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.

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.

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.

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.

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.

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.

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.

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.

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.

CRISPR/Cas9 system and its applications in human hematopoietic cells.

Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, ß-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation.

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.

Design of a high-quality optical conjugate structure in optical tweezers.

We propose an approach to realize a high-quality optical conjugate of a piezo-driven mirror (PM) in optical tweezers. Misalignments between the optical beam and the steering center of the PM are analyzed mathematically. The decentrations in different directions cause different changes, either a position change of the conjugate plane or a spot variation of the beam during PM steering. On the other hand, these misalignment-introduced problems provide the information to check the assembling errors. Thus a wanted conjugate plane of the PM can be effectively and precisely achieved according to the detection signals. This approach is also available to deal with multifactor coupling error. At the end, the procedure for error analysis is given by testing homebuilt optical tweezers.

Geometric Parameters Estimation and Calibration in Cone-Beam Micro-CT.

The quality of Computed Tomography (CT) images crucially depends on the precise knowledge of the scanner geometry. Therefore, it is necessary to estimate and calibrate the misalignments before image acquisition. In this paper, a Two-Piece-Ball (TPB) phantom is used to estimate a set of parameters that describe the geometry of a cone-beam CT system. Only multiple projections of the TPB phantom at one position are required, which can avoid the rotation errors when acquiring multi-angle projections. Also, a corresponding algorithm is derived. The performance of the method is evaluated through simulation and experimental data. The results demonstrated that the proposed method is valid and easy to implement. Furthermore, the experimental results from the Micro-CT system demonstrate the ability to reduce artifacts and improve image quality through geometric parameter calibration.

A Unique Self-Sensing, Self-Actuating AFM Probe at Higher Eigenmodes.

With its unique structure, the Akiyama probe is a type of tuning fork atomic force microscope probe. The long, soft cantilever makes it possible to measure soft samples in tapping mode. In this article, some characteristics of the probe at its second eigenmode are revealed by use of finite element analysis (FEA) and experiments in a standard atmosphere. Although the signal-to-noise ratio in this environment is not good enough, the 2 nm resolution and 0.09 Hz/nm sensitivity prove that the Akiyama probe can be used at its second eigenmode under FM non-contact mode or low amplitude FM tapping mode, which means that it is easy to change the measuring method from normal tapping to small amplitude tapping or non-contact mode with the same probe and equipment.

[Differential Reflectance Spectroscopy for In-Situ Monitoring of Organic Thin Films Growth in Vacuum Environment].

For realizing the real-time monitoring of organic thin film preparation process in vacuum environment, the present paper proposes a high precision measurement approach based on differential reflectance spectroscopy (DRS). An optical system was constructed with off the shelf optical components, such as off-axis parabolic mirror, optical flat and optical fiber. A differential algorithm was employed to analyze the spectral signals. Based on the homebuilt setup, instability induced by variation of temperature was investigated. It was concluded that with the good control of temperature and air flow, the measurement repeatability of this system is better than 2 per thousand for a long-term period. Furthermore, an initial stage of organic thin film growth of pentacene molecules on the surface of Au was studied. As compared with the data of film thickness gauge and atomic force microscope, DR spectra accurately recorded the fine optical evolution with sub-monolayer resolution, which is related to the growth of the thin film. As a result, the DR optical system exhibits characteristics of broad spectrum (range from 300 to 820 nm), high stability (repeatability better than 2X 10(-3)), and high precision (sub-monolayer resolution) after efforts were done to decrease the influences on the spectral quality produced by misalignments of the optical components, the defects of the optics, and the disturbances of the environmental conditions. It is indicated that the proposed DR method is suitable for real-time online monitoring of thin film growth with high precision.

Direct Observation of the Reversible Two-State Unfolding and Refolding of an α/ß Protein by Single-Molecule Atomic Force Microscopy.

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/ß protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/ß proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/ß and all-ß elastomeric proteins.

SPR phase detection for measuring the thickness of thin metal films.

In this paper, a novel method to determine the thickness of thin metal film is put forward which uses Surface Plasmon Resonance (SPR) phase detection method. The relations between the metal film thickness and the phases of the transverse magnetic (TM) and transverse electric (TE) polarization of the reflected light are shown in the simulation results. By recording the interference patterns which contain the information of the phase differences in the experiments, the values of thickness are calculated. Both of the theoretical analysis and experimental results indicate that the approach presented is feasible and reliable. Thus, it is possible to use the method of phase detection to determine the thickness of thin metal films within 100nm in SPR prism couplers directly with nanometer resolution.

Molecular calipers for highly precise and accurate measurements of single-protein mechanics.

Single-molecule atomic force spectroscopy (AFM) has evolved into a powerful technique toward elucidating conformational changes in proteins when exposed to applied force. AFM technologies that are currently available allow for precise measurements of proteins length changes during conformational transitions. However, because of systematic errors in piezo calibration as well as errors originating from fitting experimental data using a worm-like chain model of polymer elasticity, high-precision measurements of length changes do not necessarily translate into highly accurate measurements of length changes, resulting in uncertainty in obtaining structural information about protein conformational changes. Actually achieving highly precise and accurate force spectroscopy measurements remains a challenge. Here, we report a protein caliper method that eliminates systematic errors that occur during single-protein force spectroscopy measurements, and thus achieves highly precise and accurate length change measurements in protein mechanics studies. To do this, a series of loop elongation variants of the small protein GB1, which differ by 2, 5, 10, 15, and 24 amino acid residues, were engineered. Differential measurements of amino acid residue length obtained from different AFM setups result in a precise measure of the length of a single amino acid residue, which varies within different AFM setups because of systematic error between individual AFM piezoelectric calibrations. The measured length of a single amino acid residue from a given AFM setup is then used as a caliper for the given setup to eliminate systematic error, leading to highly accurate and precise measurements of the number of amino acid residues that are involved in a conformation change of a polypeptide chain. We further developed a more precise, robust, and model-free method to determine the apparent size of single amino acid residues and conformational changes of proteins. This method improves the accuracy of single protein force spectroscopy measurements, providing an accurate means of measuring force-induced protein conformational changes.