Characterization of the cutting edge of glass and diamond knives for ultramicrotomy by scanning force microscopy using cantilevers with a defined tip geometry. Part II

The cutting edge of glass as well as diamond knives was studied at high resolution using a scanning force microscope (SFM). The local shape of the cutting edge was estimated from single line profiles of the SFM topographs taking into account the exact shape of the probing tip estimated by a high‐resolution field emission scanning electron microscope (FESEM). The glass knives were prepared by ‘balanced breaking’. The radius of the investigated cutting edges was found to be 3.2–4.4 nm and 4.3–6.0 nm for the 35° and 45° diamond knife, respectively, and 3.4–4.3 nm for the glass knives. Besides the opening angle and the cutting edge radius, the friction of a knife during sectioning represents a significant factor influencing the quality of sections. Thus, the roughness of both the diamond clearance angle side and the back side was characterized as well. Corresponding RMS values of the roughness were found to be smaller on the back side (≈ 0.14 nm) than on the clearance angle side (≈ 0.26 nm).     

Calibration of the scanning (atomic) force microscope with gold particles

Scanning force microscopy (SFM) holds great promise for biological research. Two major problems that have confronted imaging with the scanning force microscope have been the distortion of the image and overestimation in measurements of lateral size due to the varying geometry and characteristics of the scanning tip. In this study, spherical colloidal gold particles (10, 20 and 40 nm in diameter) were used to determine (1) tip parameters (size, shape and semivertical angle); (2) the distortion of the image caused by the tip; and (3) the overestimation or broadening of lateral dimensions. These gold particles deviate little in size, are rigid and have a size similar to biological macromolecules. Images of the colloidal gold particles by SFM were compared with those obtained by electron microscopy (EM). The height of the gold particles as measured by SFM and EM was comparable and was little affected by the tip geometry. The measurements of the lateral dimensions of colloidal gold, however, showed substantial differences between SFM and EM in that SFM resulted in an overestimate of the lateral dimensions. Moreover, the distortion of images and broadening of lateral dimensions were specific to the SFM tip used. The calibration of the SFM tip with mica provided little clue as to the type of distortion and the amount of lateral broadening observed when the larger gold particles were scanned. The SFM image also depended on the orientation of the tip with respect to the specimen. Our results suggest that quantitative SFM imaging requires calibration to identify and account for both the distortions and the magnitude of lateral broadening caused by the cantilever tip. Calibration with gold particles is fast and nondestructive to the tip. The raw imaging data of the specimen can be corrected for the tip effect and true structural information can be derived. In summary, we present a simple and practical method for the calibration of the SFM tip using gold particles with a size in the range of biomacromolecules that allows: (1) selection of a cantilever tip that produces an image with minimal distortion; (2) quantitative determination of tip parameters; (3) reconstruction of the shape of the tip at different heights from the tip apex; (4) appreciation of the type of distortion that may be introduced by a specific tip and quantification of the overestimation of the lateral dimensions; and (5) calculation of the true structure of the specimen from the image data. The significance is that such calibration will permit quantitative and accurate imaging with SFM.     

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm, Lumbricus terrestris, were imaged in NH₄Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h₁ and h₂, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h₁ and 1.23 and 13.6 nm for h₂. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2±10.5 nm, n =75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h₁ and h₂ above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 ±3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 ±0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.     

Comparative study of a block copolymer morphology by transmission electron microscopy and scanning force microscopy

Using transmission electron microscopy (TEM) and scanning force microscopy (SFM) together, it was possible to verify important structural features of a nanostructured bulk material such as the kp‐morphology in an ABC triblock copolymer. By applying suitable imaging techniques during the SFM measurements it was possible to determine the morphology without additional manipulation steps in between. In comparison, TEM investigations on this type of material usually require selective staining procedures prior to the measurement. Also electron beam damage is often encountered during TEM measurements especially if components such as poly(methacrylates) are present. In contrast, SFM measurements can be assumed not to significantly change the phase dimensions of the components.     

Imaging an optical disc by the combined use of scanning tunnelling microscopy and scanning electron microscopy

We present the data obtained by scanning tunnelling microscopy combined with scanning electron microscopy of the digitally encoded structure on a stamper used to fabricate optical discs. The combination allows us to focus the STM tip on a preselected spot with a precision of ?0·3 μm. The data show the superiority of STM for a more detailed characterization of shape, width, length, height and fine structure appearing on the sample. We also show the influence of tip shape on STM resolution. Simultaneous use of both microscopes is possible but high electron doses produce an insulating layer of contaminants thick enough to make STM operation impossible.     

Characterization and fabrication of fully metal-coated scanning near-field optical microscopy SiO2 tips

The fabrication of silicon cantilever‐based scanning near‐field optical microscope probes with fully aluminium‐coated quartz tips was optimized to increase production yield. Different cantilever designs for dynamic‐ and contact‐mode force feedback were implemented. Light transmission through the tips was investigated experimentally in terms of the metal coating and the tip cone‐angle. We found that transmittance varies with the skin depth of the metal coating and is inverse to the cone angle, meaning that slender tips showed higher transmission. Near‐field optical images of individual fluorescing molecules showed a resolution < 100 nm. Scanning electron microscopy images of tips before and after scanning near‐field optical microscope imaging, and transmission electron microscopy analysis of tips before and after illumination, together with measurements performed with a miniaturized thermocouple showed no evidence of mechanical defect or orifice formation by thermal effects.     

High-resolution constant-height imaging with apertured silicon cantilever probes

We present high-resolution aperture probes based on non-contact silicon atomic force microscopy (AFM) cantilevers for simultaneous AFM and near-infrared scanning near-field optical microscopy (SNOM). For use in near-field optical microscopy, conventional AFM cantilevers are modified by covering their tip side with an opaque aluminium layer. To fabricate an aperture, this metal layer is opened at the end of the polyhedral probe using focused ion beams (FIB). Here we show that apertures of less than 50 nm can be obtained using this technique, which actually yield a resolution of about 50 nm, corresponding to λ/20 at the wavelength used. To exclude artefacts induced by distance control, we work in constant-height mode. Our attention is particularly focused on the distance dependence of resolution and to the influence of slight cantilever bending on the optical images when scanning at such low scan heights, where first small attractive forces exerted on the cantilever become detectable.     

Magnetic refinement of tips for magnetic force microscopy

Silicon cantilever probes with monolithically integrated tips are commercially available and are routinely used for atomic force microscopy (AFM). For such probes, amagnetic refinement of the silicon tip has been developed and results in a deposition of ferromagnetic material such as nickel or CrCoTa in the top area of the tip. The method consists of essentially three steps: (1) A broad-area sputter deposition of a ferromagnetic material; (2) a selective electron beam-induced carbon deposition at the top of the tip; (3) a broad-area ion-beam sputter etching, which removes the magnetic layer everywhere except underneath the carbon cap. The method allows to control the total amount and extension of the magnetic material left at the tip. It is applicable to all kinds of ferromagnetic materials which can be deposited as a thin layer by sputter deposition or evaporation. Experiments indicate that the method is reliable and improves the resolution of magnetic force microscopy (MFM). With such magnetically refined tips on silicon cantilevers, MFM measurements have been performed in contact mode as well as in dynamic and static noncontact modes. In this paper, the method for magnetic tip refinement is described and MFM measurements with these tips are presented.     

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.     

An edge reversal method for precision measurement of cutting edge radius of single point diamond tools

A method, which is referred to as the edge reversal method, is proposed for precision measurement of the cutting edge radius of single point diamond tools. An indentation mark of the cutting edge which replicates the cutting edge geometry is firstly made on a soft metal substrate surface. The cutting edge of the diamond tool and its indentation mark, which is regarded as the reversal cutting edge, are then measured by utilizing an atomic force microscopy (AFM), respectively. The cutting edge radius can be accurately evaluated through removing the influence of the AFM probe tip radius, which is comparable to the cutting edge radius, based on the two measured data without characterization of the AFM probe tip radius. The results of measurement experiments and uncertainty analysis are presented to demonstrate the feasibility of the proposed method.     

Resolution enhancing using cantilevered tip-on-aperture silicon probe in scanning near-field optical microscopy

Scanning near-field optical microscopy (SNOM) achieves a resolution beyond the diffraction limit of conventional optical microscopy systems by utilizing subwavelength aperture probe scanning. A problem associated with SNOM is that the light throughput decreases markedly as the aperture diameter decreases. Apertureless scanning near-field optical microscopes obtain a much better resolution by concentrating the light field near the tip apex. However, a far-field illumination by a focused laser beam generates a large background scattering signal. Both disadvantages are overcome using the tip-on-aperture (TOA) approach, as presented in previous works. In this study, a finite difference time domain analysis of the degree of electromagnetic field enhancement is performed to verify the efficiency of TOA probes. For plasmon enhancement, silver is deposited on commercially available cantilevered SNOM tips with 20nm thicknesses. To form the aperture and TOA in the probes, electron beam-induced deposition and focused ion beam machining were applied at the end of the sharpened tip. The results show that cantilevered TOA probes were highly efficient for improvements of the resolution of optical and topological measurement of nanostructures.     

The tetrahedral tip as a probe for scanning near-field optical microscopy at 30 nm resolution

The tetrahedral tip is introduced as a new type of a probe for scanning near-field optical microscopy (SNOM). Probe fabrication, its integration into a scheme of an inverted photon scanning tunnelling microscope and imaging at 30 nm resolution are shown. A purely optical signal is used for feedback control of the distance of the scanning tip to the sample, thus avoiding a convolution of the SNOM image with other simultaneous imaging modes such as force microscopy. The advantages of this probe seem to be a very high efficiency and its potential for SNOM at high lateral resolution below 30 nm.     

Exploring the structure of a hydrogen cyanide polymer by electron spin resonance and scanning force microscopy

Aqueous solutions of potassium cyanide and ammonium hydroxide are known to yield a heterogeneous cyanide polymer, containing paramagnetic sites and biologically significant substructures including polypeptides. Here, such solutions were used to prepare various samples of polymer for study by X-band and W-band electron spin resonance (ESR), scanning electron microscopy (SEM), and scanning force microscopy (SFM). Elemental composition of a typical sample of the polymer was C-35.2%, N-38.47%, 0-14.51%, and H-4.13%, exposing the polymer to 6M HCl hydrolyzed portions of the polymer and released glycine and traces of other amino acids. The X-band ESR spectra consist of a single slightly asymmetric line centered at g = 2.003; spin concentration measurements made at X-band using a nitroxide radical standard yield approximate radical concentrations of 10(18) spins/gm. W-band ESR indicates the presence of a single rhombic paramagnetic site with g(x) = 2.0025, g(y) = 2.0030, and g(z) = 2.0048 and the possibility of small 14N hyperfine splittings. The ESR spin echo studies yield a longitudinal relaxation time, Tl of 75 microS and a short-phase memory relaxation time, Tm, of about 300 nS. Scanning electron microscopy studies of the polymer show that it is made of ellipsoidal particles about one micron in size. The particles tend to clump together when suspended in aqueous solution. The particles disperse and dissolve in dimethyl sulfoxide (DMSO); when these solutions dry on microscope slides, optical microscopy shows a branched island morphology for the polymer. This morphology is reminiscent of snowflakes and is identified as dendritic. Phase contrast SFM of the dendritic arms show a striking segregation and ordering of various components of the polymer. Paramagnetic sites are conserved in the series of steps leading to dendritic structures.     

Scanning electron microscopic technique for imaging a diamond tool edge

A technique is described to measure the edge radius of diamond cutting tools using the scanning electron microscope (SEM). This method attempts to overcome two major limitations of the SEM in this application: low image contrast and lack of quantitative topographic information. A line of electron beam contamination, viewed at an angle, provides improved contrast for focusing and a means of obtaining the tool profile from the geometry.     

Spectroscopic scanning near-field optical microscopy with a free electron laser: CH2 bond imaging in diamond films

Hydrogen chemistry in thin films and biological systems is one of the most difficult experimental problems in today's science and technology. We successfully tested a novel solution, based on the spectroscopic version of scanning near-field optical microscopy (SNOM). The tunable infrared radiation of the Vanderbilt free electron laser enabled us to reveal clearly hydrogen-decorated grain boundaries on nominally hydrogen-free diamond films. The images were obtained by SNOM detection of reflected 3.5 µm photons, corresponding to the C–H stretch absorption, and reached a lateral resolution of 0.2 µm, well below the λ/2 (λ= wavelength) limit of classical microscopy.     

Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy

Tobacco mosaic virus (TMV) has been deposited on freshly cleaved mica substrates. The topography was investigated by contact, non-contact and lateral-force microscopy under ambient conditions in air. The results were in accord with known dimensions of TMV (i.e. 18 nm in diameter and 300 nm in length). However, convolution of tip shape with TMV morphology resulted in an apparent width of 80–140 nm in the lateral plane, a factor of 4–7 greater than the known diameter. Other artefacts - broadening and double images - were observed and ascribed to tip anomalies. High force loadings and slow repetitive scanning resulted in controlled removal of parts of the TMV structure. Accordingly, it was possible to reveal and image the central core channel of the TMV. The precision and resolution of dissection induced by AFM is currently limited by the shape of the tip, having a 40-nm radius of curvature for standard Si₃N₄ tips. It is estimated that sharper tips, with a radius of curvature of less than 10 nm, should be able to resolve, non-destructively, the protein subunits in the non-contact mode, and selectively remove single subunits in the contact mode.     

Microstructure of Monoplacophora (Mollusca) shell examined by low-voltage field emission scanning electron and atomic force microscopy

The shell of Micropilina arntzi (Mollusca: Monoplacophora), a primitive molluscan class, was examined by using field emission scanning electron microscopy (FESEM) at low voltage and atomic force microscopy (AFM). The use of these two techniques allowed the observation of fine details of Micropilina arntzi shell and contributed to bring new features concerning the study of molluscan shell microtexture. Imaging with low-voltage FESEM provided well-defined edge contours of shell structures, while analyzing the sample with AFM gave information about the step height of stacked internal structures as well as the dimension of the particles present in their surface at a nanometric level. The shell microstructure of Monoplacophora species presents different patterns and may be a taxonomic implication in the systematic studies of the group.     

A compact sensor head for simultaneous scanning force and near-field optical microscopy

A compact sensor head based on scanning force microscopy (SFM) using cantilever probes has been developed. The idea is to replace the microscope objective of a conventional optical microscope by this compact module and turn the optical microscope into a scanning force and near-field optical microscope with subwavelength resolution. We describe our concept and present initial results showing images of the object’s optical properties and surface topography recorded simultaneously.     

Resonant excitation of tip plasmons for tip-enhanced Raman SNOM

The conditions necessary for the optimisation of tip-enhanced scanning near-field optical microscopy have been determined. The Raman scattering efficiency can be enormously increased by enhancements in the local field amplitude, such as that which can occur in the vicinity of a metallic nanostructure. The field enhancement in the vicinity of a silver tip is investigated theoretically here using the finite difference time domain method. Field enhancements from electron oscillations on the tip are shown to display strong maxima at resonant illumination wavelengths and the nature of these enhancements at the substrate surface beneath the tip, both on and off resonance, has been calculated. The enhancement of the Raman signal on the surface decreases exponentially as the tip–substrate separation is increased and a peak Raman enhancement of 10⁷ is theoretically achievable at a tip–surface separation of 2 nm. The resolution is also strongly related to the distance between the tip and the substrate surface narrowing to <7 nm, significantly smaller than the radius of curvature of the end of the tip.