Dark-field Detection Research Articles

Optical STEM detection for scanning electron microscopy

Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have characterized and benchmarked a novel detection technique for scanning electron microscopy: optical scanning transmission electron microscopy (OSTEM). A qualitative and quantitative comparison was performed between OSTEM, secondary and backscattered electron detection and annular dark field detection in scanning transmission electron microscopy. Our analysis shows that OSTEM produces images similar to backscattered electron detection in terms of contrast, resolution and signal-to-noise ratio. OSTEM can complement large scale imaging with (scanning) transmission electron microscopy and has the potential to speed up imaging in single-beam scanning electron microscopes.

Determining the size of the overlapping area for image stitching in dark-field detection

Dark-field microscopic imaging system has high spatial resolution, but small image field. When an optical surface with a large-diameter is tested, the image stitching is necessary to obtain the full-aperture detection results. In this paper, Harris corner detection algorithm is used to achieve the full aperture testing result by stitching multiple detection images. According to the Shannon sampling theorem, how the size of the overlapping area influences the stitching results is analysed in detail. Combined with the spatial scale of surface defects, a method for determining the size of the overlapping area is given. The standard scratch patterns are used to simulate the stitching process. On this basis, an actual stitching processing is carried out on the detection images of surface scratches of four different spatial scales. Both the simulation and experimental results show that it is reasonable to use the scale of the smallest defect to determine the number of sampling points in the stitching area.

Hyperbolic material enhanced scattering nanoscopy for label-free super-resolution imaging

Fluorescence super-resolution microscopy has, over the last two decades, been extensively developed to access deep-subwavelength nanoscales optically. Label-free super-resolution technologies however have only achieved a slight improvement compared to the diffraction limit. In this context, we demonstrate a label-free imaging method, i.e., hyperbolic material enhanced scattering (HMES) nanoscopy, which breaks the diffraction limit by tailoring the light-matter interaction between the specimens and a hyperbolic material substrate. By exciting the highly confined evanescent hyperbolic polariton modes with dark-field detection, HMES nanoscopy successfully shows a high-contrast scattering image with a spatial resolution around 80 nm. Considering the wavelength at 532 nm and detection optics with a 0.6 numerical aperture (NA) objective lens, this value represents a 5.5-fold resolution improvement beyond the diffraction limit. HMES provides capabilities for super-resolution imaging where fluorescence is not available or challenging to apply.

Development of a confocal line-scan laser scattering probe for dark-field surface defects detection of transmissive optics.

Dark-field detection has long been used to identify micron/submicron-sized surface defects benefiting from the broadening effect of the actual defect size caused by light scattering. However, the back-side scattering of a transmissive optical slab is inevitably confused with the front-side scattering phenomenon, resulting in deterioration of the signal-to-noise ratio (SNR) of the scattering signal and false alarms for real defect detection. To this end, a confocal line-scan laser scattering probe equipped with optical sectioning ability is proposed to separate the back-side scattering from the front-side scattering. The optical sectioning ability is realized through a confocal light scattering collector, which overcomes the restriction imposed on the numerical aperture (NA) and the field of view (FOV), reaching an FOV length of 90mm and NA of 0.69. The line-scan principle of the probe protects itself from crosstalk because it produces only a laser spot on the tested surface in an instant. Experimental results verified that the probe has a line-scan length of 90mm with a uniformity better than 98%, an rms electronic noise of 3.4 mV, and an rms background noise of 6.4 mV with laser on. The probe can reject the false back-side scattering light for a 2mm thick fused silica slab at 17.1 dB SNR and operate at a high imaging efficiency of 720mm2/s with a minimum detectability limit of 1.4 µm at 12 dB SNR. This work put forward an effective method with great application value for submicron-sized defect detection in transmissive optics.

Influence of light polarization state on the imaging quality of dark-field imaging system

Annular linear polarized light is used as the illumination source of a reflective dark-field detection system in this paper. According to the theories of the bidirectional reflectance distribution function and multi-beam interference, the influence of the light polarization state on the intensity distribution of the scattered light is analyzed in detail. For surface defects, a simulation model of dark-field imaging is established based on the finite-difference time-domain method. Both the near-field and the far-field scattering intensity distributions caused by surface defects are calculated under different illumination conditions. The incidence angle and polarization state of the illuminated light are optimized. Simulation and experimental results show that the image quality will be minimally affected by the interference effect, while P-polarized light illuminates with an incident angle of 45°. The higher measurement accuracy of the dark-field imaging detection system can be obtained when the optimized illumination scheme is used.

In-situ upgrading of heavy crude oils via solvent deasphalting using of nickel oxide nanoparticles as asphaltene co-precipitants

The use of NiO nanoparticles as asphaltene co-precipitant additives is studied to improve the upgraded oil's properties and potentially reduce the solvent-to-oil ratio for the in-situ upgrading of heavy oils via solvent deasphalting. Asphaltene content, solubility profile, and C7-deasphalting laboratory experiments were carried out to evaluate the efficiency of the nanomaterial. Results showed that nickel oxide nanoparticles increased the amount of asphaltenes in the 15–18% range with respect to the case without additive at the same solvent-to crude ratio. In the presence of the NiO nanoparticles, improvements on the upgraded crude oil properties were found with an average 17.1°API gravity (16% increase) and a viscosity of ∼2370 cSt (∼16% reduction) vs. the case without additive. These results demonstrate the usefulness of using nickel oxide nanoparticles to further enhance the upgraded crude oil properties for heavy oil upgrading via solvent deasphalting. Based on elemental analysis and spectroscopic techniques (scanning transmission electron microscopy with high angle annular dark field detection, energy dispersive X-ray analysis, Mid- and Far-FT-IR, and X-ray photoelectron spectroscopy), it was found that NiO nanoparticles acted as nucleation sites (agglomerants). A carbon-containing layer from the asphaltene fraction encapsulates the nickel oxide nanoparticles. A plausible mechanism for the interaction of the nanostructured NiO with the C7-asphaltenes was proposed that involves the formation of nanosized Ni-O-carboxylate or phenolate species on the surface of the nickel oxide nanoparticles.

Application of light scattering tomography for Si(111) samples.

The detection of bulk micro-defects in Czochralski-grown silicon (Si) 〈100〉 wafers has significant importance in wafer quality control. Light Scattering Tomography (LST) is an industry standard technique for this purpose. This optical non-contact metrology requires destructive sample preparation: Samples have to be cleaved into half. One particular feature of the method is a dark field detection arrangement, which is achieved by separating the light detection part (microscope unit) from the illumination. Illumination is applied to the front surface of the sample, and the light scattered off of the defects is collected via the cleaved surface. The technique requires the perpendicularity of the cleaved surface to the front surface, which is fulfilled for Si(100) wafers. However, the nominally cleaved surface for Si(111) wafers is not perpendicular to the front surface but has an angle of 70.5°. This significant difference in cleavage results in the fact that Si(111) wafers cannot be measured by standard LST systems. Fortunately, the standard LST system can be modified by tilting the detection part under a proper angle allowing the measurements of Si(111) samples. In this article, we present this new technique in detail, showing the design and measurement capability of the new system. The measurement results are validated by a direct comparison to standard LST measurements on the same samples after proper sample preparation.

极紫外光刻掩模缺陷暗场检测系统优化设计

极紫外光刻掩模缺陷暗场检测系统优化设计

Confocal epifluorescence sensor with an arc-shaped aperture for slide-based PCR quantification

The increasing needs of point-of-care diagnostics, quarantine of epidemic pathogens, and prevention of terrorism's bio-attacks have promised the future of portable real-time quantitative polymerase chain reaction (qPCR) sensors. This work aims at developing a highly sensitive and low-cost light emitting diode (LED)-based epifluorescence sensor module for qPCR sensor development and relevant bioassay applications. Inspired by the light stop design and dark-field detection of microscopes, this paper first reports a compact confocal LED epifluorescence sensor using a light stop with an arc-shaped aperture for enhancing the flexibility of quick DNA and PCR detection. The sensor features the advantages of the dichroic mirror-free and confocal (shared-focus) characteristics, which benefits size reduction and minimal optics used. It also allows extension to integrate with in situ real-time PCR thermal cycling since the sample slide is placed apart from the epi-sensing module. The epifluorescence sensor can detect as low as sub-ng/μL standard DNA and 101 copies of Salmonella typhimurium InvA gene sequences (cloned in E. coli and after 30-cycle PCR) with SYBR® Green I from non-purified culture samples, having highly sensitive and specific signal responses comparable with that of a commercial qPCR instrument.

Fluorescence and Scattering Dual-Mode Multiplexed Imaging with Gold–Silver Alloy Core Silica Shell Nanoparticles

Gold–silver alloy core silica shell nanoparticles (Au/Ag@SiO2 NPs) with fully accordable photophysical properties were developed and used as contrast agent for multiplexed cell-imaging applications. Using a seed growth strategy, these nanoparticular labels can be designed to suit specific experimental needs and be clearly identified based on their distinctive combination of scattering and fluorescence colors. In this Article, the multiplexed cell-imaging capabilities of Au/Ag@SiO2 NPs are presented using four different combinations of core composition and fluorescence colors. In a proof of concept experiment, 86% of total nanoparticles were correctly identified by an optical microscope with a system offering fluorescence and darkfield detection capabilities. This multiplatform identification strategy was successfully applied for the detection of four different nanoparticular architectures in cell-imaging experiments.

Dark-field detection method of shallow scratches on the super-smooth optical surface based on the technology of adaptive smoothing and morphological differencing

There exist some shallow scratch defects on the super-smooth optical surface. Their detection has a low efficiency with the existing technologies. So a new detection method, dark-field detection of adaptive smoothing and morphological differencing (DFD-ASMD), is proposed. On one hand, the information of shallow scratches can be kept in dark-field images. On the other hand, their weak characteristics can be separated and protected from being overly reduced during the elimination of noise and background in the image. Experiments show the detection rate of shallow scratches is around 82%, and DFD-ASMD can lay a foundation for quality control of defects on the high-quality optical surface.

Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy.

Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

Optical Detection of Dust and Scratches on Photographic Film

Today’s information society needs efficient and economic solutions for the digital restoration of the photographic heritage. Different methods have been adopted up to now for the automatic detection of dust and scratches; each method has pros and cons, and a limited field of effectiveness. The use of infrared radiation and the spatiotemporal image analysis are among the most effective methods, although they have their limits. The infrared radiation only works for dye-based material, while the spatiotemporal image analysis is not applicable for still images and is limited due to motion in the scene. The present work defines in detail a set of methods for optical dust and scratches detection applicable on any type of transparent photographic material (silver-based as well as dye-based material, still images as well as moving images). The term “optical” refers to the fact that the considered methods seek physical evidence of the presence of foreign bodies or irregularities on the film; this allows avoiding the typical digital artifacts produced by “nonoptical” methods, for which certain elements of the scenes are erroneously obliterated because they resemble dust grains or scratches. “PDD” (Polarized Dark-field Detection) detects the flaws using an image acquired in a polarized dark-field setup; “DCD” (Dual Collimation Detection) takes advantage of the Callier effect to locate the flaws; “ n -MDD” (Multiple Direction Detection) entails the acquisition of n images in dark-field setups with different directions of illumination, and the extraction of the differences between the images through multivariate analysis. A numerical evaluation of the performances of the MDD method with an eightfold acquisition (8-MDD) is carried out by comparing its flaw detection with the flaw detection provided by commercial software based on spatiotemporal image analysis.

Channelling contrast analysis of lattice images: Conditions for probe-insensitive STEM

Quantitative analysis of lattice resolved images generated by scanning transmission electron microscopy (STEM) requires specification of probe characteristics, such as defocus, aberration and source distribution. In this paper we show that knowledge of such characteristics is unnecessary for quantitative interpretation, if the signal is integrated over a unit cell. Such a condition, whether the result of experimental setup or post-processing of lattice resolved images, reduces the intensity distribution to that of channelling contrast, where the signal for plane wave incidence is averaged over the angular range of the probe, and the result is independent of the probe characteristics. We use a Bloch wave model to show analytically how this applies to all forms of STEM imaging, such as that formed by annular dark field or backscatter detection, as well as characteristic X-ray fluorescence or electron energy loss. As a specific example, we consider how the signal from an annular dark field detector can be used to determine specimen thickness via a transfer curve for the zone axis and scattering geometries employed. This method has advantages over matching lattice images with calculations since these are sensitive to probe coherence and aberration, and saturation of the on-column intensity is approached more rapidly.

Abstract 3670: Spectroscopy imaging platform differentiates pathology diagnoses in breast surgical specimens

Abstract Optical spectroscopy probes increasingly have been employed for diagnostic sensing during breast conserving surgery to aid surgeons in complete resection while minimizing damage to healthy tissues; however, traditional fiber probe-based systems rely on the assumption that ultra-structural changes associated with malignancy yield disease-specific contrast in a single, volume-averaged measure. A scanning-beam spectroscopy platform was designed to efficiently realize the imaging extension of probe-based spectroscopy methods and to selectively sample the scattering response of breast surgical specimens. The imaging system employs dark-field illumination and confocal detection to rapidly sample broadband spectra at 100μm lateral resolution over a 1cm2 field of view. Optical scattering is exquisitely sensitive to the morphological features observed in pathology, the diagnostic gold standard, and has not been studied sufficiently in thick tissues in a waveband that avoids absorption. In this study, 29 fresh breast tissue specimens procured during conservative surgery were imaged and returned to pathology for standard histological processing. A protocol was developed for accurate co-registration between the imaged field and histology. Over 300,00 broadband spectra were sampled and parameterized according to an empirical approximation to Mie theory. Further, the gray-level co-occurrence matrix representation of texture features was used to mathematically represent intensity-level spatial dependence in the scattering images. Spatially, the intra and inter-patient scattering response is quite heterogeneous; but imaging accounts for this natural variance so that diagnostic classification improved. The average scattering power per 100x100 pixel field of view was sufficient to discriminate between benign and malignant pathologies with a positive and negative predictive value of 1.00 and 0.90 respectively, using a simple, threshold-based classification. As compared to classification on a per-spectrum basis, which yielded positive and negative predictive values of just 0.71 and 0.75 respectively. Further, textural features yielded discriminated between invasive and in situ carcinomas with p<0.05, suggesting potential to discriminate between these surgically undifferentiable pathologies. The scanning spectroscopy platform was designed to strike a balance between sensitivity to tissue ultra-structure and imaging the macroscopic fields encountered during surgery. It allows for high throughput imaging of light scattering from the surface of breast surgical specimens, and the sheer number of spectra collected dramatically improves diagnostic classification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3670. doi:1538-7445.AM2012-3670

Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) -based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207) blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira) culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21%) out of 100 culture positive blood samples, three (2.8%) out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001). A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001) signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

빔의 입사모드가 금 나노입자의 국소표면플라즈몬 산란광에 미치는 영향

Quantitative analysis of optical scattering intensities from a Au nanoparticle with a diameter of 100 nm, which is effected by the localized surface plasmon resonance (LSPR), were numerically carried out by using a dark-field detection scheme on prism basal plane for two different beam incident modes of reflectance (R-mode) and transmittance (T-mode). Two-dimensional finite difference time domain (FDTD) algorithm was adopted, and its applicabilibility was verified by comparing the simulation results with the theoretical ones. Simulation results of the scattered light intensities from a Au nanoparticle revealed that the scattered intensity of the T-mode was much stronger than that of R-mode. Comparison of the calculated results with the theoretical intensity distribution on the prism showed that the scattered intensity is marimized when the evanescent field, which is generated from the interface of prism and air at TIR angle, is coupled with Au nanoparticle.

Varying Collimation for Dark-Field Extraction

Although x-ray imaging is widely used in biomedical applications, biological soft tissues have small density changes, leading to low contrast resolution for attenuation-based x-ray imaging. Over the past years, x-ray small-angle scattering was studied as a new contrast mechanism to enhance subtle structural variation within the soft tissue. In this paper, we present a detection method to extract this type of x-ray scattering data, which are also referred to as dark-field signals. The key idea is to acquire an x-ray projection multiple times with varying collimation before an x-ray detector array. The projection data acquired with a collimator of a sufficiently high collimation aspect ratio contain mainly the primary beam with little scattering, while the data acquired with an appropriately reduced collimation aspect ratio include both the primary beam and small-angle scattering signals. Then, analysis of these corresponding datasets will produce desirable dark-field signals; for example, via digitally subtraction. In the numerical experiments, the feasibility of our dark-field detection technology is demonstrated in Monte Carlo simulation. The results show that the acquired dark field signals can clearly reveal the structural information of tissues in terms of Rayleigh scattering characteristics.

Highly sensitive detection technique of buried defects in extreme ultraviolet masks using at-wavelength scanning dark-field microscopy

A technique to probe defects buried inside extreme ultraviolet (EUV) masks has been implemented using a dark-field microscopy detection setup. Specific samples have been fabricated to evaluate the sensitivity of this technique. They consist of silicon oxide gratings of a few nanometers height, coated with 40 layer pairs of molybdenum–silicon. We observed images with a good contrast on samples with defects as low as 3nm. However, the imaging mechanism of scanning dark-field microscopy is not linear and can produce image distortions. Conditions of correct imaging have been analyzed, and simulations have been performed that show good agreement with the experimental data. This work opens the way for a better understanding of the capability of at-wavelength inspection technique for EUV mask.

Study of a Σ13 SrTiO3 grain boundary : Comparison between HREM, Exit Wave Reconstruction and Z-contrast analysis

Author(s): Ayache, J.; Kisielowski, C.; Kilaas, R.; Passerieux, G.; Lartigue, S. | Abstract: We an investigation of a *13 grain boundary in a SrTi03 with a misorientation of 67?4 by HREM, Exit Wave Reconstruction and Z-contrast microscopy. The complementary character of phase contrast imaging and High Angular Dark Field detection is pointed out.