Dynamic volume change measurements of cereal materials by environmental scanning electron microscopy and videomicroscopy

Moisture migration induced volume change in cereal materials was studied by environmental scanning electron microscopy, videomicroscopy and stereoscopy. It is shown that the in situ volume change can be monitored by the changes of projection area with the assumption of isotropic swelling/shrinkage. The projection area change from videomicroscopy matches well with the volume change by stereoscopy after dimensional normalization. The arbitrary volume values in between two relative humidity levels are available by interpolating the equilibrium volumes at beginning and ending. The sorption/desorption curves were fitted by the 'parallel exponential kinetics' model, which described two independent, parallel processes as 'slow' and 'fast'. In the low relative humidity range, sorption curves match well with the 'parallel exponential kinetics' model and the two parallel processes tend to be identical when relative humidity increases. The diffusivity of water moisture in a cracker, which was estimated by the half-equilibrium principle, has a strong dependence on relative humidity.     

Three-dimensional diffusion measurements by scanning microphotolysis

Fluorescence microphotolysis techniques have been used widely to measure lateral diffusion in two-dimensional microsystems such as cell membranes. However, a general microphotolysis method for the analysis of truly three-dimensional diffusion processes has not been developed so far. Here we combine microphotolysis with confocal laser scanning microscopy and numerical data evaluation in such a manner that small volumes (≥ 0.5 μm³) can be photolysed within extended three-dimensional samples and that fluorescence changes can be monitored at high time resolution (measuring interval 0.5 ms) and evaluated for lateral diffusion coefficients. We show furthermore that diffusion measurements can be performed according to three different experimental modes: (i) the instantaneous mode, (ii) the continuous mode and (iii) the mixed mode. For the evaluation of experimental data in terms of diffusion coefficients we have developed and thoroughly characterized a theoretical framework which is based on the numerical simulation of appropriate reaction–diffusion systems. The theoretical framework is rather general and flexible and can be applied to any microphotolysis geometry, makes provision for photolysis during fluorescence monitoring and takes into account the convolution of concentration distributions with imaging point spread functions. The new method was tested employing simple well-defined model systems.     

Human chromosome structure studied by scanning force microscopy after an enzymatic digestion of the covering cell material

In standard preparations, metaphase human chromosomes are covered by a cell material film composed mainly of proteins and RNA. This film (approximately 30 nm thickness) hides the chromosome structure to the tip of a scanning force microscope. In this work, a mild enzymatic treatment is applied to remove the cell material film. After treatment, the individual chromatin fibers at the surface were resolved. Furthermore, the chromosome shows a thickness modulation, in which thicker/thinner regions could be associated with G/R bands. Finally, the topography of the chromosomes in solution is presented. The chromosome volume swelled about five-fold and chromatin packaging in bands and coils was observed.     

Characterization of self-assembling isolated ferroelectric domains by scanning force microscopy

Lead zirconate titanate (PZT) thin films were prepared by a sol–gel process on platinized Si substrate. Their microstructure and surface morphology were characterized by XRD and Scanninn Force Microscopy. Phase transformation of the prepared PZT films from pyrochlore to ferroelectric was observed by XRD and PFM (piezoresponse force microscopy), respectively. Self-assembling nano-structured ferroelectric phases are fabricated by solution deposition technique followed by the controlling kinetics of the transformation. Complex structures of ferroelectric domains in the isolated ferroelectric phases were found in the furnace annealed PZT films in the temperature range of 400–500°C. Single ferroelectric domain structure in the isolated ferroelectric phases could be found in thinner PZT films and used to study the size effect of laterally confined ferroelectric domains.     

Ribosome substructure investigated by scanning force microscopy and image processing

Scanning force microscopy was used to study the ultrastructure of eukaryotic ribosomes from Chironomus pallidivittatus in the polysomal complex. Positively stained polysomes were imaged, and the resulting three-dimensional ribosomal structures were further processed by statistical analyses of virtual cross-sections parallel to the substrate plane. Structural investigations were based on parameters which are minimally influenced by the tip geometry, like section plane centre or axis ratio. In the lower part of the structure a shift of the section centres was observed, suggesting an attached structure. The geometry of the sections revealed an elliptical shape in the upper part (axis ratio 1.52 ± 0.22), with a less elongated shape in the lower region (axis ratio 1.41 ± 0.18). A model for the surface topography of a positively stained ribosome exhibiting a small subunit attached along the long side of an elliptical large structure is proposed.     

Macroscopic water deposits on polycrystalline gold measured by scanning force microscopy

Data of water adsorption on polycrystalline gold show the formation of a multilayer film of several nanometers with the increase of relative humidity. We have measured this adsorption process by scanning force microscopy in both dynamic and jumping modes. We find interesting differences in the adsorption of water on the terraces and at grain boundaries. Measurements of adhesion force are also reported.     

High-resolution, very broadband force measurements by solid-state laser transducers

Novel high-performance force transducers are discussed. Force measurement is based on the photoelastic effect in solid-state lasers (diode-pumped Nd:YAG) and provides an electrical frequency output. Our experiments demonstrate that the dynamic range of a single monolithic laser transducer covers at least 9 decades in which the output frequency is strictly proportional to the input force magnitude. Within the measurement bandwidth from DC up to at least 100 kHz force modulation frequency, static sensitivity of the transducer equals its dynamic sensitivity. Furthermore, input signals with modulation frequencies up to the MHz range can also be detected. The total measurement range covered by the applied laser technology ranges from nano-newton to mega-newton depending on the size of the laser crystals.     

Limits of topographic measurement by the scanning tunnelling and atomic force microscopes

Parameters describing the topographic character of a surface (height, surface wavelength, slope and curvature) can be derived from equivalent sinusoidal profiles. The response of a surface-measuring instrument may be modelled in terms of instrument parameters such as stylus radius, and scanning range and resolution. The performance of the instrument may then be mapped as a zone in amplitude-wavelength (AW) space to show the sinusoidal profiles it is capable of measuring. In a first-order analysis the STM and AFM are considered as equivalent to contact-stylus instruments with a notional stylus radius equal to the tip radius plus the gap. Comparisons between different instruments and types of instrument are readily made by mapping in AW space. The error arising from convolution of the sinusoidal profile with that of the finite tip may be quantified and plotted as contours in AW space.     

Nanoscale potential measurements in liquid by frequency modulation atomic force microscopy

We have developed a method for local potential measurements in liquid using frequency modulation atomic force microscopy. In this method, local potential is calculated from the first and second harmonic vibrations of a cantilever induced by applying an ac bias voltage between a tip and a sample. The use of an ac bias voltage with a relatively high frequency prevents uncontrolled electrochemical reactions and redistribution of ions and water. The nanoscale resolution of the method is demonstrated by imaging potential distribution of a dodecylamine thin film deposited on a graphite surface in 1 mM NaCl solution.     

Atomic force microscopy study of chromosome surface structure changed by protein extraction

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.     

A method for measuring the size distribution of latex particles by scanning force microscopy

A methodology has been developed to accurately determine the size distribution of latex particles using the scanning force microscope (SFM). Unlike other workers, who have generally measured the lateral dimensions of monolayers of latex particles using a global quantification method, we have measured the heights of individual latex particles located at the edges of latex monolayers that were immobilised onto mica substrates. In agreement with other work, we noted that the edges of monolayers of latex particles provided stable and reproducible scanning force imaging.

Whilst SFM imaging noise, image processing artifacts, tip/sample forces and variations in the mica substrate are sources of measurement error that should not be overlooked, our experience has been that the variation over time of the sensitivity of the Z actuator is the greatest potential uncertainty in determining the heights of latex particles. The methodology that we used requires frequent calibration of the Z actuator of the SFM, typically before and after two or three images, in order to ensure that the uncertainties in the Z sensitivity are known and minimised. This methodology was developed for an SFM instrument that was equipped with open loop piezoelectric actuators following a careful study of the behaviour of those actuators.

Using this methodology, we have measured the size distributions of populations of 300–400 latex particles from each of several different latex samples, with the maximum variation in the Z-actuator calibration experienced during the measurement of a sample being less than 2%, often about 1% and occasionally better still. In so doing, we have demonstrated that SFMs equipped with open loop actuators can be used for high confidence quantitative measurements of step heights.     

Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy

Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex.     

Microelectrical characterizations of junctions in solar cell devices by scanning Kelvin probe force microscopy

Scanning Kelvin probe force microscopy was applied to the microelectrical characterizations of junctions in solar cell devices. Surface Fermi-level pinning effects on the surface potential measurement were avoided by applying a bias voltage (V_b) to the device and taking the V_b-induced potential and electric field changes. Two characterizations are presented: the first is a direct measurement of Bi-induced junction shift in GaInNAs(Bi) cells; the second is a junction-uniformity measurement in a-Si:H devices. In the first characterization, using Bi as a surfactant during the molecular beam epitaxy growth of GaInNAs(Bi) makes the epitaxial layer smoother. However, the electrical potential measurement exhibits a clear Bi-induced junction shift to the back side of the absorber layer, which results in significant device degradation. In the second characterization, the potential measurement reveals highly non-uniform electric field distributions across the n–i–p junction of a-Si:H devices; the electric field concentrates much more at both n/i and i/p interfaces than in the middle of the i-layer. This non-uniform electric field is due possibly to high defect concentrations at the interfaces. The potential measurements further showed a significant improvement in the electric field uniformity by depositing buffer layers at the interfaces, and this indeed improved the device performance.     

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10(-5).     

Optical microcantilever consisting of channel waveguide for scanning near-field optical microscopy controlled by atomic force

We develop a novel optical microcantilever for scanning near-field optical microscopy controlled by atomic force mode (SNOM/AFM). The optical microcantilever has the bent channel waveguide, the corner of which acts as aperture with a large tip angle. The resonance frequency of the optical microcantilever is 9 kHz, and the spring constant is estimated to be 0.59 N/m. The optical microcantilever can be operated in contact mode of SNOM/AFM and we obtain the optical resolution of about 200 nm, which is as same size as the diameter of aperture. We confirm that the throughput of optical microcantilever with an aperture of 170 nm diameter would be improved to be more than 10⁻⁵.     

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.     

Measurement by vertical scanning profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine slices

The resorption pit assay is classically used to evaluate osteoclast activity on bone or dentine slices that can be eroded by these cells. Two different types of cells were generated from peripheral blood mononuclear cells cultured in the presence of M-CSF + sRANKL or with M-CSF + LPS. At the end of the culture period (21 days), cells were discarded and the dentine slices stained with toluidine blue and examined with an NT9100 Wyco vertical scanning profilometer. The images of the dentine surface were corrected for tilt and the eroded volume was calculated on the whole images. The depth of the eroded pits was determined. The data files were used to reconstruct the surface of the slices by standardizing the ground level to compare both conditions. Osteoclasts generated with M-CSF + sRANKL were capable of resorbing a more important volume than those generated with M-CSF + LPS. In addition, the formers were able to resorb the dentine matrix more deeply. Data provided by the microscope were used to reconstruct three-dimensional images of the dentine slices with pseudo colours varying with the depth of erosion. Vertical scanning profilometry, a technique used to measure the roughness of polished or etched surfaces in metallurgic industry, can be used to accurately measure the eroded volume and the mean erosion depth done by osteoclasts in the resorption pit assay.     

Measurements of thickness dispersion in biolayers by scanning force microscopy and comparison with spectroscopic ellipsometry analysis

Measuring the thickness of biological films remains a difficult task when using differential measurements by atomic force microscopy (AFM). The use of microstructured substrates combined with a selective adsorption constitutes an alternative to tribological measurements. The statistical thickness analysis of biological layers, especially via the dispersion measurements, can provide a way to quantify the molecular orientation. AFM thicknesses were then compared with those obtained optically by spectroscopic ellipsometry (SE) and surface plasmon resonance enhanced ellipsometry (SPREE). The biolayers could then be modeled using a vertical gradient of optical index, which reflects height dispersions. Thiol-modified DNA strands of various lengths account for a good biological model for the study of the strand motion in air and in liquid.