Measuring the multilayer silicon based microstructure using differential reflectance spectroscopy.

The yield of a large-area ultra-thin display panel depends on the realization of designed thickness of multilayer films of all pixels. Measuring the thicknesses of multilayer films of a single pixel is crucial to the accurate manufacture. However, the thinnest layer is reaching the sub-20nm level, and different layers feature remarkable divergence in thickness with similar optical constants. This turns to a key obstruction to the thickness characterization by optical spectroscopy. Based on the tiny differences in absorptivity, a fast method for measuring the film thickness in a single pixel is proposed which combines the layer number reducing model and micro-area differential reflectance spectroscopy. The lower layers can be considered as semi-infinite in the corresponding spectral range whose thickness is infinite in the fitting algorithm. Hence, the thickness of the upper layer is fitted in a simplified layer structure. For demonstration, a multilayer silicon microstructure in a single pixel, p-Si/a-Si/n-Si (10nm/950nm/50nm) on complex substrate, is measured. The light spot diameter is about 60 microns with measuring-time in 2 seconds. The measurement deviation is 3% compared by a commercial ellipsometer. To conclude, the proposed method realizes the layer number reduction for fitting multilayer thickness with large thickness difference and similar optical constants, which provides a powerful approach for multilayer microstructure characterizations.

The formation principle of micro-droplets induced by using optical tweezers.

Utilizing droplets as micro-tools has become a valuable method in biology and chemistry. In previous work, we have demonstrated a novel droplet generation-manipulation method in a conventional optical tweezer system. Here, a further study of the droplet composition and its formation principle is performed. First, it is proved through Raman spectra that the principal component of the droplets is HPO4 2- solution. Considering that the generated droplet size is at the µm level, we have adopted a variety of methods in experiments to reduce external interference. Second, using a confocal microscopic video camera, the growth process of the droplet is completely recorded in a common glass-based chamber. The finite element simulations help us to further understand that the droplet generation process using optical tweezers can be divided into two stages: "capture" caused by optical force field and "aggregation" induced by a photothermal phenomenon and thermal acceleration. Through these studies, the nature of the optical tweezer-generated droplets is revealed. As a general principle for the droplet generation, this method will provide inspiration and prospects in the fields of microfluidics and biophysics-chemistry.

Imaging layer thickness of large-area graphene using reference-aided optical differential reflection technique.

Transparent layers are critical for enhancing optical contrast of graphene on a substrate. However, once the substrate is fully covered by large-area graphene, there are no accurate transparent layer and reference for optical contrast calculations. The thickness uncertainty of the transparent layer reduces the analytical accuracy of graphene. Thus, in this Letter, we propose a reference-aided differential reflection (DR) method with a dual-light path. The accurate thickness of the transparent layer is obtained by improving the DR spectrum sensitivity using a designable reference. Hence, the analytical accuracy of graphene thickness is guaranteed. To demonstrate this concept, a centimeter-scale chemical-vapor-deposition-synthesized graphene was measured on a SiO2/Si substrate. The thickness of underlying SiO2 was first identified with the 1 nm resolution by the DR spectrum. Then, the thickness distribution of graphene was directly deduced from a DR map with submonolayer resolution at a preferred wavelength. The results were also confirmed by ellipsometry and atomic force microscopy. As a result, this new method provides an extra degree of freedom for the DR method to accurately measure the thickness of large-area two-dimensional materials.

Initial stage of MBE growth of MoSe2 monolayer.

An atomically thin MoSe2 layer has been synthesized on mica using molecular beam epitaxy (MBE). The polymorphous of the MoSe2 layer depends on the coverage and the growth temperature. At low coverages and low growth temperature, 1T-MoSe2 forms in addition to a comparable quantity of 2H-MoSe2. The metastable 1T-MoSe2 transfers gradually to the stable 2H-MoSe2 before the completion of the first monolayer. The current result sheds some light on the complexity of the nucleation and growth of transition metal dichalcogenide (TMDC) monolayers and implies a possible route for a phase selective synthesis using MBE.

Growth oscillation of MoSe2 monolayers observed by differential reflectance spectroscopy.

The precisely controlled growth of transition metal dichalcogenide (TMDC) monolayers requires sensitive and nondestructive techniques to monitor the morphology and coverage in situ and in real time. In the current work, differential reflectance spectroscopy (DRS) was applied to monitor the molecular beam epitaxy (MBE) growth of atomically thin MoSe2 layers on mica. The optical evolution exhibits an oscillation with monolayer periodicity, revealing a two-dimensional (2D) layer-by-layer growth of the MoSe2 thin films. The observed sensitivity of DRS to the step density is associated to the modified electronic structures at the edges of TMDC monolayers. As DRS works in any transparent ambient, we speculate that it could be of great use for realizing precisely controlled growth of TMDC monolayers using not only MBE but also chemical vapor deposition (CVD).

Mechanical Unfolding and Folding of a Complex Slipknot Protein Probed by Using Optical Tweezers.

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

High-speed atomic force microscope with a combined tip-sample scanning architecture.

A high-speed atomic force microscope (HS-AFM) based on a tip-sample combined scanning architecture is presented. In this system, the X-scanner, which is separated from the AFM head, carries the sample and scans along the fast-axis. The Y and Z scanners integrated in the AFM head oscillate an ultrashort cantilever probe and scan in the other two dimensions. The optical beam deflection method is improved to enable the laser to track the probe over a wide scan range. A novel probe holder realizes easy exchange and alignment of the probe. Due to the separation of the X and Y scanners, both appear with better dynamic performance and carrying capacity. Experiments show that the HS-AFM established in this work can achieve a line rate of up to 100 Hz with the basic proportional-integral-derivative control algorithm and linear driving. The permissible sample size and mass can be as large as several centimeters and above 40 g.

Direct observation of the fast and robust folding of a slipknotted protein by optical tweezers.

Understanding the folding mechanism of knotted and slipknotted proteins has attracted considerable interest. Due to their topological complexity, knotted and slipknotted proteins are predicted to fold slowly and involve large topological barriers. Molecular dynamics simulation studies suggest that a slipknotted conformation can serve as an important intermediate to help greatly reduce the topological difficulty during the folding of some knotted proteins. Here we use a single molecule optical tweezers technique to directly probe the folding of a small slipknotted protein AFV3-109. We found that stretching AFV3-109 can lead to the untying of the slipknot and complete unfolding of AFV3-109. Upon relaxation, AFV3-109 can readily refold back to its native slipknot conformation with high fidelity when the stretching force is lower than 6 pN. The refolding of AFV3-109 occurs in a sharp two-state like transition. Our results indicate that, different from knotted proteins, the folding of a slipknotted protein like AFV3-109 can be fast, and may not necessarily involve a high topological barrier.

Black Phosphorus Nano-Polarizer with High Extinction Ratio in Visible and Near-Infrared Regime.

We study computationally the design of a high extinction ratio nano polarizer based on black phosphorus (BP). A scattering-matrix calculation method is applied to compute the overall polarization extinction ratio along two orthogonal directions. The results reveal that, with a resonance cavity of SiO2, both BP/ SiO 2 /Si and h-BN/BP/ SiO 2 /Si configurations can build a linear polarizer with extinction ratio higher than 16 dB at a polarized wavelength in the range of 400 nm⁻900 nm. The polarization wavelength is tunable by adjusting the thickness of the BP layer while the thicknesses of the isotrocpic layers are in charge of extinction ratios. The additional top layer of h-BN was used to prevent BP degradation from oxidation and strengthens the practical applications of BP polarizer. The study shows that the BP/ SiO 2 /Si structure, with a silicon compatible and easy-to-realize method, is a valuable solution when designing polarization functional module in integrated photonics and optical communications circuits.

Involvement of pRb-E2F pathway in green tea extract-induced growth inhibition of human myeloid leukemia cells.

Both inhibitory and stimulatory effect of EGCG on cancer cells have been reported, which often is linked to receptor tyrosine kinase signaling. In this study, we present evidence that green tea extract and its chemical component, Epigallocatechin-3-gallate (EGCG), inhibit growth of human myeloid leukemia cells through the regulation of pRb synthesis and formation of pRb-E2F complexes. Addition of green tea extract to the culture of TF-1a and MV4-11 myeloid leukemia cells significantly inhibited their proliferation with a substantial portion of cell death being observed. The green tea extract and EGCG had no significant effect on the expression of G1 CDKs and the CDK inhibitors but downregulated the formation of pRb-CDKs. Surprisingly, the expression of pRb was markedly upregulated while the phosphorylation of pRb downregulated. The upregulation of pRb was blocked by pre-treatment with cycloheximide, a protein synthesis inhibitor, suggesting a requirement of protein synthesis. In agreement with these results, pRb-E2F complexes were upregulated and E2F DNA binding activity decreased. Since both TF-1a and MV4-11 are factor-independent cell lines, the upregulation of pRb-E2F complexes and inhibition of DNA binding activity by green tea extract is most likely through a receptor tyrosine kinase-independent pathway. We also found that the stem/progenitor cells derived from these two leukemia cell lines are more sensitive to the inhibitory effect of green tea extract. Our result suggests that concentrated green tea extract and EGCG may have potential for clinical investigation as an inducer of cancer cell death.

Three-Dimensional Atomic Force Microscopy for Sidewall Imaging Using Torsional Resonance Mode.

This article presents an atomic force microscopy (AFM) technique for true three-dimensional (3D) characterization. The cantilever probe with flared tip was used in a home-made 3D-AFM system. The cantilever was driven by two shaking piezoceramics and oscillated around its vertical or torsional resonance frequency. The vertical resonance mode was used for upper surface imaging, and the torsional resonance mode was used for sidewall detecting. The 3D-AFM was applied to measure standard gratings with the height of 100 nm and 200 nm. The experiment results showed that the presented 3D-AFM technique was able to detect the small defect features on the steep sidewall and to reconstruct the 3D topography of the measured structure.

Residual vibration control based on a global search method in a high-speed white light scanning interferometer.

To achieve high-speed measurements using white light scanning interferometers, the scanning devices used need to have high feedback gain in closed-loop operations. However, flexure hinges induce a residual vibration that can cause a misidentification of the fringe order. The reduction of this residual vibration is crucial because the highly nonlinear distortions in interferograms lead to clearly incorrect measured profiles. Input shaping can be used to control the amplitude of the residual vibration. The conventional method uses continuous wavelet transform (CWT) to estimate parameters of the scanning device. Our proposed method extracts equivalent modal parameters using a global search algorithm. Due to its simplicity, ease of implementation, and response speed, this global search method outperforms CWT. The delay time is shortened by searching, because fewer modes are needed for the shaper. The effectiveness of the method has been confirmed by the agreement between simulated shaped responses and experimental displacement information from the capacitive sensor inside the scanning device, and the intensity profiles of the interferometer have been greatly improved. An experiment measuring the surface of a silicon wafer is also presented. The method is shown to be effective at improving the intensity profiles and recovering accurate surface topography. Finally, frequency localizations are found to be almost stable with different proportional gains, but their energy distributions change.

Film thickness measurement based on nonlinear phase analysis using a Linnik microscopic white-light spectral interferometer.

Based on white-light spectral interferometry and the Linnik microscopic interference configuration, the nonlinear phase components of the spectral interferometric signal were analyzed for film thickness measurement. The spectral interferometric signal was obtained using a Linnik microscopic white-light spectral interferometer, which includes the nonlinear phase components associated with the effective thickness, the nonlinear phase error caused by the double-objective lens, and the nonlinear phase of the thin film itself. To determine the influence of the effective thickness, a wavelength-correction method was proposed that converts the effective thickness into a constant value; the nonlinear phase caused by the effective thickness can then be determined and subtracted from the total nonlinear phase. A method for the extraction of the nonlinear phase error caused by the double-objective lens was also proposed. Accurate thickness measurement of a thin film can be achieved by fitting the nonlinear phase of the thin film after removal of the nonlinear phase caused by the effective thickness and by the nonlinear phase error caused by the double-objective lens. The experimental results demonstrated that both the wavelength-correction method and the extraction method for the nonlinear phase error caused by the double-objective lens improve the accuracy of film thickness measurements.

Resolving the optical anisotropy of low-symmetry 2D materials.

Optical anisotropy is one of the most fundamental physical characteristics of emerging low-symmetry two-dimensional (2D) materials. It provides abundant structural information and is crucial for creating diverse nanoscale devices. Here, we have proposed an azimuth-resolved microscopic approach to directly resolve the normalized optical difference along two orthogonal directions at normal incidence. The differential principle ensures that the approach is only sensitive to anisotropic samples and immune to isotropic materials. We studied the optical anisotropy of bare and encapsulated black phosphorus (BP) and unveiled the interference effect on optical anisotropy, which is critical for practical applications in optical and optoelectronic devices. A multi-phase model based on the scattering matrix method was developed to account for the interference effect and then the crystallographic directions were unambiguously determined. Our result also suggests that the optical anisotropy is a probe to measure the thickness with monolayer resolution. Furthermore, the optical anisotropy of rhenium disulfide (ReS2), another class of anisotropic 2D materials, with a 1T distorted crystal structure, was investigated, which demonstrates that our approach is suitable for other anisotropic 2D materials. This technique is ideal for optical anisotropy characterization and will inspire future efforts in BP and related anisotropic 2D nanomaterials for engineering new conceptual nanodevices.

Wavelength tunable polarizer based on layered black phosphorus on Si/SiO2 substrate.

We report on both the theoretical and experimental design of a black phosphorus (BP)-based reflective linear polarizer on Si/SiO2 substrate in visible range using the Fabry-Perot cavities method. Thanks to the optical anisotropy of BP, polarization wavelength regulation and a high extinction ratio are achievable via optimizing the thickness of BP. Using azimuth-dependent reflectance difference microscopy, we directly measured a huge optical anisotropy of 1.58, corresponding to an extinction ratio of ∼9 dB, from a 96 nm BP on a silicon substrate capped by 260 nm thermally oxidized silicon at a wavelength of 690 nm for the first time, to the best of our knowledge. Our results not only provide a new route to designing nanoscale polarizers based on anisotropic two-dimensional (2D) materials, promoting the application of 2D materials in integrated optoelectronics and system-on-chip, but also suggest a modulation technique for optical anisotropy by integrating the BP film with cavity structures.

Real-time identification of the singleness of a trapped bead in optical tweezers.

Beads trapped in optical tweezers are aligned along the optical propagation direction, which makes it difficult to determine the number of beads with bright-field microscopy. This problem also dramatically influences the measurement of the optical trapping based single-molecule force spectroscopy. Here, we propose a video processing approach to count the number of trapped micro-objects in real time. The approach uses a normalized cross-correlation algorithm and image enhancement techniques to amplify a slight change of the image induced by the entry of an exotic object. As tested, this method introduces a ∼10% change per bead to the image similarity, and up to four beads, one-by-one falling into the trap, are identified. Moreover, the feasibility of the above analysis in a moving trap is investigated. A movement of the trap leads to a fluctuation of less than 2% for the similarity signal and can be ignored in most cases. The experimental results prove that image similarity measurement is a sensitive way to monitor the interruption, which is very useful, especially during experiments. In addition, the approach is easy to apply to an existing optical tweezers system.

Real-time monitoring of 2D semiconductor film growth with optical spectroscopy.

Real-time monitoring of the growth is essential for synthesizing high quality two dimensional (2D) transition-metal dichalcogenides with precisely controlled thickness. Here, we report the first real time in situ optical spectroscopic study on the molecular beam epitaxy of atomically thin molybdenum diselenide (MoSe2) films on sapphire substrates using differential reflectance spectroscopy. The characteristic optical spectrum of MoSe2 monolayer is clearly distinct from that of bilayer allowing a precise control of the film thickness during the growth. Furthermore, the evolution of the characteristic differential reflectance spectrum of the MoSe2 thin film as a function of the thickness sheds light on the details of the growth process. Our result demonstrates the importance and the great potential of the real time in situ optical spectroscopy for the realization of controlled growth of 2D semiconductor materials.

Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy.

Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding-folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron-sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding-refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding-unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro.

Single-Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2.

Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.

Normal-incidence reflectance difference spectroscopy based on a liquid crystal variable retarder.

We propose liquid crystal variable retarder-based reflectance difference spectroscopy for normal-incidence measurements. Principles, instrumentation, data collection and reduction, and calibration procedures are provided. The signal noise is better than 10-3, and the spectral range is from 1.6 to 2.4 eV with 346 photon energy channels. As a demonstration, reflectance difference signals of a multilayer pentacene film on poly (ethylene terephthalate) (PET) film are presented with different polarization azimuths. The characteristic peaks at 1.8 and 1.97 eV, corresponding to the Davydov splitting of pentacene crystal, are observed, which indicate well-ordered in-plane anisotropic structure of pentacene crystal film on PET. Thanks to normal incidence, this design is immune to adjusting the optical structure for the measurements with different working distances, and the objective lens is easily integrated to realize microarea measurements.