Near-field Fluorescence Microscopy Research Articles

Direct imaging of fluorescence enhancement in the gap between two gold nanodisks

We present an analysis of the optical coupling between two gold nanodisks by near-field fluorescence microscopy. This is achieved by simultaneously scanning and measuring the light emitted by a single Er3+/Yb3+ doped nanocrystal glued at the end of an atomic force microscope tip. The excitation of the nanocrystal was performed at λ = 975 nm via upconversion, and fluorescence was detected in the visible part of the spectrum at λ = 550 nm. For an isolated nanodisk, the near-field presents a two-lobe pattern oriented along the direction of the incident polarization. For two nanodisks with a sizable separation distance (385 nm) illuminated with the polarization along the interparticle axis, we observe a negative effect of the coupling with a slight decrease in fluorescence in the gap. For smaller gap values (195, 95, and 55 nm), a strong increase in fluorescence is observed as well as a reduced spatial localization of the field as the distance decreases. Finally, when the disks touch each other (0 nm), the dipolar–dipolar interaction between them disappears and no fluorescence enhancement occurs. A new plasmon mode is created at another wavelength. Our experimental results are in good agreement with numerical simulations of the near-field intensity distribution at the excitation wavelength on the surface of the structures. Combining fluorescence mapping and far-field scattering spectroscopy should be of strong interest to develop bio-chemical sensors based on field enhancement effects.

Scanning Near-Field Fluorescence Microscopy Applied to ESEM

A scanning near-field fluorescence microscopy for in situ test is demonstrated in this letter. The scanning near-field fluorescence microscopy can be applied to commercial environmental scanning electron microscopy (ESEM) without changing the ESEM performance. The designed scanning near-field fluorescence microscopy combines the advantage of the ESEM and scanning near-field optical microscopy (SNOM). The CdSe/ZnS quantum dot samples are prepared to verify system performance. The system offers fluorescence image and topography image simultaneously with a high depth and high resolution, thus permitting further ingredient and functional research. The topography image spatial resolution and the fluorescence image spatial resolution are below 130 nm and 115 nm, respectively, with a tip aperture of ~100 nm. The image range reaches $300\,\,\times \,\,300\,\,\mu \text{m}$ by virtue of the micromanipulators and image stitching technology.

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

Probing the Plasmonic Properties of Heterometallic Nanoprisms with Near-Field Fluorescence Microscopy

In an effort to optimize both the optical properties and chemical stability of plasmonic platforms for surface-enhanced fluorescence spectroscopy, a series of multilayered heterometallic structures were prepared. Gold–silver multilayered array of prisms were designed, where the gold layers acts as protective layers for the silver and extends the spectral range of the resulting localized surface plasmon resonances. Herein, we report an experimental study of the enhanced near-field fluorescence from quantum dots deposited at the surface of these platforms. This was performed using near-field scanning optical microscopy yielding simultaneous measurements of topographical features and near-field fluorescence of the prismatic arrays. Hence, the individual localized fluorescent hot-spots could be imaged and spatially correlated with the geometry of the nanostructures for distinct polarization configuration of the excitation light with respect to the orientation of the structures. Furthermore, the fluorescence enhancement was evaluated spatially and temporally for the series of the nanoprism platforms. In particular, the quenching of the near-field fluorescence due to the metal structures was carefully evaluated for the metal–fluorophore hybrid systems.

Nanoscale Assembly of Proteins into Amyloid Oligomers, Pores and Fibrils

Protein amyloid aggregates are implicated in a variety of debilitating human disorders such as Alzheimer's, Parkinson's and prion diseases. The transition from a normal functional protein to an abnormal misfolded form involves a profound conformational change that serves as the key step in amyloid assembly leading to nanoscopic oligomers, pores and fibrils. Using Raman spectroscopy in combination with atomic force microscopy (Figure 1a,b), we have been able to delineate the key structural transitions during amyloid formation (Bhattacharya et al. J. Phys. Chem. Lett. 2013,4, 480-485). Moreover, the underlying molecular mechanism by which amyloids are involved in inducing cellular toxicity remains elusive because the conventional optical microscopy does not allow one to monitor these processes directly at a high spatial resolution due to the diffraction-limit. Using near-field scanning fluorescence microscopy, we have been able to image the fibrils far beyond the diffraction-limit and interrogate individual fibrils by simultaneously monitoring both nanoscale topography and fluorescence brightness along the length of the fibrils (Figure 1c,d). Our nanoscale imaging results provide structural underpinnings of the supramolecular packing within the nanoscopic amyloids (Dalal & Bhattacharya et al. J. Phys. Chem. Lett. 2012, 3, 1783-1787).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Nanoscale Fluorescence Imaging of Single Amyloid Fibrils.

Amyloid formation is implicated in a variety of human diseases. It is important to perform high-resolution optical imaging of individual amyloid fibrils to delineate the structural basis of supramolecular protein assembly. However, amyloid fibrils do not lend themselves to the conventional microscopic resolution, which is hindered by the diffraction limit. Here we show super-resolution fluorescence imaging of fluorescently stained amyloid fibrils derived from disease-associated human β2-microglobulin using near-field scanning fluorescence microscopy. Using this technique, we were able to resolve the fibrils that were spatially separated by ∼75 nm. We have also been able to interrogate individual fibrils in a fibril-by-fibril manner by simultaneously monitoring both nanoscale topography and fluorescence brightness along the length of the fibrils. This method holds promise to detect conformational distributions and heterogeneity that are believed to correlate with the supramolecular packing of misfolded proteins within the fibrils in a diverse conformationally enciphered prion strains and amyloid polymorphs.

Nanoapertures for 60 nm Resolution of Membrane Organization and Dynamics

Dynamic nanoscale domains in the plasma membrane may be responsible for a variety of cellular signaling processes via membrane receptor clustering and lipid phase partitioning. However, current experimental approaches are limited in spatial and/or temporal resolution to address many membrane domain hypotheses. We have developed a new approach utilizing an array of nanoapertures to examine membrane organization and dynamics with near-field optical fluorescence microscopy without incorporating a scanning probe or disturbing the membrane.

Simultaneous near-field and far-field fluorescence microscopy of single molecules

A new microscope objective is presented for the parallel fluorescence detection below and above the critical angle of total internal reflection with single molecule sensitivity. The collection of supercritical angle fluorescence (SAF) leads to a strongly surface confined detection volume whereas the collection of undercritical angle fluorescence (UAF) allows for the observation of deeper axial sections of the specimen. By simultaneous detection of the near-field-mediated SAF and the far-field UAF emission modes the z-position of emitters can be obtained on the nanometer scale. We investigate the point spread function of the optics and demonstrate nanoscopic z-localization of single molecules. The oil immersion objective, developed for use on common microscope bodies, opens up new possibilities for the study of topographies and dynamics at surfaces and on membranes.

Arrays of Nanoapertures for Examining Membrane Organization and Dynamics

Nanoscale domains in the plasma membrane may be responsible for a variety of cellular signaling processes via membrane receptor clustering and lipid phase partitioning. However, current experimental approaches are limited in spatial and/or temporal resolution to address many membrane domain hypotheses. We have developed a new approach utilizing an array of nanoapertures to examine membrane organization and dynamics with near-field optical fluorescence microscopy without incorporating a scanning probe or disturbing the membrane. These nanoapertures are glass-filled, cylindrical pores (>50 nm diameter) in a thin aluminum film on a fused silica support, and they provide a planar surface for unperturbed cell adherence and growth. A nanoaperture confines the transmitted excitation light to a sub-diffraction limited spot directly above aperture, providing a 40-fold decrease in the illuminated area versus diffraction-limited illumination of the plasma membrane. Otherwise conventional microscopy excitation sources and fluorescent probes are used to enable fluorescence correlation spectroscopy (FCS) with 50 nanometer and 1 microsecond resolution. Further, these apertures provide two key benefits for FCS in addition to improved resolution: assured alignment of numerous illumination spots for cross-correlations and assured focusing of illumination on the cellular membrane as opposed to focusing within the cytoplasm. Chromatic aberrations and slight laser misalignment that may complicate far-field, two-color FCS are not of concern here because the illumination profile is determined by the aperture directly. This technique has been applied to both model and living cell membranes to examine the diffusion of lipids and proteins in nanoscale dimensions. In particular, results will be presented demonstrating the effectiveness of this technique to observe diffusion of membrane proteins and lipids in varying phases and cross-correlation that occurs after cross-linking a selected component.

Microscopy with Nanoapertures to Reveal Membrane Organization with 1 Microsecond and 100 Nanometer Resolution

Current hypotheses for plasma membrane organization depend critically on a variety of length and time scales that are outside the resolution of conventional experimental techniques. Our novel approach utilizes nanoapertures to examine membrane organization and dynamics with near-field optical fluorescence microscopy without incorporating a scanning probe or perturbing the membrane. Conventional microscopy excitation sources and fluorescent probes are used to enable fluorescence correlation spectroscopy (FCS) on living cell membranes with super-resolution. The nanoapertures confine the excitation light to a sub-diffraction limit length scale, providing a 25-fold decrease in the illuminated area of the membrane as compared to diffraction-limited illumination.View Large Image | View Hi-Res Image | Download PowerPoint Slide

One-photon and two-photon excited fluorescence microscopies based on polarization-control: Applications to tip-enhanced microscopy

One-photon and two-photon excited fluorescence microscopies using either radial or azimuthal polarization have been developed and applied to the imaging of quantum dots. In both cases (one-photon and two-photon excitations), the fluorescence image profile of each quantum dot is in good agreement with the electric field intensity distribution of a tightly focused spot using a high numerical aperture objective lens. While this polarization dependence of the absorption/emission of quantum dots (or other dye molecules) is useful for characterizing the orientation of the quantum dots, most of the biological applications that employ quantum dots or dye molecules as labels require the information describing not only the orientation but also the precise position of each dot. In order to improve the sensing accuracy of the dot’s position, we employ a modified near-field fluorescence microscopy system that utilizes a tip-enhancement technique and radially polarized two-photon excitations. For the tip enhancement, a commercially available silicon cantilever tip has been successfully utilized instead of metallic tips, as the latter tip can drastically quench the near-field fluorescence. Our tip-enhanced two-photon excited fluorescence microscopy technique enables visualization of the quantum dots distributed on a cover slip beyond the diffraction limit of light. We demonstrate that our approach is advantageous not only due to its high spatial resolution but also due to its high sensitivity by showing that the fluorescence signal is not detectable without the aid of the tip enhancement in some cases.

Near‐field scanning optical microscopy detects nanoscale glycolipid domains in the plasma membrane

The localization of asialo-GM1 in ordered membrane raft domains in HeLa cells has been examined using a combination of membrane fractionation and fluorescence imaging. The glycolipid is enriched in Triton X-100 insoluble membrane fractions that contain high concentrations of cholesterol and caveolin-1 but is also found in detergent soluble membrane fractions. Near-field fluorescence microscopy shows that a fraction of the asialo-GM1 is localized in small nanoscale clusters that have an upper limit for the average diameter of approximately 90 nm and are partially colocalized with caveolae membrane domains. In addition to clusters, a diffuse, non-clustered population of asialo-GM1 is observed and is hypothesized to correspond to glycolipid isolated in detergent soluble membrane fractions.

Nanoscale Fluorescence Microscopy Using Carbon Nanotubes

We demonstrate the first reported use of single-walled carbon nanotubes as nano-optical probes in apertureless near-field fluorescence microscopy. We show that, in contrast to silicon probes, carbon nanotubes always cause strong fluorescence quenching when used to image dye-doped polystyrene spheres and Cd-Se quantum dots. For quantum dots, the carbon nanotubes induce very strong near-field contrast with a spatial resolution of 20 nm. Images of dye-doped spheres exhibit crescent-shaped artifacts caused by distortions in the surface water layer found in ambient conditions.

Can scanning near‐field optical microscopy be compared with confocal laser scanning microscopy? A preliminary study on α‐sarcoglycan and β1D‐integrin in human skeletal muscle

The dystrophin-glycoprotein complex and the vinculin-talin-integrin system constitute, together a protein machinery, called costameres. The dystrophin-glycoprotein complex contains, among other proteins, also dystrophin and the sarcoglycans subcomplex, proteins playing a key role in the pathogenesis of many muscular dystrophies and linking the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix, also providing structural support to the sarcolemma. The vinculin-talin-integrin system connects some components of the extracellular matrix with intermediate filaments of desmin, forming transverse bridges between Z and M lines. In our previous reports we always studied these systems by confocal laser scanning microscopy (CLSM). In this paper we report on the first applications of optical near-field fluorescence microscopy to the spatial localization of alpha-sarcoglycan and beta1D-integrin in human skeletal muscle fibres in order to better compare and test the images obtained with conventional CLSM and with scanning near-field optical microscopy (SNOM). In addition, the analysis of the surface morphology, and the comparison with the fluorescence map is put forward and analyzed for the first time on human muscle fibres. In aperture-SNOM the sample is excited through the nanometre-scale aperture produced at the apex of an optical fibre after tapering and subsequent metal coating. The acquisition of the topography map, simultaneously to the optical signal, by SNOM, permits to exactly overlap the fluorescence images obtained from the two consecutive scans needed for the double localization. Besides, the differences between the topography and the optical spatial patterns permit to assess the absence of artefacts in the fluorescence maps. Although the SNOM represented a good method of analysis, this technique remains a complementary method to the CLSM and it can be accepted in order to confirm the hypothesis advanced by CLSM.

Coherent scattering phenomena in apertureless scanning near-field fluorescence microscopy

We study near-field fluorescence images of samples composed of aggregates of 100 nm dye-doped latex spheres. These images have been performed by a reflection Apertureless Scanning Near-field Optical Microscope (A-SNOM). We show that the near-field distribution in fluorescence A-SNOM images arises from coherent scattering phenomena between all spheres. This is a consequence of the coherent nature of the fluorescence emission of each single sphere. The results shown here are significant for all fluorescent samples characterised by a nonnegligible topography.

Layer-by-Layer Self-Assembled Polyelectrolyte Multilayers with Embedded Liposomes: Immobilized Submicronic Reactors for Mineralization

The development of chemical reactions in nanospaces is of paramount importance for the development of active nanodevices, particularly in nanofluidics. It has been shown in a previous paper that phospholipid vesicles can be incorporated without spontaneous bilayer rupture into poly-L-glutamic acid/poly(allylamine) (PGA/PAH) multilayered polyelectrolyte films. The aim of the present study was to use such a system as an "embedded submicronic reactor" able to trigger precipitation of calcium phosphates within closed spaces through an enzymatic reaction, the enzyme also being encapsulated in the vesicle interior. To this aim, large unilamellar vesicles (LUVs) were produced containing calcium ions as active ions in the mineralization process, spermine as an activator of crystal growth, and alkaline phosphatase as a catalyst to convert phosphate esters into phosphates. After stabilization by adding a layer of poly-(D-lysine), these vesicles were embedded in a (PGA-PAH)n film. A paranitrophenyl phosphate containing solution was then put in contact with this film. It is shown by means of infrared spectroscopy in the attenuated total reflection mode that, consecutively to this contact, calcium phosphates are growing inside the embedded vesicles. By using scanning near-field fluorescence microscopy, it is demonstrated that the alkaline phosphatase enzymes are most probably located inside the vesicles after their embedding. In addition, atomic force microscopy was used to show, after chemical removal of the organic top layer of the film, that the inorganic platelets produced after the precipitation reaction are localized in volumes of similar size and shape as that of the vesicles into which the phosphate ester hydrolysis and subsequent precipitation reaction did occur.

Near-Field Fluorescence Microscopy An Optical Nanotool to Study Protein Organization at the Cell Membrane

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods capable of resolving structures with nanometer-scale resolution in intact or living cells. Although fluorescence microscopy has proven to be an extremely versatile tool in cell biology, its diffraction-limited resolution prevents the investigation of membrane compartmentalization at the nanometer scale. Near-field scanning optical microscopy (NSOM) is a relatively unexplored technique that combines both enhanced spatial resolution of probing microscopes and simultaneous measurement of topographic and optical signals. Because of the very small nearfield excitation volume, background fluorescence from the cytoplasm is effectively reduced, enabling the visualization of nano-scale domains on the cell membrane with single molecule detection sensitivity at physiologically relevant packing densities. In this article we discuss technological aspects concerning the implementation of NSOM for cell membrane studies and illustrate its unique advantages in terms of spatial resolution, background suppression, sensitivity, and surface specificity for the study of protein clustering at the cell membrane. Furthermore, we demonstrate reliable operation under physiological conditions, without compromising resolution or sensitivity, opening the road toward truly live cell imaging with unprecedented detail and accuracy.

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.

Apertureless scanning near-field fluorescence microscopy in liquids

We show that apertureless scanning near-field optical microscopes that use sharp vibrating conical tips can be operated in liquid environments. We have investigated the damping of the tip oscillation as a function of its shape and as a function of its depth under the liquid surface. The degradation of the quality factor from 150 in air down to 15 in liquid does not impede to perform topographic and optical measurements with a very good sensitivity. As an example of application, we present near-field fluorescence images of dye-doped polystyrene spheres immersed in a liquid.

Two-Color Near-Field Fluorescence Microscopy Studies of Microdomains (“Rafts”) in Model Membranes

The distribution of glycolipid GM1 in supported phospholipid monolayers of a ternary lipid mixture has been studied by near-field scanning optical microscopy (NSOM). Monolayers of equimolar amounts of sphingomyelin, dioleoylphosphatidylcholine, and cholesterol show clear phase separation to give large condensed domains surrounded by a fluid phase, as visualized by both atomic force microscopy (AFM) and addition of a Texas Red labeled lipid that localizes preferentially in the fluid phase. A combination of AFM and one- and two-color NSOM experiments indicates that addition of GM1-Bodipy results in small glycolipid domains within the fluid phase. This is in contrast to previous results demonstrating that GM1 is localized in the condensed phase for similar lipid mixtures and is attributed to dye-induced changes in the distribution of the glycolipid. However, NSOM experiments for GM1-containing monolayers doped with low loadings of GM1-Bodipy resulted in the observation of small fluorescent microdomains within the condensed phase. The high spatial resolution of NSOM allows the detection of small domains that have not been observed previously by fluorescence and demonstrates that similar, although not identical, distributions of glycolipid are detected by AFM and fluorescence. The relevance of these results to rafts in natural membranes is discussed.