Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Nanoscopic observation of a gold nanoparticle-conjugated protein using near-field scanning optical microscopy

This study describes a single gold nanoparticle (AuNP)-based observation of biomolecular interaction using a near-field scanning microscope (NSOM) in transmission mode. To observe streptavidin molecules, a glass surface was first patterned with a micro-scale line of (3-aminopropyl)trimethoxysilane (APTMS) by micro-contact printing (μCP) with a subsequent reaction of N-hydroxysuccinimide (NHS)-biotin. The AuNP-conjugated streptavidin was then applied to the biotin-modified glass surface and NSOM was employed to detect the resulting specific interaction between streptavidin and biotin on the glass surface. Using the optical and topological images generated from the NSOM analysis, the interaction could be observed at the nanoscopic scale. This study demonstrates that the NSOM is a powerful tool for the detection of protein interactions at the nanoscopic level when the protein is conjugated with AuNPs.     

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1–4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1–2°. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.     

突破衍射极限的成像方法综述

"衍射极限"实际上不是一个真正的障碍,除非处理远场和定位精度。这种衍射障碍并不是坚不可摧的,可以利用一些智能技术来突破光学衍射极限。讨论了四种技术,近场扫描光学显微镜(NSOM)法,受激发射损耗(STED)显微镜法,光激活定位显微镜(PALM)法或随机光学重建显微镜(STORM)法和结构照明显微镜(SIM)法,并且介绍了各自的基本原则与优劣。NSOM利用纳米级探测器检测通过光纤的极小汇聚光斑,从而获得单个像素的分辨率;PALM和STORM利用荧光探针,实现暗场和荧光的转换,从而观察到极小的荧光团;SIM则是利用栅格图案与样品叠加成像来实现。其中,STORM具有相对较高的潜力,能够更为有效地突破衍射极限。     

Near-field fluorescence imaging of single molecules with a resolution in the range of 10 nm

We demonstrate fluorescence imaging of single molecules, by near-field scanning optical microscopy (NSOM), using the illumination-collection mode of operation, with an aperture probe. Fluorescence images of single dye molecules were obtained with a spatial resolution of 15 nm, which is smaller than the diameter of the aperture (20 nm) of the probe employed. Such super-resolution may be attributable to non-radiative energy transfer from the molecules to the coated metal of the probe since the resolution obtained in the case of conventional NSOM is limited to 30–50 nm due to penetration of light into the metal.     

Nanoscale organization of CD4 molecules of human T helper cell mapped by NSOM and quantum dots

CD4 molecule, the surface marker of T helper cell, has been confirmed to be the main cellular receptor for the human immunodeficiency viruses HIV-1, HIV-2 and SIV. Recent research demonstrated the importance of the spatial arrangement of CD4 on the cell membrane to its binding efficiency to virus. In this article, the combined near-field scanning optical microscopy (NSOM) and quantum dots (QDs) fluorescent labeling technology were performed to investigate the nanoscale organization of CD4 molecules with a spatial resolution about 100 nm. Simultaneous topographic image of the T helper cell and fluorescent image of QDs have been directly gained by NSOM/QDs-based system. Intensity- and size-distribution histograms of the QDs fluorescent spots verify that approximately 80% of the CD4 molecules are organized in nanosized domains randomly distributed on the cell surface. Intensity-size correlation analysis revealed heterogeneity in the molecular packing density of the domains. Our results also illustrated the combination of NSOM imaging and QDs labeling is an ultrasensitive, high-resolution technique to probe nanoscale organization of molecules on the cell surface.     

Single molecule mapping of the optical field distribution of probes for near-field microscopy

The most difficult task in near-field scanning optical microscopy (NSOM) is to make a high quality subwavelength aperture probe. Recently, we have developed high definition NSOM probes by focused ion beam (FIB) milling. These probes have a higher brightness, better polarization characteristics, better aperture definition and a flatter end face than conventional NSOM probes. We have determined the quality of these probes in four independent ways: by FIB imaging and by shear-force microscopy (both providing geometrical information), by far-field optical measurements (yielding throughput and polarization characteristics), and ultimately by single molecule imaging in the near-field. In this paper, we report on a new method using shear-force microscopy to study the size of the aperture and the end face of the probe (with a roughness smaller than 1.5 nm). More importantly, we demonstrate the use of single molecules to measure the full three-dimensional optical near-field distribution of the probe with molecular spatial resolution. The single molecule images exhibit various intensity patterns, varying from circular and elliptical to double arc and ring structures, which depend on the orientation of the molecules with respect to the probe. The optical resolution in the measurements is not determined by the size of the aperture, but by the high optical field gradients at the rims of the aperture. With a 70 nm aperture probe, we obtain fluorescence field patterns with 45 nm FWHM. Clearly, this unprecedented near-field optical resolution constitutes an order of magnitude improvement over far-field methods like confocal microscopy.     

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.     

Epifluorescence, confocal and total internal reflection microscopy for single-molecule experiments: a quantitative comparison

Epifluorescence, confocal and total internal reflection microscopy are the most widely used techniques for optical single‐molecule experiments. Employing these methods, we recorded the emission intensity of the same single molecule as a function of the excitation rate under otherwise identical experimental conditions. Evaluation of these data provides a quantitative comparison of the signal‐to‐background ratios that can be achieved for the three microscopic techniques.     

Terminal protein-induced stretching of bacteriophage φ29 DNA

Stretching of DNA molecules helps to resolve detail during the fluorescence microscopy of both single DNA molecules and single DNA–protein complexes. To make stretching occur, intricate procedures of specimen preparation and manipulation have been developed in previous studies. By contrast, the present study demonstrates that conventional procedures of specimen preparation cause DNA stretching to occur, if the specimen is the double‐stranded DNA genome of bacteriophage φ29. Necessary for this stretching is a protein covalently bound at both 5′ termini of φ29 DNA molecules. Some DNA molecules are attached to a cover glass only at the two ends. Others are attached at one end only with the other end free in solution. The extent of stretching varies from ～50% overstretched to ～50% understretched. The understretched DNA molecules are internally mobile to a variable extent. In addition to stretching, some φ29 DNA molecules also undergo assembly to form both linear and branched concatemers observed by single‐molecule fluorescence microscopy. The assembly also requires the terminal protein. The stretched DNA molecules are potentially useful for observing DNA biochemistry at the single molecule level.     

Near-field and confocal surface-enhanced resonance Raman spectroscopy at cryogenic temperatures

For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near‐field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano‐particles or nano‐clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm^?1 and 3000 cm^?1 at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.     

Fundamental differences between micro- and nano-Raman spectroscopy

Electric field polarization orientations and gradients close to near-field scanning optical microscope (NSOM) probes render nano-Raman fundamentally different from micro-Raman spectroscopy. With x -polarized light incident through an NSOM aperture, transmitted light has x, y and z components allowing nano-Raman investigators to probe a variety of polarization configurations. In addition, the strong field gradients in the near-field of a NSOM probe lead to a breakdown of the assumption of micro-Raman spectroscopy that the field is constant over molecular dimensions. Thus, for nano-Raman spectroscopy with an NSOM, selection rules allow for the detection of active modes with intensity dependent on the field gradient. These modes can have similar activity as infra-red absorption modes. The mechanism can also explain the origin and intensity of some Raman modes observed in surface enhanced Raman spectroscopy.     

Domain formation in thin lipid films probed with near-field scanning optical microscopy

High-resolution near-field scanning optical microscopy (NSOM) fluorescence and topographic images of l -α-dipalmitoylphosphatidylcholine (DPPC) monolayers doped with a fluorescent dye are presented. DPPC monolayers are deposited onto mica substrates from the air–water interface at several surface pressures using the Langmuir–Blodgett technique. Sub-diffraction limit phase domain structures are observed in both fluorescence and topographic NSOM images of the lipid films. The morphology of the resulting monolayers depends strongly on the surface pressure and composition of the subphase used in the film transfer. Mechanisms for lipid domain formation and growth are discussed.     

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.     

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near-field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength-sized light source for high-resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near-field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near-field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near-field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.     

Atomic force microscopy to characterize the molecular size of prion protein

The conformational transition of α‐helix‐rich cellular prion protein (PrP^C) to an isomer with high β‐sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long‐term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)₁₀‐tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu²⁺ resulted in an enhanced PrP immobilization, whereas Ni²⁺ reduced coverage of the surface by PrP. High‐resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm³. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm³ for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.     

Mapping the distribution of emissive molecules in human ocular lipofuscin granules with near-field scanning optical microscopy

Several high resolution imaging techniques are utilized to probe the structure of human ocular lipofuscin granules. Atomic force microscopy reveals typical granule sizes to be about one micrometre in diameter and hundreds of nanometres in height, in agreement with previous electron microscopy results. For issues concerning the role of lipofuscin in age-related macular degeneration, recent attention has focused on the orange-emitting fluorophore, A2E. Confocal microscopy measurements are presented which reveal the presence of a highly emissive component in the granules, consistent with the presence of A2E. It is shown, however, that the interpretation of these results is complicated by the lack of structural details about the particles. To address these issues, near-field scanning optical microscopy (NSOM) measurements are presented which measure both the lipofuscin fluorescence and topography, simultaneously. These measurements reveal distinct structure in the fluorescence image which do not necessarily correlate with the topography of the granules. Moreover, direct comparison between the NSOM fluorescence and topography measurements suggests that A2E is not the major component in lipofuscin. These measurements illustrate the unique capabilities of NSOM for probing into the microstructure of lipofuscin and uncovering new insights into its phototoxicity.     

Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions

We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.     

Microscopy for recognition of individual biomolecules

One frontier challenge in microscopy and analytical chemistry is the analysis of soft matter at the single molecule level with biological systems as most complex examples. Towards this goal we have developed two novel microscopy methods. Both employ highly specific molecular recognition schemes used by nature-the recognition of specific protein sites by antibodies and ligands. One method uses fluorescence labeled ligands for detecting single molecules in fluid systems like membranes (Fig. 1B). Unitary signals are reliably resolved even for millisecond illumination periods. The knowledge of the unitary signal from single molecules permits the determination of stoichiometries of component association (Fig. 3). Direct imaging of the diffusional path of single molecules became possible for the first time (Fig. 4). Using linear polarized excitation, the angular orientation of single molecules can be analyzed (single molecule linear dichroism, (Fig. 5), which opens a new perspective for detecting conformational changes of single biomolecules. In the other method, an antibody is flexibly linked to the tip of an atomic-force microscope. This permits the identification of receptors in multi-component systems. Molecular mapping of biosurfaces and the study of molecular dynamics in the ms to s range become possible with atomic force microscopy.