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.     

ApoTome光学成像系统

荧光显微镜已被广泛地应用于生物样本的荧光成像。然而在观察较厚样本时,来自非焦平面的荧光信息会使荧光图像变得模糊、对比度不够、背景较亮,甚至有的时候一些很重要的结构因此被掩盖而无法被观察到。目前有很多种光学切片方法可以用来去除这种非焦平面荧光的影响,例如激光共聚焦扫描显微技术或3D去卷积技术等。本文介绍了一种可以在普通荧光显微镜上实现光学切片成像的方法,并着重介绍了ApoTome光学成像系统的构造和原理。     

Total internal reflection microscopy for live imaging of cellular uptake of sub-micron non-fluorescent particles

This paper presents a simple, high-resolution, non-fluorescent imaging technique called total internal reflection microscopy (TIRM) and demonstrates its potential application to real-time imaging of live cellular events. In addition, a novel instrument is introduced that combines the simplicity of TIRM with the specificity afforded by dual-colour total internal reflection fluorescence (TIRF) microscopy and allows sequential imaging with the two modalities. The key design considerations necessary to apply these imaging modes in a single instrument are discussed. The application of TIRM alone yielded high-resolution live images of cell adherence to a poly- l -lysine modified substrate, whereby fine cellular structures are imaged. Non-fluorescent imaging of the uptake of sub-micron–sized polymeric particles by live cells is also demonstrated. Finally, images of fluorescently labelled cells were obtained in TIRF mode, sequentially to images obtained of the same cell in TIRM mode. Visual information gained using TIRF is compared with TIRM to demonstrate that the level of cell structure information obtainable with our total internal reflection microscope is comparable with the TIRF technique.     

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.     

Mechanisms of cell-cell adhesion identified by immunofluorescent labelling with quantum dots: A scanning near-field optical microscopy approach

Scanning near-field optical microscopy (SNOM) has been employed to simultaneously acquire high-resolution fluorescence images along with shear-force atomic force microscopy from cell membranes. Implementing such a technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The application of the technique to investigate cell-cell adhesion has revealed the interactions of filopodia and their functional relationship in establishing adherens junctions. This has been achieved via the selective tagging of the cell adhesion protein, E-cadherin, by immunofluorescence labelling. Two labelling routes were explored; Alexa Fluor 488 and semiconductor quantum dots. The quantum dots demonstrated significantly enhanced photostability and high quantum yield making them a versatile alternative to the conventional organic fluorophores often used in such a study. Analysis of individual cells revealed that E-cadherin is predominantly located along the cell periphery but is also found to extend throughout their filopodia. We have demonstrated that with a fully optimised sample preparation methodology, quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.     

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.     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

Attenuation correction in confocal laser microscopes: a novel two-view approach

Confocal microscopy is a three‐dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth‐dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal‐to‐noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two‐view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180° with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model‐based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space‐filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth‐dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two‐view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.     

Near-field fluorescence imaging and simultaneous observation of surface potentials

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

Correlative fluorescence and electron microscopy in tissues: immunocytochemistry

Correlative microscopy is a collection of procedures that rely upon two or more imaging modalities to examine the same specimen. The imaging modalities employed should each provide unique information and the combined correlative data should be more information rich than that obtained by any of the imaging methods alone. Currently the most common form of correlative microscopy combines fluorescence and electron microscopy. While much of the correlative microscopy in the literature is derived from studies of model cell culture systems we have focused, primarily, on correlative microscopy in tissue samples. The use of tissue, particularly human tissue, may add constraints not encountered in cell culture systems. Ultrathin cryosections, typically used for immunoelectron microscopy, have served as the substrate for correlative fluorescence and electron microscopic immunolocalization in our studies. In this work, we have employed the bifunctional reporter FluoroNanogold. This labeling reagent contains both a fluorochrome and a gold-cluster compound and can be imaged by sequential fluorescence and electron microscopy. This approach permits the examination of exactly the same sub-cellular structures in both fluorescence and electron microscopy with a high level of spatial resolution.     

太赫兹近场光学亚表面检测技术研究

研究了太赫兹散射式扫描近场光学显微镜（Terahertz scattering-type scanning near-field optical microscopy,THz s-SNOM）对亚表面金属微纳结构的显微成像检测。首次采用自主搭建的THz s-SNOM系统对表面覆盖了六方氮化硼薄膜的金微米线进行太赫兹近场显微测量,获得了具有纳米量级空间分辨率和较高对比度的近场显微图。结合全波数值模拟,分析了THz s-SNOM探测亚表面金属微纳结构的空间分辨率、近场散射信号强度和成像对比度。研究表明,THz s-SNOM具有优良的亚表面显微成像检测能力,可应用于微纳电子器件的亚表面结构表征和缺陷检测。     

Fluorescence imaging with a laser trapping scanning near-field optical microscope

We investigated fluorescence imaging using a near-field scanning optical microscope which uses a laser-stabilized gold nanoparticle as a near-field probe. This microscope is suitable for observations of biological specimens in aqueous solutions because the probe particle is held by a noncontact force exerted by a laser beam. Theoretical calculations based on Mie scattering theory are presented to evaluate the near-field enhancement by a gold particle of 40 nm diameter. We also present fluorescence images of a single fluorescent bead and discuss the near-field contribution to the fluorescence image in this type of microscope.     

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 µm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water–polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.     

Near-field second harmonic imaging of the c/a/c/a polydomain structure of epitaxial PbZrxTi1–xO3 thin films

Near-field optical second harmonic microscopy has been applied to imaging of the c/a/c/a polydomain structure of epitaxial PbZr _x Ti_{1– x} O₃ thin films in the 0 <  x  < 0.4 range. Comparison of the near-field optical images and the results of atomic force microscopy and X-ray diffraction studies show that an optical resolution of the order of 100 nm is achieved. Symmetry properties of the near-field second harmonic signal allow us to obtain good optical contrast between the local second harmonic generation in c- and a-domains. Experimentally measured near-field second harmonic images have been compared with the results of theoretical calculations. Good agreement between theory and experiment is demonstrated.     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy

Three-dimensional maps of cellular metabolic oxidation/reduction states of rabbit cornea in situ were obtained by imaging the fluorescence of the naturally occurring reduced pyridine nucleotides (both reduced nicotinamide-adenine dinucleotide, NADH, and reduced nicotinamide-adenine dinucleotide phosphate, NADPH, denoted here as NAD(P)H). Autofluorescence images with submicrometre lateral resolution were obtained throughout the entire 400 μm thickness of the cornea. Two-photon excitation scanning laser microscopy with near-infrared excitation provided high fluorescence collection efficiency, reduced photodamage, and eliminated ultraviolet chromatic aberration, all of which have previously degraded the visualization of pyridine nucleotide fluorescence. Sharp autofluorescence images of the basal epithelium (40 μm within the cornea) show substantial subcellular detail, providing the ability to monitor autofluorescence intensity changes over time, which reflect changes in oxidative metabolism and cellular dynamics necessary for maintenance of the ocular surface. The autofluorescence was confirmed to be mostly of NAD(P)H origin by cyanide exposure, which increased the fluorescence from all cell types in the cornea by about a factor of two. Autofluorescence images of individual keratocytes in the stroma were observed only after cyanide treatment, while in the predominant extracellular collagen (> 90% of the stromal volume), fluorescence was not distinguished from the background. Observation of keratocyte metabolism demonstrates the sensitivity made available by two-photon microscopy for future redox fluorescence imaging of cellular metabolic states.     

In-situ observation of shape and atomic structure of Xe nanocrystals embedded in aluminium

An imaging technique to determine in situ the shape and atomic structure of nanosized Xe crystals embedded in Al is described using high-resolution transmission electron microscopy (HRTEM). The Xe nanocrystals, with sizes less than 5 nm were prepared by the implantation of 30 keV Xe⁺ into Al at room temperature. The fcc Xe nanocrystals are mesotactic with the Al lattice and have a lattice parameter ≈ 50% larger than that of Al. HRTEM images of the Xe were not clear in [110] zone axis illumination because of the small number of Xe atoms relative to Al atoms in any atom column. An off-axial imaging technique that consists of tilting the specimen several degrees from a zone axis and defocusing to suppress the Al lattice fringes is employed for the 110 projection of the Xe/Al system and the structure of the Xe nanocrystals is successfully imaged. The Xe images clearly represent projections of cuboctahedra with faces parallel to eight Al {111} planes truncated by six {100} planes. The results of multislice image simulations using a three-dimensional atomic model agreed well with the results obtained by the off-axial imaging technique. The usefulness of the technique is demonstrated with observations of crystal defects introduced into the Xe under intense 1000 keV electron irradiation.     

Automated tracking of migrating cells in phase-contrast video microscopy sequences using image registration

Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal microscopy techniques. The results showed that using fluorescence microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (<5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence microscopy with Volocity and ImageJ.     

Confocal imaging and second-order stereological analysis of a liquid foam

Foam structures are found in diverse fields of study; the structure of fire-fighting foam, upholstery foams and even the head on a pint of beer all share detailed similarities in their microstructure and dynamics. Despite impressive developments in the theory of two-dimensional foams the challenge in future will be to analyse and model the dynamics of three-dimensional foams. However, the myriad of gas/liquid interfaces in an aqueous foam make direct imaging of their structure difficult. In this study we circumvent this problem by using fluorescence confocal microscopy to acquire three-dimensional images of the structure of a coarsening aqueous foam. A stable aqueous foam was created by mixing commercial shaving foam with ethanol and a small amount of fluorescein solution. The foam was imaged in fluorescence mode such that the liquid fraction of the foam, containing the fluorescent dye, could be seen in optical sections. These images could be acquired in three-dimensional stacks of optical sections up to a depth of about 200 μm. Single images were also acquired as a time series. The time series of single optical section images clearly show the dynamics of the foam. Early images show a structure made mainly of spherical bubbles; later images show the polyhedral structure of the foam which coarsens as a function of time. The polyhedral nature of the foams is shown particularly clearly in stereo pair images of the three-dimensional image sets. The three-dimensional images of the foams were also analysed using second-order stereology (statistical summaries of spatial distribution). The x, y and z coordinates of the foam vertices were extracted from the images and used to compute the nearest neighbour (G-function) and reduced second moment (K-function) statistics. These statistics allow quantification of the range of length scales found in the foams. These results form part of an ongoing study of the coarsening of aqueous foams.     

Adaptive aberration correction in a two-photon microscope

We demonstrate aberration correction in two-photon microscopy. Specimen-induced aberrations were measured with a modal wavefront sensor, implemented using a ferro-electric liquid crystal spatial light modulator (FLCSLM). Wavefront correction was performed using the same FLCSLM. Axial scanned ( x z ) images of fluorescently labelled polystyrene beads using an oil immersion lens show restored sectioning ability at a depth of 28 µm in an aqueous specimen.