Nanometer Sensitivity Research Articles

Speckle-based hyperspectral imaging combining multiple scattering and compressive sensing in nanowire mats.

Encoding of spectral information onto monochrome imaging cameras is of interest for wavelength multiplexing and hyperspectral imaging applications. Here, the complex spatiospectral response of a disordered material is used to demonstrate retrieval of a number of discrete wavelengths over a wide spectral range. Strong, diffuse light scattering in a semiconductor nanowire mat is used to achieve a highly compact spectrometer of micrometer thickness, transforming different wavelengths into distinct speckle patterns with nanometer sensitivity. Spatial multiplexing is achieved through the use of a microlens array, allowing simultaneous imaging of many speckles, ultimately limited by the size of the diffuse spot area. The performance of different information retrieval algorithms is compared. A compressive sensing algorithm exhibits efficient reconstruction capability in noisy environments and with only a few measurements.

Shedding Light on Cas9 Target Search

The CRISPR/Cas adaptive immune system provides prokaryotes with a defensive mechanism against invading viruses and plasmids. The invading viral DNA fragments are incorporated into the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus in the bacterial genome and are later used to recognize and destroy the invader when it returns. In the last stage of CRISPR immunity, called the interference stage, Cas (CRISPR-associated) proteins assemble with short guide RNA molecules which are transcribed from the CRISPR locus. These guide RNA molecules can be programmed to recognize any DNA sequence. In recent years the CRISPR/Cas adaptive immune system has seen an immense growth in interest with the type II CRISPR-Cas9 system being in the center of attention. In this system, the DNA of the invading virus is recognized and cleaved by a single protein Cas9 which is guided by an RNA duplex. Due to its simplicity, CRISPR-Cas9 system is a promising tool in gene engineering as its guide RNA can be programmed to recognize virtually any sequence in the genome. The CRISPR-Cas9 has been demonstrated to work in a variety of organisms, however, despite the large interest in this system, the precise mechanism by which Cas9 finds and cleaves its target remains ambiguous. We utilize biophysical single-molecule techniques, namely total internal reflection fluorescence microscopy (TIRFM) together with Forster resonance energy transfer (FRET) to investigate the mechanics of Cas9 target search with nanometer sensitivity. We are probing one-dimensional diffusion of the protein along the DNA strand and investigating the effects different DNA sequences have on the mechanics of Cas9 target search.

Integrated magnetic tweezers and single-molecule FRET for investigating the mechanical properties of nucleic acid

Many enzymes promote structural changes in their nucleic acid substrates via application of piconewton forces over nanometer length scales. Magnetic tweezers (MT) is a single molecule force spectroscopy method widely used for studying the energetics of such mechanical processes. MT permits stable application of a wide range of forces and torques over long time scales with nanometer spatial resolution. However, in any force spectroscopy experiment, the ability to monitor structural changes in nucleic acids with nanometer sensitivity requires the system of interest to be held under high degrees of tension to improve signal to noise. This limitation prohibits measurement of structural changes within nucleic acids under physiologically relevant conditions of low stretching forces. To overcome this challenge, researchers have integrated a spatially sensitive fluorescence spectroscopy method, single molecule-FRET, with MT to allow simultaneous observation and manipulation of nanoscale structural transitions over a wide range of forces. Here, we describe a method for using this hybrid instrument to analyze the mechanical properties of nucleic acids. We expect that this method for analysis of nucleic acid structure will be easily adapted for experiments aiming to interrogate the mechanical responses of other biological macromolecules.

(Invited) Development of in situ Electrochemical Small-Angle Neutron Scattering (eSANS) for Simultaneous Structure and Redox Characterization of Nanoparticles

The demand for engineered nanomaterials in expanding research sectors, such as energy and healthcare, require materials with widely disparate physicochemical properties. The oxidation and reduction (redox) properties of nanomaterials are crucial for catalysis, matching semiconductor band gaps for photovoltaics, as well as toxicology, since they may cause oxidative stress by generating reactive-oxygen species. Optical spectroscopy analysis of molecular and nanomaterials undergoing redox reactions quantifies the electron transfer and energetics of the reaction via potential control and are the most common analytical techniques employed for this purpose. However, with conjugation to organic ligands and smaller, well-defined particle sizes, engineered nanomaterial redox characterization would benefit by techniques that provide nanometer sensitivity to nanoparticle core and ligand structure. We developed an in situ electrochemical small-angle neutron scattering (eSANS) method to measure simultaneously the redox properties and size, shape, and interactions of solution-dispersed nanomaterials [1]. By combining multi-step potentials and chronocoulometry readout with SANS, the structure and redox properties of engineered nanomaterials are followed, simultaneously in one experiment. Specifically, ZnO nanoparticles were examined as dilute dispersions in pH buffered deuterium oxide solutions under negative electrode potentials. The ZnO disk-shaped nanoparticles undergo an irreversible size transformation upon reduction at the vitreous carbon electrode. The decrease in average nanoparticle size near a current maximum shows the reduction reaction from ZnO to Zn occurs. The eSANS method provides nanometer scale sensitivity to the nanoparticle size and shape changes due to an electrochemical reaction that is crucial to understand in energy, healthcare, and other applications. These experiments utilized the EQ-SANS beamline at the Spallation Neutron Source, Oak Ridge National Laboratory that uniquely provides the ability to follow the redox kinetics under quasi-stationary potential step-scans. The methodology, contrast variation, and scattering properties of the micro and nanoporous carbon electrodes will also be described. [1] V.M. Prabhu and V. Reipa, J. Phys. Chem. Lett. 3, 646 (2012)

Phase calibration target for quantitative phase imaging with ptychography.

Quantitative phase imaging (QPI) utilizes refractive index and thickness variations that lead to optical phase shifts. This gives contrast to images of transparent objects. In quantitative biology, phase images are used to accurately segment cells and calculate properties such as dry mass, volume and proliferation rate. The fidelity of the measured phase shifts is of critical importance in this field. However to date, there has been no standardized method for characterizing the performance of phase imaging systems. Consequently, there is an increasing need for protocols to test the performance of phase imaging systems using well-defined phase calibration and resolution targets. In this work, we present a candidate for a standardized phase resolution target, and measurement protocol for the determination of the transfer of spatial frequencies, and sensitivity of a phase imaging system. The target has been carefully designed to contain well-defined depth variations over a broadband range of spatial frequencies. In order to demonstrate the utility of the target, we measure quantitative phase images on a ptychographic microscope, and compare the measured optical phase shifts with Atomic Force Microscopy (AFM) topography maps and surface profile measurements from coherence scanning interferometry. The results show that ptychography has fully quantitative nanometer sensitivity in optical path differences over a broadband range of spatial frequencies for feature sizes ranging from micrometers to hundreds of micrometers.

A study of ultra-precision ball rotating displacement measurement using laser focus deviation probes

An ultra-precision ball rotating displacement measurement setup based on laser focus deviation was presented. The setup is capable of measuring ultra-precision ball rotating displacement at nanometer sensitivity. The experiment results indicated that the method has high repeatability and potentially with high throughput. A dual-probe setup is also studied to evaluate diameter variation in a ball with errors introduced by misalignment, while spindle radial vibration being automatically eliminated. Optical probe-based ultra-precision ball displacement and diameter variation measurement are suitable for in situ application with high throughput and repeatability.

Measuring the mechanical properties of polymer–carbon nanotube composites by artificial intelligence

Polymer-Carbon nanotube composites (PCNCs) are a new class of composite materials, which is receiving significant attention both in academia and industry. In this paper, the application of Artificial Intelligence (AI) to the measurement of the elastic modulus of PCNCs was investigated. This is the first time that an AI-based modeling algorithm is used as an alternative tool for nano-indentation methods such as depth-sensing indentation (DSI). To build the model, 138 pair input-target data were gathered from the literature, randomly divided into 88 and 50 data sets and then were respectively trained and tested by the proposed AI model. In the set of the models, matrix type, nanofiller type, processing method, nanofiller content and method of analysis were introduced to the AI model as input layers, while the elastic modulus (either quasi-static E or dynamic [Formula: see text]) is used as output parameter. Although the modeling process is done with a nanometer sensitivity, the training and testing set results of the explicit formulations obtained by the AI model showed that artificial intelligent methods have strong potential and can be applied for the measurement of the elastic modulus of PCNCs as a non-destructive testing.

Full-field speckle interferometry for non-contact photoacoustic tomography

A full-field speckle interferometry method for non-contact and prospectively high speed Photoacoustic Tomography is introduced and evaluated as proof of concept. Thermoelastic pressure induced changes of the objects topography are acquired in a repetitive mode without any physical contact to the object. In order to obtain high acquisition speed, the object surface is illuminated by laser pulses and imaged onto a high speed camera chip. In a repetitive triple pulse mode, surface displacements can be acquired with nanometre sensitivity and an adjustable sampling rate of e.g. 20 MHz with a total acquisition time far below one second using kHz repetition rate lasers. Due to recurring interferometric referencing, the method is insensitive to thermal drift of the object due to previous pulses or other motion. The size of the investigated area and the spatial and temporal resolution of the detection are scalable. In this study, the approach is validated by measuring a silicone phantom and a porcine skin phantom with embedded silicone absorbers. The reconstruction of the absorbers is presented in 2D and 3D. The sensitivity of the measurement with respect to the photoacoustic detection is discussed. Potentially, Photoacoustic Imaging can be brought a step closer towards non-anaesthetized in vivo imaging and new medical applications not allowing acoustic contact, such as neurosurgical monitoring or burnt skin investigation.

Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders.

Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed.

Optical assay of erythrocyte function in banked blood.

Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time.

Development of in Situ Electrochemical Small-Angle Neutron Scattering (eSANS) for Simultaneous Structure and Redox Characterization of Nanoparticles

The demand for engineered nanomaterials in expanding research sectors, such as energy and healthcare, require materials with widely disparate physicochemical properties. The oxidation and reduction (redox) properties of nanomaterials are crucial for catalysis, matching semiconductor band gaps for photovoltaics, as well as toxicology, since they may cause oxidative stress by generating reactive-oxygen species. Optical spectroscopy analysis of molecular and nanomaterials undergoing redox reactions quantifies the electron transfer and energetics of the reaction via potential control and are the most common analytical techniques employed for this purpose. However, with conjugation to organic ligands and smaller, well-defined particle sizes, engineered nanomaterial redox characterization would benefit by techniques that provide nanometer sensitivity to nanoparticle core and ligand structure. We developed an in situ electrochemical small-angle neutron scattering (eSANS) method to measure simultaneously the redox properties and size, shape, and interactions of solution-dispersed nanomaterials [1]. By combining multi-step potentials and chronocoulometry readout with SANS, the structure and redox properties of engineered nanomaterials are followed, simultaneously in one experiment. Specifically, ZnO nanoparticles were examined as dilute dispersions in pH buffered deuterium oxide solutions under negative electrode potentials. The ZnO disk-shaped nanoparticles undergo an irreversible size transformation upon reduction at the vitreous carbon electrode. The decrease in average nanoparticle size near a current maximum shows the reduction reaction from ZnO to Zn occurs. The eSANS method provides nanometer scale sensitivity to the nanoparticle size and shape changes due to an electrochemical reaction that is crucial to understand in energy, healthcare, and other applications. These experiments utilized the EQ-SANS beamline at the Spallation Neutron Source, Oak Ridge National Laboratory that uniquely provides the ability to follow the redox kinetics under quasi-stationary potential step-scans. The methodology, contrast variation, and scattering properties of the micro and nanoporous carbon electrodes will also be described.[1] V.M. Prabhu and V. Reipa, J. Phys. Chem. Lett. 3, 646 (2012)

Biophysical Study of Babesia Infected Red Blood Cell using Diffraction Phase Microscopy

Babesiisa microti is a protozoan parasite which causes a tick-borne zoonotic disease through blood meditated infection. Conventionally, it is hard to detect the location of parasite infection inside a host blood cell, its progress and physical properties simultaneously with invasive staining method. Thus, we present the biophysical study of Babesia infected red blood cell (RBC) using non-invasive diffraction phase microscopy (DPM) (1, 2). DPM is a quantitative phase imaging technique employing a common-path laser interferometry that measures dynamics of biological samples with nanometer sensitivity. In addition, three -dimensional refractive-index map is also measured simply by tomographic DPM reconstruction with various incident illumination angles (3). Since the hemoglobin is a major optical contrast inside a RBC, the refractive- index map allows visualizing the babesia- infected sites, and that it can be used to determine the infection stage. Secondly, the dynamic membrane fluctuations of RBCs, measured by DPM, are analyzed to address the cell deformability by Babesia infection. Other physiological characteristics such as morphology and dry mass, which were not revealed in conventional method, are also investigated. These results lead us to non-invasively and quantitatively investigate new biological insights for internal structures as well as physiological condition of parasite- infected RBC. 1. Park, Y. K., et al. 2006. Diffraction phase and fluorescence microscopy. Optics Express 14:8263-8268. 2. Popescu, G., et al. 2006. Diffraction phase microscopy for quantifying cell structure and dynamics. Optics. Letters. 31:775-777. 3. Park, Y., et al, and S. Suresh. 2008. Refractive index maps and membrane dynamics of human red blood cells parasitized by Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 105:13730-13735.

Entire 3-Dimensional Image of Red Blood Cells using Defocusing Microscopy

We present a simple bright-field optical microscopy technique, which we termed Defocusing Microscopy (DM) [1-3], able to actually reconstruct the 3-dimensional (3D) structure of phase objects. For this reconstruction it is only necessary to measure the object DM contrast at two different objective focal positions. We apply the developed method to red blood cells and obtain the shapes of the upper and lower surfaces, the last one deformed by the substrate. We reconstruct cells in isotonic, hypotonic conditions, as shown in the figure below. Additionally, we obtain the height fluctuations, with nanometer sensitivity, and the elastic modulii of each surface separately. For entire 3D imaging of phase objects in motion, one can use two cameras focused on two different positions. Therefore, DM technique can also be applied to obtain 3D reconstruction of cells flowing in micro-fluidic devices.[1] U. Agero, C.H. Monken, C. Ropert, R.T. Gazzinelli, and O.N. Mesquita, Phys. Rev. E 67, 051904 (2003).[2] L.G. Mesquita, U. Agero, and O.N. Mesquita, Applied Phys. Lett. 88, 133901 (2006).[3] G. Glionna, C.K. Oliveira; L.G. Siman, Moyses HW, D.M.U. Prado, C.H. Monken, and O.N. Mesquita, Applied Phys. Lett. 94, 193701 (2009).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Localized cell stiffness measurement using axial movement of an optically trapped microparticle

A simple optical tweezers design is proposed to manipulate particles in the axial direction and estimate particle position with nanometer sensitivity. Balb3T3 cell is probed using two different-sized particles, and the localized cell stiffness is evaluated using Hertz model. A series of experiments are performed to obtain the necessary parameters for the cell stiffness computation: particle displacement, trapping stiffness, force exertion, and cell deformation. The computed cell stiffness measurements are 17 and 40 Pa using 4 μm- and 2 μm-sized particles, respectively. Results suggest that the proposed optical tweezers scheme can measure the stiffness of a particular cell locale using Hertz model, offering insights about how cells respond to outside mechanical stimulus.

Molecular angiogenic events of a two‐herb wound healing formula involving MAPK and Akt signaling pathways in human vascular endothelial cells

The emergence of electric cell-substrate impedance sensing (ECIS) technology has provided new insight in advanced cell behavioral study by its nanometer sensitivity, precise electrical wounds generation, and high reproducibility that can be monitored in real time in a noninvasive way. However, little is known regarding pro-angiogenic agents in wound healing studies using endothelial cells evaluated with ECIS technology. Our previous studies showed a prominent wound healing effect of a two-herb formula (NF3) comprising of Astragali Radix and Rehmanniae Radix in a rat chronic wound model through actions including angiogenesis. Here we further investigated the angiogenic effect and its underlying molecular mechanism through proliferation, motility, and tubule formation of human vascular endothelial cells (HECV) using ECIS technology. It was first shown that HECV treated with NF3 had a higher resistance than that of control using ECIS cell attachment and cell migration model (p < 0.01). We further validated in a scratch assay that NF3 treatment significantly stimulated HECV cell migration (p < 0.01-0.05). Also, NF3-treated HECV were observed to develop into a significantly more branched tubular structure when compared with control (p < 0.05-0.01). Meanwhile, Western blot analysis of NF3-treated HECV revealed the activated expression of p-Akt, and mitogen-activated protein (MAP) kinases for p-ERK, p-p38, and p-JNK. We propose that the effect of NF3 in the promotion of endothelial cell migration and tubule formation could be mediated through pathways involving p-Akt and activated MAP kinases. Hence, we demonstrated the complexity of the angiogenic effect activated by NF3 molecularly and functionally. NF3 treatment could offer therapeutic value to chronic wound healing for its pro-angiogenic efficacy.

Characterization of phase separation of polystyrene/poly(vinyl methyl ether) blends using fluorescence

We have investigated the fluorescence emission spectra of pyrene and anthracene dyes covalently bonded to polystyrene (PS) upon phase separation from poly(vinyl methyl ether) (PVME). The specific chemical structure of the fluorescent labels is found to affect the measured phase separation temperature TS, with fluorophores covalently attached in closer proximity to the PS backbone identifying phase separation a few degrees earlier. The sharp increase in fluorescence intensity upon phase separation that occurs for all fluorophores with little change in spectral shape is consistent with a mechanism of static fluorescence quenching resulting from the specific interaction with a nearby quenching molecular unit. Based on recent work that has identified a weak hydrogen bond occurring between the aromatic hydrogens of PS and the ether oxygen of PVME, we believe a similar weak hydrogen bond is likely occurring between the PVME oxygen and the aromatic dyes providing a local (few nanometer) sensitivity to phase separation. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011

Material sensitive scanning probe microscopy of subsurface semiconductor nanostructuresvia beam exit Ar ion polishing

Whereas scanning probe microscopy (SPM) is highly appreciated for its nanometre scaleresolution and sensitivity to surface properties, it generally cannot image solid statenanostructures under the immediate sample surface. Existing methods of cross-sectioning(focused ion beam milling and mechanical and Ar ion polishing) are either prohibitivelyslow or cannot provide a required surface quality. In this paper we present a novel methodof Ar ion beam cross-section polishing via a beam exiting the sample. In this approach, asample is tilted at a small angle with respect to the polishing beam that enters fromunderneath the surface of interest and exits at a glancing angle. This creates analmost perfect nanometre scale flat cross-section with close to open angle prismaticshape of the polished and pristine sample surfaces ideal for SPM imaging. Usingthe new method and material sensitive ultrasonic force microscopy we mappedthe internal structure of an InSb/InAs quantum dot superlattice of 18 nm layerperiodicity with the depth resolution of the order of 5 nm. We also report using thismethod to reveal details of interfaces in VLSI (very large scale of integration) lowk dielectric interconnects, as well as discussing the performance of the new approach for SPMas well as for scanning electron microscopy studies of nanostructured materials and devices.

Elementary Mechanisms of Force Generation

Elementary mechanisms of force generationAmin L.1, Shahapure R.1, Ercolini E.1,2, & Torre V.1,31International School for Advanced Studies (SISSA-ISAS), Trieste, Italy. 2 Cluster in Biomedicine (CBM), Area Science Park Basovizza, Trieste, Italy. 3Italian Institute of Technology, ISAS Unit, Italy.By using optical tweezers, we have characterized in details the mechanism of force generation in neuronal filopodia and lamellipodia with a nanometer sensitivity and sub-millisecond temporal resolution. Filopodia of dorsal root ganglia (DRG) neurons exert forces up to 2 pN, while those of hippocampal neurons a larger force up to 4-6 pN. When lamellipodia of DRG or hippocampal growth cones grow pushing a trapped bead, the autocorrelation of bead fluctuations decays with multiple time constants, while during Brownian fluctuations a single time constant of approximately 1 msec is observed. During push the power spectrum density of bead fluctuations is not fitted by a Lorentzian distribution and the energy content of low frequencies is higher than during Brownian fluctuations. Bead fluctuations along the three spatial components during push exhibit a higher cross-correlation than during Brownian fluctuations. Thus, during push the underlying dynamics is spatially and temporally correlated. Several algorithms show that during push, forward and backward jumps of 2-20 nm are detected. The net protrusion in a given time window is equal to the sum of all forward and backward jumps in the same window. These forward and backward jumps are identified as bursts of actin polymerization and depolymerization respectively and represent the elementary mechanisms of force generation.

Detrended fluctuation analysis of membrane flickering in discocyte and spherocyte red blood cells using quantitative phase microscopy

Dynamic analyses of vibrational motion in cell membranes provide a lot of information on the complex dynamic motilities of a red blood cell (RBC). Here, we present the correlation properties of membrane fluctuation in discocyte and spherocyte RBCs by using quantitative phase microscopy (QPM). Since QPM can provide nanometer sensitivity in thickness measurement within a millisecond time scale, we were able to observe the membrane flicking of an RBC in nanometer resolution up to the bandwidth of 50 Hz. The correlation properties of the vibrational motion were analyzed with the detrended fluctuation analysis (DFA) method. Fractal scaling exponent α in the DFA method was calculated for the vibrational motion of a cell surface at various surface points for normal discocyte and abnormal spherocyte RBCs. Measured α values for normal RBCs are distributed between 0.7 and 1.0, whereas those for abnormal spherocyte RBCs are within a range from 0.85 to 1.2. We have also verified that the vibrational motion of background fluid outside of a cell has an α value close to 0.5, which is a typical property of an uncorrelated white noise.

ChemInform Abstract: Thinner, Smaller, Faster: IR Techniques to Probe the Functionality of Biological and Biomimetic Systems

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