absorption (TPA) cross section for single Au25 NCs that can be attributed to few-atom local field effects and to local field-induced microscopic cascading, indicating their potential for use in ultrasensitive sensing, disease diagnostics, cancer cell therapy, and molecular computers. Finally, we report room-temperature aperture-based TPEF NSOM imaging of these NCs for the first time at 30 nm point resolution, which is a ∼5-fold improvement compared to the previous best result for the same technique. This report unveils the unique combination of an unusually large TPA cross section and the high photostability of Au NCs to (non-destructively) investigate stable isolated single NCs using TPEF NSOM. This is the first reported optical study of monolayer-protected single quantum clusters, opening some very promising opportunities in spectroscopy of nanosized objects, bioimaging, ultrasensitive sensing, molecular computers, and high-density data storage.

Topological organization of NADPH‐oxidase in haematopoietic stem cell membrane: preliminary study by fluorescence near‐field optical microscopy

The aim of this study was to characterize the local distribution and organization of the plasma membrane NADPH-oxidase (NOX) in human haematopoietic stem cell (HSC) by means of the fluorescence scanning near-field optical microscopy approach. The presence of NOX in haematopoietic stem cells is thought to have a functional role as O(2) sensor and/or as low-level reactive oxygen species (ROS) producer to be used as redox messenger for controlling cell growth and differentiation. Given the harmful potential of ROS, a fine-tuning of NOX activity is needed. The high resolution imaging of haematopoietic stem cell membrane obtained in this study combined with the immunodetection of NOX indicates for this the occurrence of a cluster-organized structure. These membrane 'rafts'-like micro-compartments may constitute localized protein aggregates whereby the assembly/activation of the NOX components are functionally integrated with upstream factors constituting signal-transduction platforms.

Speciation of Europium (III) Surface Species on Monocrystalline Alumina Using Time-Resolved Laser-Induced Fluorescence—Scanning Near-Field Optical Microscopy

The aim of this work was to perform highly localized spectroscopic surface measurements by combining time-resolved laser spectroscopy and scanning near-field optical microscopy. The final purpose of that was to study surface sorption at the molecular level of trivalent ions in the framework of nuclear waste disposal assessment. Time-resolved laser spectroscopy presents the advantages of being selective, sensitive, and noninvasive and scanning near-field optical microscopy is a promising technique for high resolution surface speciation. Investigation of the interaction between trivalent europium and a monocrystalline alumina (1102) surface was made using different conditions of concentration and pH. We found that the distribution of sorbed europium was always homogeneous with a decay time of europium (III) equal to 350 micros+/-15 micros. On the other hand, carbonate species with a decay time of 210 micros+/-10 micros or other hydroxide species with a decay time of 180 micros+/-10 micros were detected on the surface when a higher concentration or a higher pH solution, respectively, were used. Distribution of these species was heterogeneous and their associated fluorescence signal was relatively high, evoking a precipitated form. X-ray photoelectron spectroscopy (XPS) was also used on the same samples as a complementary technique. A binding energy of 1135.1 eV was obtained for the sorbed europium and another binding energy of 1134.4 eV was obtained for the hydroxide species, thus confirming the presence of two kinds of species on the surface.

Investigation of the Doughnut Effect in Cy3-Labeled Protein Microarrays using Scanning Near-Field Optical Microscope

We demonstrate the fluorescence mapping of protein microarrays by the technique of scanning near-field optical microscopy (SNOM) and confocal microscopy. Micron sized spots (300 μm) of human Immunoglobulin G (hIgG) protein with and without a Cy3 dye labeling have been fabricated on glass substrates by an immobilization method which makes use of calixcrown derivatives termed Prolinker. We have also tried to probe into the well-known “doughnut effect” observed in fluorescence images of proteins using the SNOM technique. The topographic and fluorescence SNOM images revealed that the number of proteins at the boundary of the spot were more than at the center in the case of the microarray spot which showed brighter luminescence at the edge than at the center in the confocal image.

Scanning near-field optical microscopy on rough surfaces: Applications in chemistry, biology, and medicine

Shear-force apertureless scanning near-field optical microscopy (SNOM) with very sharp uncoated tapered waveguides relies on the unexpected enhancement of reflection in the shear-force gap. It is the technique for obtaining chemical (materials) contrast in the optical image of “real world” surfaces that are rough and very rough without topographical artifacts, and it is by far less complicated than other SNOM techniques that can only be used for very flat surfaces. The experimental use of the new photophysical effect is described. The applications of the new technique are manifold. Important mechanistic questions in solid-state chemistry (oxidation, diazotization, photodimerization, surface hydration, hydrolysis) are answered with respect to simultaneous AFM (atomic force microscopy) and detailed crystal packing. Prehistoric petrified bacteria and concomitant pyrite inclusions are also investigated with local RAMAN SNOM. Polymer beads and unstained biological objects (rabbit heart, shrimp eye) allow for nanoscopic analysis of cell organelles. Similarly, human teeth and a cancerous tissue are analyzed. Bladder cancer tissue is clearly differentiated from healthy tissue without staining and this opens a new highly promising diagnostic tool for precancer diagnosis. Industrial applications are demonstrated at the corrosion behavior of dental alloys (withdrawal of a widely used alloy, harmless substitutes), improvement of paper glazing, behavior of blood bags upon storage, quality assessment of metal particle preparations for surface enhanced RAMAN spectroscopy, and determination of diffusion coefficient and light fastness in textile fiber dyeing. The latter applications include fluorescence SNOM. Local fluorescence SNOM is also used in the study of partly aggregating dye nanoparticles within resin/varnish preparations. Unexpected new insights are obtained in all of the various fields that cannot be obtained by other techniques.

Near-Field Scanning Fluorescence Microscopy Study of Ion Channel Clusters in Cardiac Myocyte Membranes

Near-field scanning optical microscopy (NSOM) has been used to study the nanoscale distribution of voltage-gated L-type Ca 2+ ion channels, which play an important role in cardiac function. NSOM fluorescence imaging of immunostained cardiac myocytes (H9C2 cells) demonstrates that the ion channel is localized in small clusters with an average diameter of 100 nm. The clusters are randomly distributed throughout the cell membrane, with some larger fluorescent patches that high-resolution images show to consist of many small closely-spaced clusters. We have imaged unstained cells to assess the contribution of topography-induced artifacts and find that the topography-induced signal is <10% of the NSOM fluorescence intensity. We have also examined the dependence of the NSOM signal intensity on the tip-sample separation to assess the contributions from fluorophores that are significantly below the cell surface. This indicates that chromophores >∼200 nm below the probe will have negligible contributions to the observed signal. The ability to quantitatively measure small clusters of ion channels will facilitate future studies that examine changes in protein localization in stimulated cells and during cardiac development. Our work illustrates the potential of NSOM for studying membrane domains and protein localization/colocalization on a length scale which exceeds that available with optical microscopy.

Hybrid near-field scanning optical microscopy tips for live cell measurements

We report a near-field scanning optical microscopy (NSOM) probe that enables high-resolution imaging of living cells under physiological buffered conditions. The hybrid design combines a conventional fiber optic near-field probe with a standard atomic force microscopy cantilever. Imaging of fluorescent latex spheres suspended in an acetate matrix demonstrates the subdiffraction limited fluorescence and topography capabilities of the tips. The reduced spring constant of the hybrid tip is also shown to be amenable to measurements on living cells. NSOM fluorescence and topography measurements on living human arterial smooth muscle cells under buffered conditions are demonstrated.

Near-field microscopy and lithography of light-emitting polymers.

We describe the application of scanning near-field optical microscopy (SNOM) to the study of the photophysical and self-organization properties of thin films of blends of conjugated polymers, and also to the lateral nanoscale patterning of conjugated-polymer structures. Such thin-film plastic semiconductor nanostructures offer significant potential for use in opto-electronic devices. The implementation of SNOM we employ is the most established form in which a probe with a sub-wavelength aperture is scanned in close proximity to the sample surface. We consider the nature of the near-field optical distribution, which decays within the first ca. 100 nm of these semiconductor materials, and address the identification of topographic artefacts in near-field optical images. While the topographic information obtained simultaneously with optical data in any SNOM experiment enables an easy comparison with the higher-resolution tapping-mode atomic force microscopy, the spectroscopic contrast provided by fluorescence SNOM gives an unambiguous chemical identification of the different phases in a conjugated-polymer blend. Both fluorescence and photoconductivity SNOM indicate that intermixing of constituent polymers in a blend, or nanoscale phase separation, is responsible for the high efficiency of devices employing these materials as their active layer. We also demonstrate a scheme for nano-optical lithography with SNOM of conjugated-polymer structures, which has been employed successfully for the fabrication of poly(-phenylene vinylene) nanostructures with 160 nm feature sizes.

Prolonged Excited-State Lifetime of Porphyrin Due to the Addition of Colloidal SiO2 to Triton X-100 Micelles

A water-insoluble porphyrin derivative was studied in Triton X-100 micelles by steady-state spectroscopy and picosecond photolysis. The porphyrin in micelles formed J aggregates, and, consequently, the excited-state lifetime of such a porphyrin was very short. After the addition of colloidal SiO2, the B-band absorption blue-shifted, forming a broad absorption peak. This peak can be ascribed to the monomer-dominated absorption of porphyrin. The photolysis experiment showed that the dynamic curve of the porphyrin with SiO2 exhibited a rapid recovery plus an offset resulting from the S1 state absorption. The long lifetime part constituted about half of the total absorption. Such a long-lifetime component could be ascribed to the absorption of monomeric porphyrin. The fluorescence near-field scanning optical microscopy showed that the porphyrin in the presence of SiO2 exhibited stronger fluorescence images compared with that in the absence of SiO2, though the topography images of the these samples were simila...

High-resolution Studies of Lung Surfactant Collapse¶

Survanta is a replacement lung surfactant (LS) used in the treatment of respiratory distress syndrome (RDS), the fourth leading cause of infant mortality in the United States. It consists of purified LS from bovine sources and retains the surfactant proteins (SP) SP-B and SP-C, both thought to be important in proper respiratory function. As such, it provides a useful and biologically relevant model system to probe the structure and function of natural LS. Here, we report results from high-resolution studies on model monolayers formed from Survanta to probe the mechanism of collapse at high surface pressure. Our results show the formation of two different collapse structures. At 62 mN/m, slightly below the collapse pressure, monolayer collapse occurs through buckling. Confocal fluorescence measurements on supported films reveal regions of overlapping phase structure in the films that mark the transition from monolayer to multilayer. Simultaneous near-field scanning optical microscopy fluorescence and force measurements show that the transition seen in the fluorescence measurements accompanies corresponding approximately 4-5 nm changes in membrane topography. This change in height is consistent with bilayer formation on monolayer collapse. Analysis of the phase structure near the transitions also suggests that the buckling occurs from a continuous film. However, when the film is compressed to its collapse pressure of 65 mN/m, buckling is no longer evident in the collapsed region. In addition, multilayers and lipid-protein aggregates that are up to 40 nm higher than the monolayer are observed in the collapsed film at this pressure.

Fully time-resolved near-field scanning optical microscopy fluorescence imaging

Time-correlated single photon counting has been coupled with near-field scanning optical microscopy (NSOM) to record complete fluorescence lifetime decays at each pixel in an NSOM image. The resulting three-dimensional data sets can be binned in the time dimension to create images of photons at particular time delays or images of the fluorescence lifetime. Alternatively, regions of interest identified in the topography and fluorescence images can be used to bin the data in the spatial dimensions resulting in high signal to noise fluorescence decays of particular regions of the sample. The technique has been demonstrated on films of poly(vinylalcohol), doped with the fluorescent dye, cascade blue (CB). The CB segregates into small circular regions of high concentration within the films during the drying process. The lifetime imaging shows that the spots have slightly faster excited state decays due to quenching of the luminescence as a result of the higher concentration. The technique is also used to image the fluorescence lifetime of an annealed film of poly(dihexylfluorene). The samples show high contrast in the total intensity fluorescence image, but the lifetime image reveals the sample to be extremely uniform.

Phase Separation in Supported Phospholipid Bilayers Visualized by Near-Field Scanning Optical Microscopy in Aqueous Solution

An experimental approach for near-field scanning optical microscopy (NSOM) of biological samples in an aqueous environment with a resolution of <100 nm has been developed. This was accomplished by using high-throughput bent optical fiber probes prepared by a two-step chemical etching method followed by focused ion beam milling to fabricate a reproducible aperture. The utility of the method for high-resolution fluorescence imaging of biological samples in liquid was demonstrated using phase separated, supported phospholipid bilayers as models for natural membranes. Bilayers with a phase separated dipalmitoyl−phosphatidylcholine/dilauroyl−phosphatidylcholine (DPPC/DLPC) mixture in one or both leaflets were imaged by both atomic force microscopy and NSOM. The addition of dihexadecanoyl-sn-glycero-3-phosphoethanolamine−Texas Red (DHPE−TR) was used to visualize fluid and gel phases for the NSOM fluorescence measurements. Hybrid bilayers with 7:3 DLPC/DPPC in the bottom leaflet and DPPC on top showed phase sepa...

S-Nitrosoglutathione-induced mouse thymocyte apoptosis studied by fluorescence near-field scanning optical microscopy

This study is an attempt to deeply understand the mechanisms ensuring self-tolerance of T cells via clonal deletion of thymocytes and exploring T lymophocyte homeostasis by observing the apoptosis of single mouse thymocyte induced by S-nitrosoglutathione (GSNO, a nitric oxide donor) using fluorescence near-field scanning optical microscopy (NSOM) in illumination mode. The GSNO-induced thymocytes were stained with propidium iodide containing 0.01% Triton X-100 and excited with light of 488 nm and the emitting fluorescence at 525 nm. According to the NSOM fluorescence image and the simultaneously obtained topography image, the feature of mouse thymocyte apoptosis was characterized by scattering pattern of the fluorescence spots with the size 0.2–2.1 μm at the full width at half-maximum of fluorescence intensity 78–80 kHz in the GSNO-treated thymocyte nucleus. Whereas there is no fluorescence from the untreated thymocyte. The intensity of the fluorescence from the dexamethasone-treated thymocyte was much stronger than that from GSNO-induced thymocytes. Furthermore, the fluorescence distribution in the latter were concentrated in the nucleus. Those results also demonstrate the advantages of NSOM such as high spatial resolution and the topography of biology samples.

Nanoscale morphology of polyelectrolyte self-assembled films probed by scanning force and near-field scanning optical microscopy

We applied shear force microscopy, an analog to attractive-mode atomic force microscopy, and near-field scanning optical microscopy (NSOM) to the study of surface roughness and nanoscale morphology in polyelectrolyte self-assembled layers. Our data show that the surface roughness of multilayer films on glass grows linearly with the number of polyelectrolyte bilayers for the first 10 bilayers, and then asymptotes at a surface roughness of 4 nm. The surface of these films is characterized by bumps of 100–500 nm in diameter and 25–50 nm tall. In addition, the size and density of the bumps for each bilayer are uncorrelated to the previous surface. This result is in sharp contrast to what is observed for other self-assembled layer structures, such as metal-phosphonate and Langmuir–Blodgett self-assembly, where the surface roughness linearly increases with the number of layers. Subsurface morphology in thin films was observed via fluorescence NSOM of a dye-doped polyelectrolyte film. The NSOM images show domains of higher and lower fluorescence intensity, which could be assigned to local changes in the film thickness, suggesting that the dye molecules are uniformly distributed within the film. We also show that both the initial and asymptotic surface roughness can be dramatically decreased if the bare glass surface is treated with a high charge-density polyelectrolyte, such as poly(ethyleneimine) (PEI), prior to the bilayer formation. Finally, we observed that if the initial substrate is rough, growth of the polyelectrolyte layers acts to smooth the surface until the equilibrium value is reached.

Near-Field Scanning Optical Microscopy Studies of a Fluorescent Polyelectrolyte−Surfactant Complex

Thin films prepared from a complex formed between poly(vinyl sulfate) (PVS) and a cationic indocarbocyanine surfactant dye (DiI) are investigated by near-field scanning optical microscopy (NSOM) and atomic force microscopy (AFM). Local film morphology and chemical composition are studied as a function of dye/anionic-site stoichiometry. AFM images show that overall film morphology changes modestly with dye content. Fluorescence NSOM images reveal that at lowest PVS loading, the films are extremely heterogeneous, exhibiting round fluorescent regions 100−250 nm in size. As the dye content is increased, the fluorescent regions increase in number but remain of similar sizes. NSOM fluorescence spectra prove these regions contain aggregated DiI and indicate they are likely polymer−surfactant micelles entrapped in the films.

Photovoltaic Performance and Morphology of Polyfluorene Blends: A Combined Microscopic and Photovoltaic Investigation

The interplay between phase separation in polyfluorene blends which show photoinduced charge transfer and photovoltaic performance in photodiodes has been investigated. Phase separation length scales have been varied from several microns to tens of nanometers by limiting the time allowed for solvent-enhanced self-organization through several different processing routes. Concurrent with the decrease in feature size, an increase in maximum photovoltaic efficiency of nearly 1 order of magnitude was observed in photodiodes incorporating the phase-separated blends as the active layer. The structure of the blend films was investigated using fluorescence microscopy, fluorescence scanning near-field optical microscopy, and atomic force microscopy. In some cases, a hierarchy of micron- and nanometer-scale phase separation was observed which may explain the unexpectedly high photoresponse in devices with up to micron-scale phase separation structure. This result along with in situ fluorescence microscopy studies of...

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.

Simultaneous Near-Field Optical Birefringence and Fluorescence Contrast Applied to the Study of Dye-Doped Polymer-Dispersed Liquid Crystals

The local optical and electrooptical properties of dye-doped polymer-dispersed liquid crystal (PDLC) thin films are explored by near-field scanning optical microscopy (NSOM). Detailed information on the mechanisms for dye alignment and electric-field-induced dye reorientation in localized sample regions is sought. Dye reorientation is predicted to occur by two distinct processes: (i) via its interactions with the liquid crystal host and (ii) via the direct interactions of its permanent and induced dipole moments with the applied field. The liquid crystal is doped with a nearly isotropically aligned BODIPY dye so that contributions of both reorientation mechanisms can be explored. An order parameter of 0.10 ± 0.02 is measured for BODIPY in bulk aligned samples, suggesting that host−guest interactions are very weak. Simultaneously recorded birefringence (transmission) and fluorescence NSOM images confirm that the dye is apparently not well aligned, even on submicrometer length scales. Surprisingly, NSOM studies of dye reorientation prove that host−guest interactions still play a substantial role in controlling the dye reorientation process. The field dependence of dye reorientation is found to be correlated with liquid crystal reorientation, varying spatially across individual droplets. Analysis of the simultaneously recorded birefringence and fluorescence data shows that the dye is strongly reoriented via its interactions with the reorienting liquid-crystal host and also via its independent interactions with the applied field. The former mechanism is manifested as a rotation of the dye alignment axis while the latter leads to a field-dependent increase in the local dye order parameter. “Bistable” alignment of the dye in the nematic host is proposed as a possible explanation for the apparent coupling of the two reorientation mechanisms.

Near-Field Scanning Optical Microscopy (NSOM) Studies of the Relationship between Interchain Interactions, Morphology, Photodamage, and Energy Transport in Conjugated Polymer Films

It is becoming increasingly clear that the way in which a conjugated polymer film is cast affects the interactions between polymer chains and thus the optical and electrical properties of the film. Given that conjugated polymer films cast in different ways also show different nanometer-scale surface topographies, the question that arises is: What is the correlation between surface topography, local chain packing, and the local electronic properties of a conjugated polymer film? In this paper, we address this question using fluorescence near-field scanning optical microscopy (NSOM) to examine films of poly(2-methoxy-5-(2‘-ethylhexyloxy)-1,4-phenylene vinylene) (MEH-PPV) that were prepared in different ways. The spatially resolved photoluminescence (SRPL) spectra collected on top of the nanometer-scale topographic features (“bumps”) exhibited by spin-cast MEH-PPV films show an enhancement of the red portion of the emission relative to spectra collected from flat regions of the film. Moreover, photooxidativ...