Study of Phase Imaging Contrast Optimization in Tapping Mode Atomic Force Microscopy

Phase contrast and attraction–repulsion transition in tapping mode atomic force microscopy

Atomic force microscopy investigation of filled elastomers and comparison with transmission electron microscopy — application to silica-filled silicone elastomers

Topographic and phase-contrast imaging in atomic force microscopy

In this study, atomic force microscopy (AFM) is used to investigate the heterogeneity of blended films of poly(methyl methacrylate) (PMMA) and polybutadiene (PB) on silicon substrates before and after annealing. The blended films with different ratios of PMMA to PB are prepared by spin casting onto silicon substrates from solution. The surface morphology and composition of these cast films are investigated using tapping mode and force mode AFM. Annealing the samples in air at 75 ± 5 °C causes changes in the relative chemical and mechanical differences between PMMA and PB, so the phase image contrast is studied as a function of annealing time. The effect of tapping force level on phase image contrast is also explored. To identify the different components in polymer blends and to understand the influence of relative surface stiffness on the phase images, nanoscale indentation measurements are made on the observed phase-separated regions. Interpretation of AFM results is aided by data collected from both con...

Atomic force microscopy (AFM) was used in tapping mode to probe morphological changes in surface-confined cells resulting from adhesion-modifying chemicals. Bacteria (Burkholderia cepacia G4 and Pseudomonas stutzeri KC) were exposed to Tween 20, heparin, disodium tetraborate, sodium pyrophosphate, low ionic strength water, lysozyme/ethylenediaminetetraacetic acid (EDTA), and 3-(4-morpholino)propanesulfonic acid sodium salt (MOPS) buffer (as a control), and the surface topography of the cells was examined after exposure to each chemical. Cells were attached to glass slides for AFM imaging by a new method of cross-linking carboxyl groups on the bacterial surfaces with amine groups that had been coupled to glass slides. Topographic images, phase images, traces of surface topography, and analyses of surface roughness were performed on all samples. We are not aware of any studies besides the present one in which phase imaging has been used on bacteria. Height traces illustrated the effect of different chemical treatments on the cells by showing whether the topography of the cell was altered by the different treatments. The surface roughness was quantified in terms of the root-mean-square (RMS) average of the height deviations. All of the treatment chemicals except disodium tetraborate caused higher RMS values to be measured for G4 and KC. Disodium tetraborate flattened the cells and, therefore, resulted in slightly lower or equal RMS values as the control (MOPS buffer). RMS values were correlated with the qualitative shapes of the cells. Lysozyme/EDTA, sodium pyrophosphate, and disodium tetraborate produced the most damage to cellular morphology, as observed by topographic images and surface traces, and decreased cellular viability. These data show that AFM operated in tapping mode can provide a useful method for investigating the consequences of bacterial exposure to surface-modifying chemicals.

Tapping mode atomic force microscopy (TMAFM) measurements were performed for blends of two elastomers, cis-1,4-butadiene rubber (BR) and styrene-co-butadiene rubber (SBR) containing silica filler particles. To help interpret the TMAFM phase and height images of the BR/SBR blends, transmission electron microscopy (TEM) measurements were carried out for the BR/SBR blends, and dynamic mechanical analysis (DMA) as well as frequency-sweep/force-probe TMAFM measurements were carried out for BR and SBR homopolymers. TEM images show that silica filler particles of BR/SBR blends are present mainly in the SBR component, and DMA results reveal that BR has a lower glass transition temperature than does SBR. In the phase images of BR/SBR blends the less stiff component BR is brighter than is the stiffer component SBR. For the rational interpretation of TMAFM phase images of viscoelastic materials, it is crucial to consider the indentation depth of the tip into samples as well as the reduced tip−sample energy dissipati...

Under energy-dissipative cantilevered tip-sample interaction, phase imaging using tapping-mode atomic force microscopy enables compositional mapping of composites containing a harder inorganic phase at the nanometer scale, embedded in a polymer matrix. The contrast in the phase images is shown to be dependent on the variation in the elastic properties of the diblock copolymer reverse micelles loaded with zinc acetate. Tapping conditions are also shown to determine whether the contrast is positive or negative for the harder core of the loaded micelles, based on the competition between attractive and repulsive tip-sample interaction forces. The broader implications are significant for scanning probe microscopy of other soft materials systems containing the segregation of a harder phase.

OA248] Computer-based platform for phase contrast breast imaging

Quantitative differential phase-contrast (DPC) imaging is one of the commonly used methods for phase retrieval. However, quantitative DPC imaging requires several pairwise intensity measurements, which makes it difficult to monitor living cells in real-time. In this study, we present a single-shot quantitative DPC imaging method based on the combination of deep learning (DL) and color-encoded illumination. Our goal is to train a model that can generate an isotropic quantitative phase image (i.e., target) directly from a single-shot intensity measurement (i.e., input). The target phase image was reconstructed using a linear-gradient pupil with two-axis measurements, and the model input was the measured color intensities obtained from a radially asymmetric color-encoded illumination pattern. The DL-based model was trained, validated, and tested using thirteen different cell lines. The total number of training, validation, and testing images was 264 (10 cells), 10 (1 cell), and 40 (2 cells), respectively. Our results show that the DL-based phase images are visually similar to the ground-truth phase images and have a high structural similarity index (>0.98). Moreover, the phase difference between the ground-truth and DL-based phase images was smaller than 13%. Our study shows the feasibility of using DL to generate quantitative phase imaging from a single-shot intensity measurement.

Imaging recognition events between human IgG and rat anti-human IgG by atomic force microscopy

In available synchrotron radiation facilities all over the world, the size of electron beam bunch has distinct difference between the horizontal direction and the vertical direction at the cross section vertical to electron moving direction. It will cause low symmetry of source size in horizontal and vertical directions, and result in big difference in the spacial resolution and phase contrast between these two directions in synchrotron radiation X-ray in-line phase contrast imaging. The superior resolution and phase contrast of one direction will couple with the inferior ones of the other direction in actual object imaging, this interaction will degrade both spacial resolution and phase contrast of the whole image and lead to poor image quality. In this paper, a four-knife white beam slit is used in Beijing synchrotron radiation facility (BSRF) 4W1A beamline to adjust source size in horizontal and vertical directions and restrict the point spread function (PSF) of source, the source size is limited from 1.23 mm (horizontal)×0.46 mm (vertical) to 0.35 mm×0.35 mm. By this way, a highly symmetric effective X-ray source is obtained. The symmetry of source size is improved from 2.7 to about 1. Although the total X-ray flux loses 99.95%, the X-ray flux density is only reduced to 22%. It because total flux is taken account of the expanded spot size due to divergence, while flux density is mainly related to effective source size, which is limited not severely as expanded spot size by white beam slit. The flux density reduction by this method is not a critical defect in high resolution X-ray phase contrast imaging and can be improved by other ways. The polyamide fiber net is imaged using X-ray in-line phase contrast imaging at 4W1A, BSRF. Diameter of fiber is about 50 microns, X-ray energy is 13 keV, the effective pixel size is 0.325 micron, the experiment images are obtained at object-to-detector distances of 4, 10, 16, 36, 100, 200 and 300 mm with source-to-object distance of about 43 m. The results show that when source size in horizontal direction is limited from 1.23 to 0.35 mm (symmetry of source size is about 1 at this condition), the maximal phase contrast of image is advanced from 0.057 to 0.35. It means the density sensitivity is promoted about 5 times than in ordinary source size conditions. The spacial resolutions in different source size symmetry conditions are also primary analyzed based on Rayleigh criterion. The result shows that spacial resolution can be improved notably in horizontal direction by limiting horizontal source size to a nearly symmetric source size condition. Our work demonstrates that the spacial resolution and phase contrast can be promoted at the same level by source size symmetry improved, which markedly improved the imaging quality in synchrotron radiation X-ray in-line phase-contrast imaging. It will have great benefit to high-resolution X-ray in-line phase contrast imaging. The reasons of these improvements are analyzed and developments in future are also discussed.

Recent results show that bone vasculature is a major contributor to local tissue porosity, and therefore can be directly linked to the mechanical properties of bone tissue. With the advent of third generation synchrotron radiation (SR) sources, micro-computed tomography (?CT) with resolutions in the order of 1 ?m and better has become feasible. This technique has been employed frequently to analyze trabecular architecture and local bone tissue properties, i.e. the hard or mineralized bone tissue. Nevertheless, less is known about the soft tissues in bone, mainly due to inadequate imaging capabilities. Here, we discuss three different methods and applications to visualize soft tissues. The first approach is referred to as negative imaging. In this case the material around the soft tissue provides the absorption contrast necessary for X-ray based tomography. Bone vasculature from two different mouse strains was investigated and compared qualitatively. Differences were observed in terms of local vessel number and vessel orientation. The second technique represents corrosion casting, which is principally adapted for imaging of vascular systems. The technique of corrosion casting has already been applied successfully at the Swiss Light Source. Using the technology we were able to show that pathological features reminiscent of Alzheimer"s disease could be distinguished in the brain vasculature of APP transgenic mice. The third technique discussed here is phase contrast imaging exploiting the high degree of coherence of third generation synchrotron light sources, which provide the necessary physical conditions for phase contrast. The in-line approach followed here for phase contrast retrieval is a modification of the Gerchberg-Saxton-Fienup type. Several measurements and theoretical thoughts concerning phase contrast imaging are presented, including mathematical phase retrieval. Although up-to-now only phase images have been computed, the approach is now ready to retrieve the phase for a large number of angular positions of the specimen allowing application of holotomography, which is the three-dimensional reconstruction of phase images.

In scanning transmission electron microscopy (STEM), one can obtain a variety of STEM images such as bright‐field (BF) and annular dark‐field (ADF) STEM images by changing the shape of the scintillator. However, the intensity distribution of convergent beam electron diffraction (CBED) patterns at the detector plane is yet to be fully utilized. Meanwhile, direct electron detectors with fast frame rate have recently been commercialized and used in electron microscopy. Such detectors, when used for recording CBED pattern images for each STEM probe position, are called pixelated STEM detectors. With the obtained 4‐dimensional (4D) dataset, any shape of STEM detector can be synthesized in a post processing by a free selection of the integration area. Therefore, we can synthesize variety of STEM images such as differential phase contrast (DPC) and annular bright field (ABF) images, if we once record the image signal with a pixelated detector. The 4D dataset can also be used for the advanced image processing techniques such as ptychography, which has been shown to provide high efficiency for reconstructing the phase image of an object [1,2]. Using ptychography, not only the phase contrast can be enhanced but also the effect of lens aberrations to the image such as defocus can be corrected by the post processing using the information collected by a pixelated detector. Experiments were performed using an aberration corrected microscope (JEOL JEM‐ARM200F) equipped with a pixelated detector (pnDetector pnCCD), the fast direct electron detector, which can record images at a speed of 1,000 fps in a full frame mode (264 x 264 pixels). Binning or windowing can increase the speed. The camera was placed below the ADF detector to enable simultaneous recording. Figure 1 shows STEM images of a monolayer graphene obtained at 80 kV. Fig. 1a shows an ADF image obtained with the probe current of approximately 0.2 pA and the dwell time for a pixel of 0.5 ms. Because of a combination of low dose and residual uncorrected aberrations, the lattice contrast of graphene is almost buried in the noise originated from 50 Hz commercial frequency. Fig. 1b shows a reconstructed phase image using ptychography. The image contrast is significantly improved compared to the simultaneous ADF image, but the contrast transfer of the image (see the Fourier transform displayed in the inset) is anisotropic, resulting in uncertain positions of carbon atoms. This is because there remains large two‐fold astigmatism and defocus in the image shown in Fig. 1(b), because we could not adjust those by observation of the ADF image due to the weak image signal. Fig. 1c is a phase image in which the aberrations are corrected through post processing using the same 4D dataset by applying correction functions in the spatial frequency domain. The image is no longer anisotropic and the carbon atomic positions can be unambiguously determined. Although the same amount of electron dose is used to form the ADF and the corrected phase images, the result clearly shows the benefit of ptychographic phase reconstruction in improving image signal to noise and being able to correct aberrations through post processing. Figure 2 shows the ptychographic phase maps of the 4D dataset at a certain spatial frequency. With an aberration‐free electron probe, they would be flat phase on the two sidebands, and the phase difference between the bands be π. In Fig. 2a, the map of the original dataset, there is a phase gradient inside each sideband because the aberrations were present in the electron probe. Fig. 2b is the correction function that compensates for the aberration seen in Fig. 2a. Fig. 2c is the corrected phase map and corresponding to the image in Fig. 1c. Here, only defocus and two‐fold astigmatism are corrected but corrections of other higher order aberrations are in principle possible. Figure 3 shows through focus images created in the same way as above. As we know the full information on the electron wave at the condenser aperture plane, we can induce any aberrations such as defocus.

Portal vein imaging is an important method for investigating portal venous disorders. However, the diagnostic requirements are not usually satisfied when using single imaging techniques. Diagnostic accuracy can be improved by combining different imaging techniques. Contrast agents that can be used for combined imaging modalities are needed. In this study, the feasibility of using microbubbles containing gadolinium (MCG) as contrast agents for both phase contrast imaging (PCI) and magnetic resonance imaging (MRI) are investigated. MCG were made by encapsulating sulfur hexafluoride (SF6) gas with gadolinium and lyophilized powder. Absorption contrast imaging (ACI) and PCI of MCG were performed and compared in vitro. MCG were injected into the main portal trunk of living rats. PCI and MRI were performed at 2 min and 10 min after MCG injection, respectively. PCI exploited the differences in the refractive index and visibly showed the MCG, which were not detectable by ACI. PCI could facilitate clear revelation of the MCG-infused portal veins. The diameter of the portal veins could be determined by the largest MCG in the same portal vein. The minimum diameter of clearly detected portal veins was about 300 µm by MRI. These results indicate that MCG could enhance both PCI and MRI for imaging portal veins. The detection sensitivity of PCI and MRI could compensate for each other when using MCG contrast agents for animals.

Cylindrical micelles prepared in aqueous solutions from cationic surfactants octadecyl trimethylammonium (OTA+) or cetyltrimethylammonium (CTA+) and parachlorobenzoate (PCB) counterion were successfully imaged after evaporation of water using tapping mode atomic force microscopy (TM-AFM) onto very smooth gold and glass substrates. With the help of the obtained topography AFM images, it was shown that the micellar structures are preserved on gold substrates after evaporation of the solvent despite the new set of stresses due mainly to capillary forces and dehydration. The influence of the substrate on the resulting micellar morphology observed in air was investigated for these two materials: cylindrical micelles were evidenced as loosely adherent on gold surface in the presence of parachlorobenzoate (PCB) and identical, geometrically speaking, to those known to exist in aqueous solutions. In this situation, topographic AFM images allowed us to determine accurately their geometrical characteristics such as diameter and length in the nanometer range. On the other hand, AFM images obtained in air on glass surfaces revealed micellar structures that are different from those existing in the bulk of the solution. Indeed, bilayer-type micelles with a thickness close to twice the surfactant monomer expected length were observed, indicating that the well-established and strong influence of glass on micelle geometry at the glass/solution interface is maintained after evaporation of water. These results have been analyzed on the basis of positive charge of gold deduced from electrochemical impedance spectroscopy (EIS) and Raman spectroscopy measurements on one hand and of the negative charge of glass on the other hand. Although these results appeal to new theoretical considerations dealing with dynamics of evaporation of micellar solution drops and/or with counterion contributions to macromolecular interactions in aqueous solutions and in air, this new AFM imaging method appears to be the more adequate one to image and measure the micelles formed in the presence of water.