Transition to Virtual Microscopy in Medical Undergraduate Pathology Education: First Experience of Turkey in Dokuz Eylül University Hospital.

PubMed

Sağol, Özgül; Yörükoğlu, Kutsal; Lebe, Banu; Durak, Merih Güray; Ulukuş, Çağnur; Tuna, Burçin; Musal, Berna; Canda, Tülay; Özer, Erdener

2015-01-01

Pathology education includes an important visual part supporting a wide range of theoretical knowledge. However, the use of traditional microscopes in pathology education has declined over the last decade and there is a lack of interest for microscopy. Virtual microscopy, which was first described in 1985 and has experienced a revolution since 2000, is an alternative technique to conventional microscopy, in which microscopic slides are scanned to form digital images and stored in the web. The aim of this study was to evaluate the use of virtual microscopy in practical pathology sessions and its effects on our students and undergraduate education at our faculty. Second and third year medical students who were used to conventional microscopes were included in the study. The practical sessions were carried out via virtual slides and the effect of the new technique was investigated by a scale at the end of each session. Academic staff from the pathology department joined sessions to promote discussion and respond to questions. Student ratings were analysed statistically. The evaluation of the ratings showed that the students were easily adapted to the use of virtual microscopy. They found it user-friendly and thought that the opportunity of viewing slides at home was advantageous. Collaboration between students and interactive discussions was also improved with this technique. It was concluded that the use of virtual microscopy could contribute to the pathology education of our students.

Two-Photon Excitation, Fluorescence Microscopy, and Quantitative Measurement of Two-Photon Absorption Cross Sections

NASA Astrophysics Data System (ADS)

DeArmond, Fredrick Michael

As optical microscopy techniques continue to improve, most notably the development of super-resolution optical microscopy which garnered the Nobel Prize in Chemistry in 2014, renewed emphasis has been placed on the development and use of fluorescence microscopy techniques. Of particular note is a renewed interest in multiphoton excitation due to a number of inherent properties of the technique including simplified optical filtering, increased sample penetration, and inherently confocal operation. With this renewed interest in multiphoton fluorescence microscopy, comes an increased demand for robust non-linear fluorescent markers, and characterization of the associated tool set. These factors have led to an experimental setup to allow a systematized approach for identifying and characterizing properties of fluorescent probes in the hopes that the tool set will provide researchers with additional information to guide their efforts in developing novel fluorophores suitable for use in advanced optical microscopy techniques as well as identifying trends for their synthesis. Hardware was setup around a software control system previously developed. Three experimental tool sets were set up, characterized, and applied over the course of this work. These tools include scanning multiphoton fluorescence microscope with single molecule sensitivity, an interferometric autocorrelator for precise determination of the bandwidth and pulse width of the ultrafast Titanium Sapphire excitation source, and a simplified fluorescence microscope for the measurement of two-photon absorption cross sections. Resulting values for two-photon absorption cross sections and two-photon absorption action cross sections for two standardized fluorophores, four commercially available fluorophores, and ten novel fluorophores are presented as well as absorption and emission spectra.

Non-contact lateral force microscopy.

PubMed

Weymouth, A J

2017-08-16

The goal of atomic force microscopy (AFM) is to measure the short-range forces that act between the tip and the surface. The signal recorded, however, includes long-range forces that are often an unwanted background. Lateral force microscopy (LFM) is a branch of AFM in which a component of force perpendicular to the surface normal is measured. If we consider the interaction between tip and sample in terms of forces, which have both direction and magnitude, then we can make a very simple yet profound observation: over a flat surface, long-range forces that do not yield topographic contrast have no lateral component. Short-range interactions, on the other hand, do. Although contact-mode is the most common LFM technique, true non-contact AFM techniques can be applied to perform LFM without the tip depressing upon the sample. Non-contact lateral force microscopy (nc-LFM) is therefore ideal to study short-range forces of interest. One of the first applications of nc-LFM was the study of non-contact friction. A similar setup is used in magnetic resonance force microscopy to detect spin flipping. More recently, nc-LFM has been used as a true microscopy technique to systems unsuitable for normal force microscopy.

Preface to Special Topic: Piezoresponse Force Microscopy

DOE PAGES

Balke, Nina; Bassiri-Gharb, Nazanin; Lichtensteiger, Céline

2015-08-19

Almost two decades beyond the inception of piezoresponse force microscopy (PFM) and the seminal papers by G€uthner and Dransfeld1 and Gruverman et al., the technique has become the prevailing approach for nanoscale functional characterization of polar materials and has been extended to the probing of other electromechanical effects through the advent of electrochemical strain microscopy (ESM). This focus issue celebrates some of the recent advances in the field and offers a wider outlook of polar materials and their overall characterization. In this paper, we cover topics that include discussions of the properties of traditional ferroelectrics, such as lead zirconate titanatemore » (PZT) and lithium niobate, relaxorferroelectrics, as well as more “exotic” ferroelectric oxides such as hafnia, ferroelectric biological matter, and multiferroic materials. Technique-oriented contributions include papers on the coupling of PFM with other characterization methods such as x-ray diffraction (XRD) and superconducting quantum interface device (SQUID), in addition to considerations on the open questions on the electromechanical response in biased scanning probe microscopy (SPM) techniques, including the effects of the laser spot placement on the readout cantilever displacement, the influence of the tip on the creation of the domain shapes, and the impact of ionic and electronic dynamics on the observed nanoscale hysteretic phenomena.« less

Digital stereo-holographic microscopy for studying three-dimensional particle dynamics

NASA Astrophysics Data System (ADS)

Byeon, Hyeokjun; Go, Taesik; Lee, Sang Joon

2018-06-01

A digital stereo-holographic microscopy (DsHM) with two viewing angles is proposed to measure 3D information of microscale particles. This approach includes two volumetric recordings and numerical reconstruction, and it involves the combination of separately reconstructed holograms. The 3D positional information of a particle was determined by searching the center of the overlapped reconstructed volume. After confirming the proposed technique using static spherical particles, the 3D information of moving particles suspended in a Hagen-Poiseiulle flow was successfully obtained. Moreover, the 3D information of nonspherical particles, including ellipsoidal particles and red blood cells, were measured using the proposed technique. In addition to 3D positional information, the orientation and shape of the test samples were obtained from the plane images by slicing the overlapped volume perpendicular to the directions of the image recordings. This DsHM technique will be useful in analyzing the 3D dynamic behavior of various nonspherical particles, which cannot be measured by conventional digital holographic microscopy.

Capturing the Surface Texture and Shape of Pollen: A Comparison of Microscopy Techniques

PubMed Central

Sivaguru, Mayandi; Mander, Luke; Fried, Glenn; Punyasena, Surangi W.

2012-01-01

Research on the comparative morphology of pollen grains depends crucially on the application of appropriate microscopy techniques. Information on the performance of microscopy techniques can be used to inform that choice. We compared the ability of several microscopy techniques to provide information on the shape and surface texture of three pollen types with differing morphologies. These techniques are: widefield, apotome, confocal and two-photon microscopy (reflected light techniques), and brightfield and differential interference contrast microscopy (DIC) (transmitted light techniques). We also provide a first view of pollen using super-resolution microscopy. The three pollen types used to contrast the performance of each technique are: Croton hirtus (Euphorbiaceae), Mabea occidentalis (Euphorbiaceae) and Agropyron repens (Poaceae). No single microscopy technique provided an adequate picture of both the shape and surface texture of any of the three pollen types investigated here. The wavelength of incident light, photon-collection ability of the optical technique, signal-to-noise ratio, and the thickness and light absorption characteristics of the exine profoundly affect the recovery of morphological information by a given optical microscopy technique. Reflected light techniques, particularly confocal and two-photon microscopy, best capture pollen shape but provide limited information on very fine surface texture. In contrast, transmitted light techniques, particularly differential interference contrast microscopy, can resolve very fine surface texture but provide limited information on shape. Texture comprising sculptural elements that are spaced near the diffraction limit of light (∼250 nm; NDL) presents an acute challenge to optical microscopy. Super-resolution structured illumination microscopy provides data on the NDL texture of A. repens that is more comparable to textural data from scanning electron microscopy than any other optical microscopy technique investigated here. Maximizing the recovery of morphological information from pollen grains should lead to more robust classifications, and an increase in the taxonomic precision with which ancient vegetation can be reconstructed. PMID:22720050

Bending the Rules: Widefield Microscopy and the Abbe Limit of Resolution

PubMed Central

Verdaasdonk, Jolien S.; Stephens, Andrew D.; Haase, Julian; Bloom, Kerry

2014-01-01

One of the most fundamental concepts of microscopy is that of resolution–the ability to clearly distinguish two objects as separate. Recent advances such as structured illumination microscopy (SIM) and point localization techniques including photoactivated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) strive to overcome the inherent limits of resolution of the modern light microscope. These techniques, however, are not always feasible or optimal for live cell imaging. Thus, in this review, we explore three techniques for extracting high resolution data from images acquired on a widefield microscope–deconvolution, model convolution, and Gaussian fitting. Deconvolution is a powerful tool for restoring a blurred image using knowledge of the point spread function (PSF) describing the blurring of light by the microscope, although care must be taken to ensure accuracy of subsequent quantitative analysis. The process of model convolution also requires knowledge of the PSF to blur a simulated image which can then be compared to the experimentally acquired data to reach conclusions regarding its geometry and fluorophore distribution. Gaussian fitting is the basis for point localization microscopy, and can also be applied to tracking spot motion over time or measuring spot shape and size. All together, these three methods serve as powerful tools for high-resolution imaging using widefield microscopy. PMID:23893718

Measurements and Diagnostics of Diamond Films and Coatings

NASA Technical Reports Server (NTRS)

Miyoshi, Kazuhisa; Wu, Richard L. C.

1999-01-01

The commercial potential of chemical-vapor-deposited (CVD) diamond films has been established and a number of applications have been identified through university, industry, and government research studies. This paper discusses the methodologies used for property measurement and diagnostic of CVD diamond films and coatings. Measurement and diagnostic techniques studied include scanning electron microscopy, transmission electron microscopy, atomic force microscopy, stylus profilometry, x-ray diffraction, electron diffraction, Raman spectroscopy, Rutherford backscattering, elastic recoil spectroscopy, and friction examination. Each measurement and diagnostic technique provides unique information. A combination of techniques can provide the technical information required to understand the quality and properties of CVD diamond films, which are important to their application in specific component systems and environments. In this study the combination of measurement and diagnostic techniques was successfully applied to correlate deposition parameters and resultant diamond film composition, crystallinity, grain size, surface roughness, and coefficient of friction.

Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques.

PubMed

Paddock, Stephen W; Eliceiri, Kevin W

2014-01-01

Confocal microscopy is an established light microscopical technique for imaging fluorescently labeled specimens with significant three-dimensional structure. Applications of confocal microscopy in the biomedical sciences include the imaging of the spatial distribution of macromolecules in either fixed or living cells, the automated collection of 3D data, the imaging of multiple labeled specimens and the measurement of physiological events in living cells. The laser scanning confocal microscope continues to be chosen for most routine work although a number of instruments have been developed for more specific applications. Significant improvements have been made to all areas of the confocal approach, not only to the instruments themselves, but also to the protocols of specimen preparation, to the analysis, the display, the reproduction, sharing and management of confocal images using bioinformatics techniques.

Room temperature chemical synthesis of lead selenide thin films with preferred orientation

NASA Astrophysics Data System (ADS)

Kale, R. B.; Sartale, S. D.; Ganesan, V.; Lokhande, C. D.; Lin, Yi-Feng; Lu, Shih-Yuan

2006-11-01

Room temperature chemical synthesis of PbSe thin films was carried out from aqueous ammoniacal solution using Pb(CH3COO)2 as Pb2+ and Na2SeSO3 as Se2- ion sources. The films were characterized by a various techniques including, X-ray diffraction (XRD), energy dispersive X-ray analysis (EDAX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HR-TEM), selected area electron diffraction (SAED), Fast Fourier transform (FFT) and UV-vis-NIR techniques. The study revealed that the PbSe thin film consists of preferentially oriented nanocubes with energy band gap of 0.5 eV.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device.

PubMed

Park, Jong Kang; Rowlands, Christopher J; So, Peter T C

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice.

Enhanced Axial Resolution of Wide-Field Two-Photon Excitation Microscopy by Line Scanning Using a Digital Micromirror Device

PubMed Central

Park, Jong Kang; Rowlands, Christopher J.; So, Peter T. C.

2017-01-01

Temporal focusing multiphoton microscopy is a technique for performing highly parallelized multiphoton microscopy while still maintaining depth discrimination. While the conventional wide-field configuration for temporal focusing suffers from sub-optimal axial resolution, line scanning temporal focusing, implemented here using a digital micromirror device (DMD), can provide substantial improvement. The DMD-based line scanning temporal focusing technique dynamically trades off the degree of parallelization, and hence imaging speed, for axial resolution, allowing performance parameters to be adapted to the experimental requirements. We demonstrate this new instrument in calibration specimens and in biological specimens, including a mouse kidney slice. PMID:29387484

Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

PubMed Central

Chung, Chao-Yu; Boik, John; Potma, Eric O.

2014-01-01

Optical imaging with spectroscopic vibrational contrast is a label-free solution for visualizing, identifying, and quantifying a wide range of biomolecular compounds in biological materials. Both linear and nonlinear vibrational microscopy techniques derive their imaging contrast from infrared active or Raman allowed molecular transitions, which provide a rich palette for interrogating chemical and structural details of the sample. Yet nonlinear optical methods, which include both second-order sum-frequency generation (SFG) and third-order coherent Raman scattering (CRS) techniques, offer several improved imaging capabilities over their linear precursors. Nonlinear vibrational microscopy features unprecedented vibrational imaging speeds, provides strategies for higher spatial resolution, and gives access to additional molecular parameters. These advances have turned vibrational microscopy into a premier tool for chemically dissecting live cells and tissues. This review discusses the molecular contrast of SFG and CRS microscopy and highlights several of the advanced imaging capabilities that have impacted biological and biomedical research. PMID:23245525

Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

PubMed

Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

2016-06-01

A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-resolution measurements of the multilayer ultra-structure of articular cartilage and their translational potential

PubMed Central

2014-01-01

Current musculoskeletal imaging techniques usually target the macro-morphology of articular cartilage or use histological analysis. These techniques are able to reveal advanced osteoarthritic changes in articular cartilage but fail to give detailed information to distinguish early osteoarthritis from healthy cartilage, and this necessitates high-resolution imaging techniques measuring cells and the extracellular matrix within the multilayer structure of articular cartilage. This review provides a comprehensive exploration of the cellular components and extracellular matrix of articular cartilage as well as high-resolution imaging techniques, including magnetic resonance image, electron microscopy, confocal laser scanning microscopy, second harmonic generation microscopy, and laser scanning confocal arthroscopy, in the measurement of multilayer ultra-structures of articular cartilage. This review also provides an overview for micro-structural analysis of the main components of normal or osteoarthritic cartilage and discusses the potential and challenges associated with developing non-invasive high-resolution imaging techniques for both research and clinical diagnosis of early to late osteoarthritis. PMID:24946278

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

PubMed

Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

Nanoscale elasticity mappings of micro-constituents of abalone shell by band excitation-contact resonance force microscopy

NASA Astrophysics Data System (ADS)

Li, Tao; Zeng, Kaiyang

2014-01-01

The macroscopic mechanical properties of the abalone shell have been studied extensively in the literature, but the in situ nanoscale elasticity of various micro-constituents in the shell have not been characterized and reported yet. In this study, the nanoscale elasticity mappings including different micro-constituents in abalone shell were observed by using the Contact Resonance Force Microscopy (CR-FM) technique. CR-FM is one of the advanced scanning probe microscopy techniques that is able to quantify the local elastic moduli of various materials in a non-destructive manner. Instead of an average value, an elasticity mapping that reveals the nanoscale variations of elastic moduli with location can be extracted and correlated with the topography of the structure. Therefore in this study, by adopting the CR-FM technique that is incorporated with the band excitation technique, the elasticity variations of the abalone shell caused by different micro-constituents and crystal orientations are reported, and the elasticity values of the aragonite and calcite nanograins are quantified.The macroscopic mechanical properties of the abalone shell have been studied extensively in the literature, but the in situ nanoscale elasticity of various micro-constituents in the shell have not been characterized and reported yet. In this study, the nanoscale elasticity mappings including different micro-constituents in abalone shell were observed by using the Contact Resonance Force Microscopy (CR-FM) technique. CR-FM is one of the advanced scanning probe microscopy techniques that is able to quantify the local elastic moduli of various materials in a non-destructive manner. Instead of an average value, an elasticity mapping that reveals the nanoscale variations of elastic moduli with location can be extracted and correlated with the topography of the structure. Therefore in this study, by adopting the CR-FM technique that is incorporated with the band excitation technique, the elasticity variations of the abalone shell caused by different micro-constituents and crystal orientations are reported, and the elasticity values of the aragonite and calcite nanograins are quantified. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05292c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balke, Nina; Bassiri-Gharb, Nazanin; Lichtensteiger, Céline

Almost two decades beyond the inception of piezoresponse force microscopy (PFM) and the seminal papers by G€uthner and Dransfeld1 and Gruverman et al., the technique has become the prevailing approach for nanoscale functional characterization of polar materials and has been extended to the probing of other electromechanical effects through the advent of electrochemical strain microscopy (ESM). This focus issue celebrates some of the recent advances in the field and offers a wider outlook of polar materials and their overall characterization. In this paper, we cover topics that include discussions of the properties of traditional ferroelectrics, such as lead zirconate titanatemore » (PZT) and lithium niobate, relaxorferroelectrics, as well as more “exotic” ferroelectric oxides such as hafnia, ferroelectric biological matter, and multiferroic materials. Technique-oriented contributions include papers on the coupling of PFM with other characterization methods such as x-ray diffraction (XRD) and superconducting quantum interface device (SQUID), in addition to considerations on the open questions on the electromechanical response in biased scanning probe microscopy (SPM) techniques, including the effects of the laser spot placement on the readout cantilever displacement, the influence of the tip on the creation of the domain shapes, and the impact of ionic and electronic dynamics on the observed nanoscale hysteretic phenomena.« less

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

PubMed

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bulk microstructure and local elastic properties of carbon nanocomposites studied by impulse acoustic microscopy technique

NASA Astrophysics Data System (ADS)

Levin, V.; Petronyuk, Yu.; Morokov, E.; Chernozatonskii, L.; Kuzhir, P.; Fierro, V.; Celzard, A.; Bellucci, S.; Bistarelli, S.; Mastrucci, M.; Tabacchioni, I.

2016-05-01

Bulk microstructure and elastic properties of epoxy-nanocarbon nanocomposites for diverse types and different content of carbon nanofiller has been studied by using impulse acoustic microscopy technique. It has been shown occurrence of various types of mesoscopic structure formed by nanoparticles inside the bulk of nanocomposite materials, including nanoparticle conglomerates and nanoparticle aerogel systems. In spite of the bulk microstructure, nanocarbon composites demonstrate elastic uniformity and negligible influence of nanofiller on elastic properties of carbon nanocomposite materials.

Bioorthogonal Chemical Imaging for Biomedicine

NASA Astrophysics Data System (ADS)

Min, Wei

2017-06-01

Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because relatively bulky fluorescent labels could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, we have developed a bioorthogonal chemical imaging platform. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes, nitriles and stable isotopes including 2H and 13C), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, multiplicity and biocompatibility for imaging small biomolecules in live systems including tissues and organisms. Exciting biomedical applications such as imaging fatty acid metabolism related to lipotoxicity, glucose uptake and metabolism, drug trafficking, protein synthesis, DNA replication, protein degradation, RNA synthesis and tumor metabolism will be presented. This bioorthogonal chemical imaging platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, further chemical and spectroscopic strategies allow for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species, bringing small molecules under the illumination of modern light microscopy.

Lensfree On-Chip Microscopy and Tomography for Bio-Medical Applications

PubMed Central

Isikman, Serhan O.; Bishara, Waheb; Mudanyali, Onur; Sencan, Ikbal; Su, Ting-Wei; Tseng, Derek; Yaglidere, Oguzhan; Sikora, Uzair; Ozcan, Aydogan

2012-01-01

Lensfree on-chip holographic microscopy is an emerging technique that offers imaging of biological specimens over a large field-of-view without using any lenses or bulky optical components. Lending itself to a compact, cost-effective and mechanically robust architecture, lensfree on-chip holographic microscopy can offer an alternative toolset addressing some of the emerging needs of microscopic analysis and diagnostics in low-resource settings, especially for telemedicine applications. In this review, we summarize the latest achievements in lensfree optical microscopy based on partially coherent on-chip holography, including portable telemedicine microscopy, cell-phone based microscopy and field-portable optical tomographic microscopy. We also discuss some of the future directions for telemedicine microscopy and its prospects to help combat various global health challenges. PMID:24478572

Multimodality optical imaging of embryonic heart microstructure

PubMed Central

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H.; Boudoux, Caroline; Vakoc, Benjamin J.; Bouma, Brett E.; Tearney, Guillermo J.

2009-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 µm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass. PMID:18163837

Multimodality optical imaging of embryonic heart microstructure.

PubMed

Yelin, Ronit; Yelin, Dvir; Oh, Wang-Yuhl; Yun, Seok H; Boudoux, Caroline; Vakoc, Benjamin J; Bouma, Brett E; Tearney, Guillermo J

2007-01-01

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

The Electron Microscopy Outreach Program: A Web-based resource for research and education.

PubMed

Sosinsky, G E; Baker, T S; Hand, G; Ellisman, M H

1999-01-01

We have developed a centralized World Wide Web (WWW)-based environment that serves as a resource of software tools and expertise for biological electron microscopy. A major focus is molecular electron microscopy, but the site also includes information and links on structural biology at all levels of resolution. This site serves to help integrate or link structural biology techniques in accordance with user needs. The WWW site, called the Electron Microscopy (EM) Outreach Program (URL: http://emoutreach.sdsc.edu), provides scientists with computational and educational tools for their research and edification. In particular, we have set up a centralized resource containing course notes, references, and links to image analysis and three-dimensional reconstruction software for investigators wanting to learn about EM techniques either within or outside of their fields of expertise. Copyright 1999 Academic Press.

HANFORD WASTE MINERALOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-29

This report lists the observed mineral phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports that used experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases observed in Hanford waste.

HANFORD WASTE MINEROLOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-18

This report lists the observed mineral phase phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports using experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases present observed in Hanford waste.

Spectroscopic study of Pbs nano-structured layer prepared by Pld utilized as a Hall-effect magnetic sensor

NASA Astrophysics Data System (ADS)

Atwa, D. M.; Aboulfotoh, N.; El-magd, A. Abo; Badr, Y.

2013-10-01

Lead sulfide (PbS) nano-structured films have been grown on quartz substrates using PLD technique. The deposited films were characterized by several structural techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Selected-area electron diffraction patterns (SAED). The results prove the formation of cubic phase of PbS nanocrystals. Elemental analysis of the deposited films compared to the bulk target was obtained via laser induced fluorescence of the produced plasma particles and the energy dispersive X-ray "EDX" technique. The Hall coefficient measurements indicate an efficient performance of the deposited films as a magnetic sensor.

Preparation of gold nanocluster bioconjugates for electron microscopy.

PubMed

Heinecke, Christine L; Ackerson, Christopher J

2013-01-01

In this chapter, we describe types of gold nanoparticle-biomolecule conjugates and their use in electron microscopy. Included are two detailed protocols for labeling an IgG antibody with gold monolayer protected clusters. The first approach is a direct bonding approach that utilizes the ligand place exchange reaction. The second approach describes NHS-EDC coupling of Au(144)(pMBA)(60) with IgG. Also included are various characterization techniques for determining labeling efficiency.

Transmission X-ray microscopy for full-field nano-imaging of biomaterials

PubMed Central

ANDREWS, JOY C; MEIRER, FLORIAN; LIU, YIJIN; MESTER, ZOLTAN; PIANETTA, PIERO

2010-01-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure (XANES) imaging. These techniques are discussed and compared in light of results from imaging of biological materials including microorganisms, bone and mineralized tissue and plants, with a focus on hard X-ray TXM at ≤ 40 nm resolution. PMID:20734414

Imaging TiO2 nanoparticles on GaN nanowires with electrostatic force microscopy

NASA Astrophysics Data System (ADS)

Xie, Ting; Wen, Baomei; Liu, Guannan; Guo, Shiqi; Motayed, Abhishek; Murphy, Thomas; Gomez, R. D.

Gallium nitride (GaN) nanowires that are functionalized with metal-oxides nanoparticles have been explored extensively for gas sensing applications in the past few years. These sensors have several advantages over conventional schemes, including miniature size, low-power consumption and fast response and recovery times. The morphology of the oxide functionalization layer is critical to achieve faster response and recovery times, with the optimal size distribution of nanoparticles being in the range of 10 to 30 nm. However, it is challenging to characterize these nanoparticles on GaN nanowires using common techniques such as scanning electron microscopy, transmission electron microscopy, and x-ray diffraction. Here, we demonstrate electrostatic force microscopy in combination with atomic force microscopy as a non-destructive technique for morphological characterization of the dispersed TiO2 nanoparticles on GaN nanowires. We also discuss the applicability of this method to other material systems with a proposed tip-surface capacitor model. This project was sponsored through N5 Sensors and the Maryland Industrial Partnerships (MIPS, #5418).

Analysis-Preserving Video Microscopy Compression via Correlation and Mathematical Morphology

PubMed Central

Shao, Chong; Zhong, Alfred; Cribb, Jeremy; Osborne, Lukas D.; O’Brien, E. Timothy; Superfine, Richard; Mayer-Patel, Ketan; Taylor, Russell M.

2015-01-01

The large amount video data produced by multi-channel, high-resolution microscopy system drives the need for a new high-performance domain-specific video compression technique. We describe a novel compression method for video microscopy data. The method is based on Pearson's correlation and mathematical morphology. The method makes use of the point-spread function (PSF) in the microscopy video acquisition phase. We compare our method to other lossless compression methods and to lossy JPEG, JPEG2000 and H.264 compression for various kinds of video microscopy data including fluorescence video and brightfield video. We find that for certain data sets, the new method compresses much better than lossless compression with no impact on analysis results. It achieved a best compressed size of 0.77% of the original size, 25× smaller than the best lossless technique (which yields 20% for the same video). The compressed size scales with the video's scientific data content. Further testing showed that existing lossy algorithms greatly impacted data analysis at similar compression sizes. PMID:26435032

Higher-eigenmode piezoresponse force microscopy: a path towards increased sensitivity and the elimination of electrostatic artifacts

NASA Astrophysics Data System (ADS)

MacDonald, Gordon A.; DelRio, Frank W.; Killgore, Jason P.

2018-03-01

Piezoresponse force microscopy (PFM) and related bias-induced strain sensing atomic force microscopy techniques provide unique characterization of material-functionality at the nanoscale. However, these techniques are prone to unwanted artifact signals that influence the vibration amplitude of the detecting cantilever. Here, we show that higher-order contact resonance eigenmodes can be readily excited in PFM. The benefits of using the higher-order eigenmodes include absolute sensitivity enhancement, electrostatic artifact reduction, and lateral versus normal strain decoupling. This approach can significantly increase the proportion of total signal arising from desired strain (as opposed to non-strain artifacts) in measurements with cantilevers exhibiting typical, few N m‑1 spring constants to cantilevers up to 1000× softer than typically used.

Laser beam shaping for biomedical microscopy techniques

NASA Astrophysics Data System (ADS)

Laskin, Alexander; Kaiser, Peter; Laskin, Vadim; Ostrun, Aleksei

2016-04-01

Uniform illumination of a working field is very important in optical systems of confocal microscopy and various implementations of fluorescence microscopy like TIR, SSIM, STORM, PALM to enhance performance of these laser-based research techniques. Widely used TEM00 laser sources are characterized by essentially non-uniform Gaussian intensity profile which leads usually to non-uniform intensity distribution in a microscope working field or in a field of microlenses array of a confocal microscope optical system, this non-uniform illumination results in instability of measuring procedure and reducing precision of quantitative measurements. Therefore transformation of typical Gaussian distribution of a TEM00 laser to flat-top (top hat) profile is an actual technical task, it is solved by applying beam shaping optics. Due to high demands to optical image quality the mentioned techniques have specific requirements to a uniform laser beam: flatness of phase front and extended depth of field, - from this point of view the microscopy techniques are similar to holography and interferometry. There are different refractive and diffractive beam shaping approaches used in laser industrial and scientific applications, but only few of them are capable to fulfil the optimum conditions for beam quality required in discussed microscopy techniques. We suggest applying refractive field mapping beam shapers πShaper, which operational principle presumes almost lossless transformation of Gaussian to flat-top beam with flatness of output wavefront, conserving of beam consistency, providing collimated low divergent output beam, high transmittance, extended depth of field, negligible wave aberration, and achromatic design provides capability to work with several lasers with different wavelengths simultaneously. The main function of a beam shaper is transformation of laser intensity profile, further beam transformation to provide optimum for a particular technique spot size and shape has to be realized by an imaging optical system which can include microscope objectives and tube lenses. This paper will describe design basics of refractive beam shapers and optical layouts of their applying in microscopy systems. Examples of real implementations and experimental results will be presented as well.

Investigation of TiO2 nanoparticles translocation through a Caco-2 monolayer

NASA Astrophysics Data System (ADS)

Brun, E.; Jugan, M.-L.; Herlin-Boime, N.; Jaillard, D.; Fayard, B.; Flank, A.-M.; Mabondzo, A.; Carrière, M.

2011-07-01

Nanoparticles (NPs) are introduced in a growing number of commercial products, including food and beverage but their effects on gastrointestinal tract are poorly investigated. Here we focused on the translocation of TiO2 NPs through Caco-2 monolayers exposed to anatase and rutile NPs up to 24 h. Internalization was followed by transmission electronic microscopy and μ-XRF elemental mapping, coupled to XAS analysis of Ti atoms environment. This innovative technique is among the best techniques to get insights on NP fate after internalization. The originality of this project relies on the panel of microscopy techniques implemented to investigate digestive barrier translocation, bringing together biologists, chemists and physicists in a pluridisciplinary research program.

Investigation and Control of "Sphere-Like" Buckminsterfullerene C60 and "Disk-Like" Copper(II) Phthalocyanine

NASA Astrophysics Data System (ADS)

McAfee, Terry Richard

Due to the growing global need for cheap, flexible, and portable electronics, numerous research groups from mechanical and electrical engineering, material science, chemistry, and physics have increasingly turned to organic electronics research over the last ˜5--10 years. Largely, the focus of researchers in this growing field have sought to obtain the next record holding device, allowing a heuristic approach of trial and error to become dominant focus of research rather than a fundamental understanding. Rather than working with the latest high performance organic semiconducting materials and film processing techniques, I have chosen to investigate and control the fundamental self-assembly interactions of organic photovoltaic thin films using simplified systems. Specifically, I focus on organic photovoltaic research using two of the oldest and well studies semiconducting materials, namely "sphere-like" electron donor material Buckminsterfullerene C60 and "disklike" electron acceptor material Copper(II) Phthalocyanine. I manufactured samples using the well-known technique of physical vapor deposition using a high vacuum chamber that I designed and built to accommodate my need of precise material deposition control, with codeposition capability. Films were characterized using microscopy and spectroscopy techniques locally at NCSU, including Atomic Force Microscopy, scanning tunneling microscopy, X-ray photoelectron spectroscopy, and Ultraviolet-visible spectroscopy, as well as at National Laboratory based synchrotron x-ray techniques, including Carbon and Nitrogen k-edge Total Electron Yield and Transmission Near Edge X-ray absorption fine structure spectroscopy, Carbon k-edge Resonant Soft x-ray Microscopy, Resonant Soft x-ray reflectivity, and Grazing Incidence Wide-Angle X-ray scattering.

High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

PubMed

Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

2015-12-01

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

X-ray microscopy of live biological micro-organisms

NASA Astrophysics Data System (ADS)

Raja Al-Ani, Ma'an Nassar

Real-time, compact x-ray microscopy has the potential to benefit many scientific fields, including microbiology, pharmacology, organic chemistry, and physics. Single frame x-ray micro-radiography, produced by a compact, solid-state laser plasma source, allows scientists to use x-ray emission for elemental analysis, and to observe biological specimens in their natural state. In this study, x-ray images of mouse kidney tissue, live bacteria, Pseudomonas aeruginosa and Burkholderia cepacia, and the bacteria's interaction with the antibiotic gentamicin, are examined using x-ray microscopy. For the purposes of comparing between confocal microscopy and x-ray microscopy, we introduced to our work the technique of gold labeling. Indirect immunofluorescence staining and immuno-gold labeling were applied on human lymphocytes and human tumor cells. Differential interference contrast microscopy (DIC) showed the lymphocyte body and nucleus, as did x-ray microscopy. However, the high resolution of x-ray microscopy allows us to differentiate between the gold particles bound to the antibodies and the free gold. A compact, tabletop Nd: glass laser is used in this study to produce x-rays from an Yttrium target. An atomic force microscope is used to scan the x-ray images from the developed photo-resist. The use of compact, tabletop laser plasma sources, in conjunction with x-ray microscopy, is a new technique that has great potential as a flexible, user-friendly scientific research tool.

Cardiovascular Imaging Using Two-Photon Microscopy

PubMed Central

Scherschel, John A.; Rubart, Michael

2008-01-01

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer. PMID:18986603

Transmission X-ray microscopy for full-field nano imaging of biomaterials.

PubMed

Andrews, Joy C; Meirer, Florian; Liu, Yijin; Mester, Zoltan; Pianetta, Piero

2011-07-01

Imaging of cellular structure and extended tissue in biological materials requires nanometer resolution and good sample penetration, which can be provided by current full-field transmission X-ray microscopic techniques in the soft and hard X-ray regions. The various capabilities of full-field transmission X-ray microscopy (TXM) include 3D tomography, Zernike phase contrast, quantification of absorption, and chemical identification via X-ray fluorescence and X-ray absorption near edge structure imaging. These techniques are discussed and compared in light of results from the imaging of biological materials including microorganisms, bone and mineralized tissue, and plants, with a focus on hard X-ray TXM at ≤ 40-nm resolution. Copyright © 2010 Wiley-Liss, Inc.

Application of scanning acoustic microscopy to advanced structural ceramics

NASA Technical Reports Server (NTRS)

Vary, Alex; Klima, Stanley J.

1987-01-01

A review is presentod of research investigations of several acoustic microscopy techniques for application to structural ceramics for advanced heat engines. Results obtained with scanning acoustic microscopy (SAM), scanning laser acoustic microscopy (SLAM), scanning electron acoustic microscopy (SEAM), and photoacoustic microscopy (PAM) are compared. The techniques were evaluated on research samples of green and sintered monolithic silicon nitrides and silicon carbides in the form of modulus-of-rupture bars containing deliberately introduced flaws. Strengths and limitations of the techniques are described with emphasis on statistics of detectability of flaws that constitute potential fracture origins.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

PubMed Central

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish. PMID:21280920

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

PubMed

Lim, Daryl; Ford, Tim N; Chu, Kengyeh K; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

NASA Astrophysics Data System (ADS)

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Asymmetric Flow-Field Flow Fractionation (AF4) of Aqueous C60 Aggregates with Dynamic Light Scattering Size and LC-MS

EPA Science Inventory

Current methods for the size determination of nanomaterials in aqueous suspension include dynamic or static light scattering and electron or atomic force microscopy techniques. Light scattering techniques are limited by poor resolution and the scattering intensity dependence on p...

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

PubMed Central

Zhou, Lulu; Cai, Mingjun; Tong, Ti; Wang, Hongda

2017-01-01

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy. PMID:28441775

Australian Red Dune Sand: A Potential Martian Regolith Analog

NASA Technical Reports Server (NTRS)

Kuhlman, K. R.; Marshall, J.; Evans, N. D.; Luttge, A.

2001-01-01

To demonstrate the potential scientific and technical merits of in situ microscopy on Mars, we analyzed a possible Martian regolith analog - an acolian red dune sand from the central Australian desert (near Mt. Olga). This sand was chosen for its ubiquitous red coating and the desert environment in which is it found. Grains of this sand were analyzed using a variety of microanalytical techniques. A database of detailed studies of such terrestrial analogs would assist the study of geological and astrobiological specimens in future missions to Mars. Potential instrument concepts for in situ deployment on Mars include local electrode atom probe nanoanalysis (LEAP), vertical scanning white light interferometry (VSWLI), scanning electron microscopies, energy dispersive x-ray microanalysis (EDX), atomic force microscopy (AFM) and X-ray diffraction (XRD). While in situ deployment of these techniques is many years away, ground-based studies using these analytical techniques extend our understanding of the data obtained from instruments to be flown in the near future.

Green synthesis and characterization of alginate nanoparticles and its role as a biosorbent for Cr(VI) ions

NASA Astrophysics Data System (ADS)

Geetha, P.; Latha, M. S.; Pillai, Saumya S.; Deepa, B.; Santhosh Kumar, K.; Koshy, Mathew

2016-02-01

Green synthesis of nanoparticles has attained considerable attention in recent years because of its myriad of applications including drug delivery, tissue engineering and water purification. In the present study, alginate nanoparticles stabilized by honey were prepared by cross-linking aqueous solution of alginate with calcium ions. Honey mediated synthesis has been reported earlier for the production of metal nanoparticles. However no literature is available on the use of this technique for polymeric nanoparticles. Highly stable nanoparticles of 10-100 nm size were generated by this technique. The synthesised nanoparticles were characterized by transmission electron microscopy, scanning electron microscopy, atomic force microscopy, dynamic light scattering and Fourier transform infrared spectroscopic techniques. Potential of using these nanoparticles for heavy metal removal was studied by using Cr(VI) from aqueous solution, where a maximum removal efficiency of 93.5% was obtained. This method was also successfully employed for the production of other polymeric nanoparticles like casein, chitosan and albumin.

Unconventional methods of imaging: computational microscopy and compact implementations

NASA Astrophysics Data System (ADS)

McLeod, Euan; Ozcan, Aydogan

2016-07-01

In the past two decades or so, there has been a renaissance of optical microscopy research and development. Much work has been done in an effort to improve the resolution and sensitivity of microscopes, while at the same time to introduce new imaging modalities, and make existing imaging systems more efficient and more accessible. In this review, we look at two particular aspects of this renaissance: computational imaging techniques and compact imaging platforms. In many cases, these aspects go hand-in-hand because the use of computational techniques can simplify the demands placed on optical hardware in obtaining a desired imaging performance. In the first main section, we cover lens-based computational imaging, in particular, light-field microscopy, structured illumination, synthetic aperture, Fourier ptychography, and compressive imaging. In the second main section, we review lensfree holographic on-chip imaging, including how images are reconstructed, phase recovery techniques, and integration with smart substrates for more advanced imaging tasks. In the third main section we describe how these and other microscopy modalities have been implemented in compact and field-portable devices, often based around smartphones. Finally, we conclude with some comments about opportunities and demand for better results, and where we believe the field is heading.

Cassette Series Designed for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast

PubMed Central

Young, Carissa L.; Raden, David L.; Caplan, Jeffrey; Czymmek, Kirk; Robinson, Anne S.

2012-01-01

During the past decade, it has become clear that protein function and regulation are highly dependent upon intracellular localization. Although fluorescent protein variants are ubiquitously used to monitor protein dynamics, localization, and abundance; fluorescent light microscopy techniques often lack the resolution to explore protein heterogeneity and cellular ultrastructure. Several approaches have been developed to identify, characterize, and monitor the spatial localization of proteins and complexes at the sub-organelle level; yet, many of these techniques have not been applied to yeast. Thus, we have constructed a series of cassettes containing codon-optimized epitope tags, fluorescent protein variants that cover the full spectrum of visible light, a TetCys motif used for FlAsH-based localization, and the first evaluation in yeast of a photoswitchable variant – mEos2 – to monitor discrete subpopulations of proteins via confocal microscopy. This series of modules, complete with six different selection markers, provides the optimal flexibility during live-cell imaging and multicolor labeling in vivo. Furthermore, high-resolution imaging techniques include the yeast-enhanced TetCys motif that is compatible with diaminobenzidine photooxidation used for protein localization by electron microscopy and mEos2 that is ideal for super-resolution microscopy. We have examined the utility of our cassettes by analyzing all probes fused to the C-terminus of Sec61, a polytopic membrane protein of the endoplasmic reticulum of moderate protein concentration, in order to directly compare fluorescent probes, their utility and technical applications. Our series of cassettes expand the repertoire of molecular tools available to advance targeted spatiotemporal investigations using multiple live-cell, super-resolution or electron microscopy imaging techniques. PMID:22473760

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

PubMed

Kraus, Max-Joseph; Seifert, Jan; Strasser, Erwin F; Gawaz, Meinrad; Schäffer, Tilman E; Rheinlaender, Johannes

2016-09-01

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

Assessment of nerve ultrastructure by fibre-optic confocal microscopy.

PubMed

Cushway, T R; Lanzetta, M; Cox, G; Trickett, R; Owen, E R

1996-01-01

Fibre-optic technology combined with confocality produces a microscope capable of optical thin sectioning. In this original study, tibial nerves have been stained in a rat model with a vital dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide, and analysed by fibre-optic confocal microscopy to produce detailed images of nerve ultrastructure. Schwann cells, nodes of Ranvier and longitudinal myelinated sheaths enclosing axons were clearly visible. Single axons appeared as brightly staining longitudinal structures. This allowed easy tracing of multiple signal axons within the nerve tissue. An accurate measurement of internodal lengths was easily accomplished. This technique is comparable to current histological techniques, but does not require biopsy, thin sectioning or tissue fixing. This study offers a standard for further in vivo microscopy, including the possibility of monitoring the progression of nerve regeneration following microsurgical neurorraphy.

Non-destructive characterization of SiC coated carbon-carbon composites by multiple techniques

NASA Astrophysics Data System (ADS)

Nixon, Thomas D.; Hemstad, Stan N.; Pfeifer, William H.

SiC coated carbon-carbon composites were evaluated using several non-destructive techniques as a means of quantifying the quality of both the coating and substrate. The techniques employed included dye penetrant infiltration, eddy current measurement, C-scan, and computed tomography (CT). The NDE results were then correlated to oxidation performance and destructive evaluations by electron and optical microscopy.

Characterization of the Roman curse tablet

NASA Astrophysics Data System (ADS)

Liu, Wen; Zhang, Boyang; Fu, Lin

2017-08-01

The Roman curse tablet, produced in ancient Rome period, is a metal plate that inscribed with curses. In this research, several techniques were used to find out the physical structure and chemical composition of the Roman curse tablet, and testified the hypothesis that whether the tablet is made of pure lead or lead alloy. A sample of Roman Curse Tablet from the Johns Hopkins Archaeological Museum was analyzed using several different characterization techniques to determine the physical structure and chemical composition. The characterization techniques used were including optical microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), and differential scanning calorimetry (DSC). Because of the small sample size, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and X-ray fluorescence (XRF) cannot test the sample. Results from optical microscopy and SEM, enlarged images of the sample surface were studied. The result revealed that the sample surface has a rough, non-uniform, and grainy surface. AFM provides three-dimensional topography of the sample surface, studying the sample surface in atomic level. DSC studies the thermal property, which is most likely a lead-alloy, not a pure lead. However, none of these tests indicated anything about the chemical composition. Future work will be required due to the lack of measures finding out its chemical composition. Therefore, from these characterization techniques above, the Roman curse tablet sample is consisted of lead alloy, not pure lead.

Nonlinear plasmonic imaging techniques and their biological applications

NASA Astrophysics Data System (ADS)

Deka, Gitanjal; Sun, Chi-Kuang; Fujita, Katsumasa; Chu, Shi-Wei

2017-01-01

Nonlinear optics, when combined with microscopy, is known to provide advantages including novel contrast, deep tissue observation, and minimal invasiveness. In addition, special nonlinearities, such as switch on/off and saturation, can enhance the spatial resolution below the diffraction limit, revolutionizing the field of optical microscopy. These nonlinear imaging techniques are extremely useful for biological studies on various scales from molecules to cells to tissues. Nevertheless, in most cases, nonlinear optical interaction requires strong illumination, typically at least gigawatts per square centimeter intensity. Such strong illumination can cause significant phototoxicity or even photodamage to fragile biological samples. Therefore, it is highly desirable to find mechanisms that allow the reduction of illumination intensity. Surface plasmon, which is the collective oscillation of electrons in metal under light excitation, is capable of significantly enhancing the local field around the metal nanostructures and thus boosting up the efficiency of nonlinear optical interactions of the surrounding materials or of the metal itself. In this mini-review, we discuss the recent progress of plasmonics in nonlinear optical microscopy with a special focus on biological applications. The advancement of nonlinear imaging modalities (including incoherent/coherent Raman scattering, two/three-photon luminescence, and second/third harmonic generations that have been amalgamated with plasmonics), as well as the novel subdiffraction limit imaging techniques based on nonlinear behaviors of plasmonic scattering, is addressed.

Biogenicity and Syngeneity of Organic Matter in Ancient Sedimentary Rocks: Recent Advances in the Search for Evidence of Past Life

NASA Astrophysics Data System (ADS)

Oehler, Dorothy Z.; Cady, Sherry L.

2014-08-01

The past decade has seen an explosion of new technologies for assessment of biogenicity and syngeneity of carbonaceous material within sedimentary rocks. Advances have been made in techniques for analysis of in situ organic matter as well as for extracted bulk samples of soluble and insoluble (kerogen) organic fractions. The in situ techniques allow analysis of micrometer-to-sub-micrometer-scale organic residues within their host rocks and include Raman and fluorescence spectroscopy/imagery, confocal laser scanning microscopy, and forms of secondary ion/laser-based mass spectrometry, analytical transmission electron microscopy, and X-ray absorption microscopy/spectroscopy. Analyses can be made for chemical, molecular, and isotopic composition coupled with assessment of spatial relationships to surrounding minerals, veins, and fractures. The bulk analyses include improved methods for minimizing contamination and recognizing syngenetic constituents of soluble organic fractions as well as enhanced spectroscopic and pyrolytic techniques for unlocking syngenetic molecular signatures in kerogen. Together, these technologies provide vital tools for the study of some of the oldest and problematic carbonaceous residues and for advancing our understanding of the earliest stages of biological evolution on Earth and the search for evidence of life beyond Earth. We discuss each of these new technologies, emphasizing their advantages and disadvantages, applications, and likely future directions.

Hybrid label-free multiphoton and optoacoustic microscopy (MPOM)

NASA Astrophysics Data System (ADS)

Soliman, Dominik; Tserevelakis, George J.; Omar, Murad; Ntziachristos, Vasilis

2015-07-01

Many biological applications require a simultaneous observation of different anatomical features. However, unless potentially harmful staining of the specimens is employed, individual microscopy techniques do generally not provide multi-contrast capabilities. We present a hybrid microscope integrating optoacoustic microscopy and multiphoton microscopy, including second-harmonic generation, into a single device. This combined multiphoton and optoacoustic microscope (MPOM) offers visualization of a broad range of structures by employing different contrast mechanisms and at the same time enables pure label-free imaging of biological systems. We investigate the relative performance of the two microscopy modalities and demonstrate their multi-contrast abilities through the label-free imaging of a zebrafish larva ex vivo, simultaneously visualizing muscles and pigments. This hybrid microscopy application bears great potential for developmental biology studies, enabling more comprehensive information to be obtained from biological specimens without the necessity of staining.

The Development of Teaching and Learning in Bright-Field Microscopy Technique

ERIC Educational Resources Information Center

Iskandar, Yulita Hanum P.; Mahmud, Nurul Ethika; Wahab, Wan Nor Amilah Wan Abdul; Jamil, Noor Izani Noor; Basir, Nurlida

2013-01-01

E-learning should be pedagogically-driven rather than technologically-driven. The objectives of this study are to develop an interactive learning system in bright-field microscopy technique in order to support students' achievement of their intended learning outcomes. An interactive learning system on bright-field microscopy technique was…

Tip-enhanced near-field optical microscopy

PubMed Central

Mauser, Nina; Hartschuh, Achim

2013-01-01

Tip-enhanced near-field optical microscopy (TENOM) is a scanning probe technique capable of providing a broad range of spectroscopic information on single objects and structured surfaces at nanometer spatial resolution and with highest detection sensitivity. In this review, we first illustrate the physical principle of TENOM that utilizes the antenna function of a sharp probe to efficiently couple light to excitations on nanometer length scales. We then discuss the antenna-induced enhancement of different optical sample responses including Raman scattering, fluorescence, generation of photocurrent and electroluminescence. Different experimental realizations are presented and several recent examples that demonstrate the capabilities of the technique are reviewed. PMID:24100541

Atomic force microscopy of lead iodide crystal surfaces

NASA Astrophysics Data System (ADS)

George, M. A.; Azoulay, M.; Jayatirtha, H. N.; Biao, Y.; Burger, A.; Collins, W. E.; Silberman, E.

1994-03-01

Atomic force microscopy (AFM) was used to characterize the surface of lead iodide crystals. The high vapor pressure of lead iodide prohibits the use of traditional high resolution surface study techniques that require high vacuum conditions. AFM was used to image numerous insulating surface in various ambients, with very little sample preparation techniques needed. Freshly cleaved and modified surfaces, including, chemical and vacuum etched, and air aged surfaces, were examined. Both intrinsic and induced defects were imaged with high resolution. The results were compared to a similar AFM study of mercuric iodide surfaces and it was found that, at ambient conditions, lead iodide is significantly more stable than mercuric iodide.

High-resolution high-sensitivity elemental imaging by secondary ion mass spectrometry: from traditional 2D and 3D imaging to correlative microscopy

NASA Astrophysics Data System (ADS)

Wirtz, T.; Philipp, P.; Audinot, J.-N.; Dowsett, D.; Eswara, S.

2015-10-01

Secondary ion mass spectrometry (SIMS) constitutes an extremely sensitive technique for imaging surfaces in 2D and 3D. Apart from its excellent sensitivity and high lateral resolution (50 nm on state-of-the-art SIMS instruments), advantages of SIMS include high dynamic range and the ability to differentiate between isotopes. This paper first reviews the underlying principles of SIMS as well as the performance and applications of 2D and 3D SIMS elemental imaging. The prospects for further improving the capabilities of SIMS imaging are discussed. The lateral resolution in SIMS imaging when using the microprobe mode is limited by (i) the ion probe size, which is dependent on the brightness of the primary ion source, the quality of the optics of the primary ion column and the electric fields in the near sample region used to extract secondary ions; (ii) the sensitivity of the analysis as a reasonable secondary ion signal, which must be detected from very tiny voxel sizes and thus from a very limited number of sputtered atoms; and (iii) the physical dimensions of the collision cascade determining the origin of the sputtered ions with respect to the impact site of the incident primary ion probe. One interesting prospect is the use of SIMS-based correlative microscopy. In this approach SIMS is combined with various high-resolution microscopy techniques, so that elemental/chemical information at the highest sensitivity can be obtained with SIMS, while excellent spatial resolution is provided by overlaying the SIMS images with high-resolution images obtained by these microscopy techniques. Examples of this approach are given by presenting in situ combinations of SIMS with transmission electron microscopy (TEM), helium ion microscopy (HIM) and scanning probe microscopy (SPM).

Integrated photoacoustic microscopy, optical coherence tomography, and fluorescence microscopy for multimodal chorioretinal imaging

NASA Astrophysics Data System (ADS)

Tian, Chao; Zhang, Wei; Nguyen, Van Phuc; Huang, Ziyi; Wang, Xueding; Paulus, Yannis M.

2018-02-01

Current clinical available retinal imaging techniques have limitations, including limited depth of penetration or requirement for the invasive injection of exogenous contrast agents. Here, we developed a novel multimodal imaging system for high-speed, high-resolution retinal imaging of larger animals, such as rabbits. The system integrates three state-of-the-art imaging modalities, including photoacoustic microscopy (PAM), optical coherence tomography (OCT), and fluorescence microscopy (FM). In vivo experimental results of rabbit eyes show that the PAM is able to visualize laser-induced retinal burns and distinguish individual eye blood vessels using a laser exposure dose of 80 nJ, which is well below the American National Standards Institute (ANSI) safety limit 160 nJ. The OCT can discern different retinal layers and visualize laser burns and choroidal detachments. The novel multi-modal imaging platform holds great promise in ophthalmic imaging.

Flow Microscopy Imaging Is Sensitive to Characteristics of Subvisible Particles in Peginesatide Formulations Associated With Severe Adverse Reactions.

PubMed

Daniels, Austin L; Randolph, Theodore W

2018-05-01

The presence of subvisible particles in formulations of therapeutic proteins is a risk factor for adverse immune responses. Although the immunogenic potential of particulate contaminants likely depends on particle structural characteristics (e.g., composition, size, and shape), exact structure-immunogenicity relationships are unknown. Images recorded by flow imaging microscopy reflect information about particle morphology, but flow microscopy is typically used to determine only particle size distributions, neglecting information on particle morphological features that may be immunologically relevant. We recently developed computational techniques that utilize the Kullback-Leibler divergence and multidimensional scaling to compare the morphological properties of particles in sets of flow microscopy images. In the current work, we combined these techniques with expectation maximization cluster analyses and used them to compare flow imaging microscopy data sets that had been collected by the U.S. Food and Drug Administration after severe adverse drug reactions (including 7 fatalities) were observed in patients who had been administered some lots of peginesatide formulations. Flow microscopy images of particle populations found in the peginesatide lots associated with severe adverse reactions in patients were readily distinguishable from images of particles in lots where severe adverse reactions did not occur. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Ex vivo confocal microscopy: an emerging technique in dermatology

PubMed Central

Perrot, Jean Luc; Labeille, Bruno; Cambazard, Frédéric; Rubegni, Pietro

2018-01-01

This review aims to give an overview of the current available applications of ex vivo confocal microscopy (EVCM) in dermatology. EVCM is a relatively new imaging technique that allows microscopic examination of freshly excised unfixed tissue. It enables a rapid examination of the skin sample directly in the surgery room and thus represents an alternative to the intraoperative micrographic control of the surgical margins of cutaneous tumors by standard microscopic examination on cryopreserved sections during Mohs surgery. Although this technique has mainly been developed for the margin’s control of basal cell carcinoma, many other skin tumors have been studied, including melanoma. Use of EVCM is continuing to evolve, and many possible applications are under investigation, such as the study of nails and hair diseases and the diagnosis of skin infections. PMID:29785327

Recent Advances in Nanodisc Technology for Membrane Proteins Studies (2012–2017)

PubMed Central

Rouck, John; Krapf, John; Roy, Jahnabi; Huff, Hannah; Das, Aditi

2017-01-01

Historically, progress in membrane protein research has been hindered due to solubility issues. The introduction of biomembrane mimetics has since stimulated the field’s momentum. One mimetic, the nanodisc, has proved to be an exceptional system for solubilizing membrane proteins. Herein, we critically evaluate the advantages and imperfections from employing nanodiscs in biophysical and biochemical studies. Specifically, we examine the techniques that have been modified to study membrane proteins in nanodiscs. Techniques discussed include fluorescence microscopy, solution state/solid state NMR, electron microscopy, SAXS, and several mass spectroscopy methods. Newer techniques such as SPR, charge sensitive optical detection, and scintillation proximity assays are also reviewed. Lastly, we cover nanodiscs advancing nanotechnology through nanoplasmonic biosensing, lipoprotein-nanoplatelets, and sortase-mediated labeling of nanodiscs. PMID:28581067

Recent advances in Lorentz microscopy

DOE PAGES

Phatak, C.; Petford-Long, A. K.; De Graef, M.

2016-01-05

Lorentz transmission electron microscopy (LTEM) has evolved from a qualitative magnetic domain observation technique to a quantitative technique for the determination of the magnetization state of a sample. Here, we describe recent developments in techniques and imaging modes, including the use of spherical aberration correction to improve the spatial resolution of LTEM into the single nanometer range, and novel in situ observation modes. We also review recent advances in the modeling of the wave optical magnetic phase shift as well as in the area of phase reconstruction by means of the Transport of Intensity Equation (TIE) approach, and discuss vectormore » field electron tomography, which has emerged as a powerful tool for the 3D reconstruction of magnetization configurations. Finally, we conclude this review with a brief overview of recent LTEM applications.« less

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

NASA Astrophysics Data System (ADS)

Cerbino, Roberto; Cicuta, Pietro

2017-09-01

Differential dynamic microscopy (DDM) is a technique that exploits optical microscopy to obtain local, multi-scale quantitative information about dynamic samples, in most cases without user intervention. It is proving extremely useful in understanding dynamics in liquid suspensions, soft materials, cells, and tissues. In DDM, image sequences are analyzed via a combination of image differences and spatial Fourier transforms to obtain information equivalent to that obtained by means of light scattering techniques. Compared to light scattering, DDM offers obvious advantages, principally (a) simplicity of the setup; (b) possibility of removing static contributions along the optical path; (c) power of simultaneous different microscopy contrast mechanisms; and (d) flexibility of choosing an analysis region, analogous to a scattering volume. For many questions, DDM has also advantages compared to segmentation/tracking approaches and to correlation techniques like particle image velocimetry. The very straightforward DDM approach, originally demonstrated with bright field microscopy of aqueous colloids, has lately been used to probe a variety of other complex fluids and biological systems with many different imaging methods, including dark-field, differential interference contrast, wide-field, light-sheet, and confocal microscopy. The number of adopting groups is rapidly increasing and so are the applications. Here, we briefly recall the working principles of DDM, we highlight its advantages and limitations, we outline recent experimental breakthroughs, and we provide a perspective on future challenges and directions. DDM can become a standard primary tool in every laboratory equipped with a microscope, at the very least as a first bias-free automated evaluation of the dynamics in a system.

Microsphere-aided optical microscopy and its applications for super-resolution imaging

NASA Astrophysics Data System (ADS)

Upputuri, Paul Kumar; Pramanik, Manojit

2017-12-01

The spatial resolution of a standard optical microscope (SOM) is limited by diffraction. In visible spectrum, SOM can provide ∼ 200 nm resolution. To break the diffraction limit several approaches were developed including scanning near field microscopy, metamaterial super-lenses, nanoscale solid immersion lenses, super-oscillatory lenses, confocal fluorescence microscopy, techniques that exploit non-linear response of fluorophores like stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, etc. Recently, photonic nanojet generated by a dielectric microsphere was used to break the diffraction limit. The microsphere-approach is simple, cost-effective and can be implemented under a standard microscope, hence it has gained enormous attention for super-resolution imaging. In this article, we briefly review the microsphere approach and its applications for super-resolution imaging in various optical imaging modalities.

Enhancing multi-spot structured illumination microscopy with fluorescence difference

NASA Astrophysics Data System (ADS)

Ward, Edward N.; Torkelsen, Frida H.; Pal, Robert

2018-03-01

Structured illumination microscopy is a super-resolution technique used extensively in biological research. However, this technique is limited in the maximum possible resolution increase. Here we report the results of simulations of a novel enhanced multi-spot structured illumination technique. This method combines the super-resolution technique of difference microscopy with structured illumination deconvolution. Initial results give at minimum a 1.4-fold increase in resolution over conventional structured illumination in a low-noise environment. This new technique also has the potential to be expanded to further enhance axial resolution with three-dimensional difference microscopy. The requirement for precise pattern determination in this technique also led to the development of a new pattern estimation algorithm which proved more efficient and reliable than other methods tested.

Atomic force microscopy of biological samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doktycz, Mitchel John

2010-01-01

The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate howmore » this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).« less

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

PubMed

Schaudinn, C; Carr, G; Gorur, A; Jaramillo, D; Costerton, J W; Webster, P

2009-08-01

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

Photocontrollable Fluorescent Proteins for Superresolution Imaging

PubMed Central

Shcherbakova, Daria M.; Sengupta, Prabuddha; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V.

2014-01-01

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization–based and nonlinear ensemble–based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine. PMID:24895855

Advances in imaging and quantification of electrical properties at the nanoscale using Scanning Microwave Impedance Microscopy (sMIM)

NASA Astrophysics Data System (ADS)

Friedman, Stuart; Yang, Yongliang; Amster, Oskar

2015-03-01

Scanning Microwave Impedance Microscopy (sMIM) is a mode for Atomic Force Microscopy (AFM) enabling imaging of unique contrast mechanisms and measurement of local permittivity and conductivity at the 10's of nm length scale. Recent results will be presented illustrating high-resolution electrical features such as sub 15 nm Moire' patterns in Graphene, carbon nanotubes of various electrical states and ferro-electrics. In addition to imaging, the technique is suited to a variety of metrology applications where specific physical properties are determined quantitatively. We will present research activities on quantitative measurements using multiple techniques to determine dielectric constant (permittivity) and conductivity (e.g. dopant concentration) for a range of materials. Examples include bulk dielectrics, low-k dielectric thin films, capacitance standards and doped semiconductors. Funded in part by DOE SBIR DE-SC0009586.

Plant cell wall characterization using scanning probe microscopy techniques

PubMed Central

Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

2009-01-01

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

New Diagnostic Aides for Melanoma

PubMed Central

Ferris, Laura K.; Harris, Ryan J.

2012-01-01

Synopsis Detection of melanoma at an early stage is crucial to improving survival rates in melanoma. Accurate diagnosis by current techniques including dermatoscopy remains difficult, and new tools are needed to improve our diagnostic abilities. This article discusses recent advances in diagnostic techniques including confocal scanning laser microscopy, MelaFind, Siascopy, noninvasive genomic detection, as well as other future possibilities to aid in diagnosing melanoma. Advantages and barriers to implementation of the various technologies are discussed as well. PMID:22800557

Enhancing multi-spot structured illumination microscopy with fluorescence difference

PubMed Central

Torkelsen, Frida H.

2018-01-01

Structured illumination microscopy is a super-resolution technique used extensively in biological research. However, this technique is limited in the maximum possible resolution increase. Here we report the results of simulations of a novel enhanced multi-spot structured illumination technique. This method combines the super-resolution technique of difference microscopy with structured illumination deconvolution. Initial results give at minimum a 1.4-fold increase in resolution over conventional structured illumination in a low-noise environment. This new technique also has the potential to be expanded to further enhance axial resolution with three-dimensional difference microscopy. The requirement for precise pattern determination in this technique also led to the development of a new pattern estimation algorithm which proved more efficient and reliable than other methods tested. PMID:29657751

Laboratory Techniques for the Blind

ERIC Educational Resources Information Center

Tombaugh, Dorothy

1972-01-01

Describes modifications of laboratory procedures for the BSCS Green Version biology, including dissection, microbiology, animal behavior, physiology, biochemistry, and genetics that make the methods suitable for direct experimentation by blind students. Discusses models as substitutes for microscopy. (AL)

Combining single-molecule manipulation and single-molecule detection.

PubMed

Cordova, Juan Carlos; Das, Dibyendu Kumar; Manning, Harris W; Lang, Matthew J

2014-10-01

Single molecule force manipulation combined with fluorescence techniques offers much promise in revealing mechanistic details of biomolecular machinery. Here, we review force-fluorescence microscopy, which combines the best features of manipulation and detection techniques. Three of the mainstay manipulation methods (optical traps, magnetic traps and atomic force microscopy) are discussed with respect to milestones in combination developments, in addition to highlight recent contributions to the field. An overview of additional strategies is discussed, including fluorescence based force sensors for force measurement in vivo. Armed with recent exciting demonstrations of this technology, the field of combined single-molecule manipulation and single-molecule detection is poised to provide unprecedented views of molecular machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transport in Nanoporous Materials Including MOFs: The Applicability of Fick's Laws.

PubMed

Titze, Tobias; Lauerer, Alexander; Heinke, Lars; Chmelik, Christian; Zimmermann, Nils E R; Keil, Frerich J; Ruthven, Douglas M; Kärger, Jörg

2015-11-23

Diffusion in nanoporous host-guest systems is often considered to be too complicated to comply with such "simple" relationships as Fick's first and second law of diffusion. However, it is shown herein that the microscopic techniques of diffusion measurement, notably the pulsed field gradient (PFG) technique of NMR spectroscopy and microimaging by interference microscopy (IFM) and IR microscopy (IRM), provide direct experimental evidence of the applicability of Fick's laws to such systems. This remains true in many situations, even when the detailed mechanism is complex. The limitations of the diffusion model are also discussed with reference to the extensive literature on this subject. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biogenicity and Syngeneity of Organic Matter in Ancient Sedimentary Rocks: Recent Advances in the Search for Evidence of Past Life

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oehler, Dorothy Z.; Cady, Sherry L.

2014-12-01

he past decade has seen an explosion of new technologies for assessment of biogenicity and syngeneity of carbonaceous material within sedimentary rocks. Advances have been made in techniques for analysis of in situ organic matter as well as for extracted bulk samples of soluble and insoluble (kerogen) organic fractions. The in situ techniques allow analysis of micrometer-to-sub-micrometer-scale organic residues within their host rocks and include Raman and fluorescence spectroscopy/imagery, confocal laser scanning microscopy, and forms of secondary ion/laser-based mass spectrometry, analytical transmission electron microscopy, and X-ray absorption microscopy/spectroscopy. Analyses can be made for chemical, molecular, and isotopic composition coupled withmore » assessment of spatial relationships to surrounding minerals, veins, and fractures. The bulk analyses include improved methods for minimizing contamination and recognizing syngenetic constituents of soluble organic fractions as well as enhanced spectroscopic and pyrolytic techniques for unlocking syngenetic molecular signatures in kerogen. Together, these technologies provide vital tools for the study of some of the oldest and problematic carbonaceous residues and for advancing our understanding of the earliest stages of biological evolution on Earth and the search for evidence of life beyond Earth. We discuss each of these new technologies, emphasizing their advantages and disadvantages, applications, and likely future directions.« less

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

PubMed

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

An Improved Fungal Mounting Technique for Nomarski Microscopy.

ERIC Educational Resources Information Center

Fairclough, Andrew; And Others

1985-01-01

Conventional sellotape techniques for fungal mounting produce interference patterns when using Normarsky microscopy. A technique is described which overcomes this problem and produces a permanent mount with a completely clear background. (Author/JN)

Qualitative and quantitative interpretation of SEM image using digital image processing.

PubMed

Saladra, Dawid; Kopernik, Magdalena

2016-10-01

The aim of the this study is improvement of qualitative and quantitative analysis of scanning electron microscope micrographs by development of computer program, which enables automatic crack analysis of scanning electron microscopy (SEM) micrographs. Micromechanical tests of pneumatic ventricular assist devices result in a large number of micrographs. Therefore, the analysis must be automatic. Tests for athrombogenic titanium nitride/gold coatings deposited on polymeric substrates (Bionate II) are performed. These tests include microshear, microtension and fatigue analysis. Anisotropic surface defects observed in the SEM micrographs require support for qualitative and quantitative interpretation. Improvement of qualitative analysis of scanning electron microscope images was achieved by a set of computational tools that includes binarization, simplified expanding, expanding, simple image statistic thresholding, the filters Laplacian 1, and Laplacian 2, Otsu and reverse binarization. Several modifications of the known image processing techniques and combinations of the selected image processing techniques were applied. The introduced quantitative analysis of digital scanning electron microscope images enables computation of stereological parameters such as area, crack angle, crack length, and total crack length per unit area. This study also compares the functionality of the developed computer program of digital image processing with existing applications. The described pre- and postprocessing may be helpful in scanning electron microscopy and transmission electron microscopy surface investigations. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Size characterization of airborne SiO2 nanoparticles with on-line and off-line measurement techniques: an interlaboratory comparison study

NASA Astrophysics Data System (ADS)

Motzkus, C.; Macé, T.; Gaie-Levrel, F.; Ducourtieux, S.; Delvallee, A.; Dirscherl, K.; Hodoroaba, V.-D.; Popov, I.; Popov, O.; Kuselman, I.; Takahata, K.; Ehara, K.; Ausset, P.; Maillé, M.; Michielsen, N.; Bondiguel, S.; Gensdarmes, F.; Morawska, L.; Johnson, G. R.; Faghihi, E. M.; Kim, C. S.; Kim, Y. H.; Chu, M. C.; Guardado, J. A.; Salas, A.; Capannelli, G.; Costa, C.; Bostrom, T.; Jämting, Å. K.; Lawn, M. A.; Adlem, L.; Vaslin-Reimann, S.

2013-10-01

Results of an interlaboratory comparison on size characterization of SiO2 airborne nanoparticles using on-line and off-line measurement techniques are discussed. This study was performed in the framework of Technical Working Area (TWA) 34—"Properties of Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS) in the project no. 3 "Techniques for characterizing size distribution of airborne nanoparticles". Two types of nano-aerosols, consisting of (1) one population of nanoparticles with a mean diameter between 30.3 and 39.0 nm and (2) two populations of non-agglomerated nanoparticles with mean diameters between, respectively, 36.2-46.6 nm and 80.2-89.8 nm, were generated for characterization measurements. Scanning mobility particle size spectrometers (SMPS) were used for on-line measurements of size distributions of the produced nano-aerosols. Transmission electron microscopy, scanning electron microscopy, and atomic force microscopy were used as off-line measurement techniques for nanoparticles characterization. Samples were deposited on appropriate supports such as grids, filters, and mica plates by electrostatic precipitation and a filtration technique using SMPS controlled generation upstream. The results of the main size distribution parameters (mean and mode diameters), obtained from several laboratories, were compared based on metrological approaches including metrological traceability, calibration, and evaluation of the measurement uncertainty. Internationally harmonized measurement procedures for airborne SiO2 nanoparticles characterization are proposed.

The 2015 super-resolution microscopy roadmap

NASA Astrophysics Data System (ADS)

Hell, Stefan W.; Sahl, Steffen J.; Bates, Mark; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J.; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Jakobs, Stefan; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J.; Eggeling, Christian; Klenerman, David; Willig, Katrin I.; Vicidomini, Giuseppe; Castello, Marco; Diaspro, Alberto; Cordes, Thorben

2015-11-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling

Back to the Future - Part 1. The medico-legal autopsy from ancient civilization to the post-genomic era.

PubMed

Cecchetto, Giovanni; Bajanowski, Thomas; Cecchi, Rossana; Favretto, Donata; Grabherr, Silke; Ishikawa, Takaki; Kondo, Toshikazu; Montisci, Massimo; Pfeiffer, Heidi; Bonati, Maurizio Rippa; Shokry, Dina; Vennemann, Marielle; Ferrara, Santo Davide

2017-07-01

Part 1 of the review "Back to the Future" examines the historical evolution of the medico-legal autopsy and microscopy techniques, from Ancient Civilization to the Post-Genomic Era. In the section focusing on "The Past", the study of historical sources concerning the origins and development of the medico-legal autopsy, from the Bronze Age until the Middle Ages, shows how, as early as 2000 BC, the performance of autopsies for medico-legal purposes was a known and widespread practice in some ancient civilizations in Egypt, the Far East and later in Europe. In the section focusing on "The Present", the improvement of autopsy techniques by Friedrich Albert Zenker and Rudolf Virchow and the contemporary development of optical microscopy techniques for forensic purposes during the 19th and 20th centuries are reported, emphasizing, the regulation of medico-legal autopsies in diverse nations around the world and the publication of international guidelines or best practices elaborated by International Scientific Societies. Finally, in "The Future" section, innovative robotized and advanced microscopy systems and techniques, including their possible use in the bio-medicolegal field, are reported, which should lead to the improvement and standardization of the autopsy methodology, thereby achieving a more precise identification of natural and traumatic pathologies.

Dictionary of Microscopy

NASA Astrophysics Data System (ADS)

Heath, Julian

2005-10-01

The past decade has seen huge advances in the application of microscopy in all areas of science. This welcome development in microscopy has been paralleled by an expansion of the vocabulary of technical terms used in microscopy: terms have been coined for new instruments and techniques and, as microscopes reach even higher resolution, the use of terms that relate to the optical and physical principles underpinning microscopy is now commonplace. The Dictionary of Microscopy was compiled to meet this challenge and provides concise definitions of over 2,500 terms used in the fields of light microscopy, electron microscopy, scanning probe microscopy, x-ray microscopy and related techniques. Written by Dr Julian P. Heath, Editor of Microscopy and Analysis, the dictionary is intended to provide easy navigation through the microscopy terminology and to be a first point of reference for definitions of new and established terms. The Dictionary of Microscopy is an essential, accessible resource for: students who are new to the field and are learning about microscopes equipment purchasers who want an explanation of the terms used in manufacturers' literature scientists who are considering using a new microscopical technique experienced microscopists as an aide mémoire or quick source of reference librarians, the press and marketing personnel who require definitions for technical reports.

High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

NASA Astrophysics Data System (ADS)

Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

NASA Astrophysics Data System (ADS)

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-08-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium-oxygen, lithium-sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

PubMed Central

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-01-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium–oxygen, lithium–sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Pulsed-Plasma Physical Vapor Deposition Approach Toward the Facile Synthesis of Multilayer and Monolayer Graphene for Anticoagulation Applications.

PubMed

Vijayaraghavan, Rajani K; Gaman, Cezar; Jose, Bincy; McCoy, Anthony P; Cafolla, Tony; McNally, Patrick J; Daniels, Stephen

2016-02-01

We demonstrate the growth of multilayer and single-layer graphene on copper foil using bipolar pulsed direct current (DC) magnetron sputtering of a graphite target in pure argon atmosphere. Single-layer graphene (SG) and few-layer graphene (FLG) films are deposited at temperatures ranging from 700 °C to 920 °C within <30 min. We find that the deposition and post-deposition annealing temperatures influence the layer thickness and quality of the graphene films formed. The films were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and optical transmission spectroscopy techniques. Based on the above studies, a diffusion-controlled mechanism was proposed for the graphene growth. A single-step whole blood assay was used to investigate the anticoagulant activity of graphene surfaces. Platelet adhesion, activation, and morphological changes on the graphene/glass surfaces, compared to bare glass, were analyzed using fluorescence microscopy and SEM techniques. We have found significant suppression of the platelet adhesion, activation, and aggregation on the graphene-covered surfaces, compared to the bare glass, indicating the anticoagulant activity of the deposited graphene films. Our production technique represents an industrially relevant method for the growth of SG and FLG for various applications including the biomedical field.

DISTRIBUTION SYSTEM SOLIDS - A RESEARCH APPROACH

EPA Science Inventory

The U.S. EPA's AWBERC research facility is equipped with capabilities to analyze a variety of solids in support many Laboratory-wide research studies. Techniques available on site include X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microsco...

Techniques for 3D tracking of single molecules with nanometer accuracy in living cells

NASA Astrophysics Data System (ADS)

Gardini, Lucia; Capitanio, Marco; Pavone, Francesco S.

2013-06-01

We describe a microscopy technique that, combining wide-field single molecule microscopy, bifocal imaging and Highly Inclined and Laminated Optical sheet (HILO) microscopy, allows a 3D tracking with nanometer accuracy of single fluorescent molecules in vitro and in living cells.

Surface Characterization.

ERIC Educational Resources Information Center

Fulghum, J. E.; And Others

1989-01-01

This review is divided into the following analytical methods: ion spectroscopy, electron spectroscopy, scanning tunneling microscopy, atomic force microscopy, optical spectroscopy, desorption techniques, and X-ray techniques. (MVL)

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes.

PubMed

Ishmukhametov, Robert R; Russell, Aidan N; Wheeler, Richard J; Nord, Ashley L; Berry, Richard M

2016-02-08

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Friction force microscopy: a simple technique for identifying graphene on rough substrates and mapping the orientation of graphene grains on copper

NASA Astrophysics Data System (ADS)

Marsden, A. J.; Phillips, M.; Wilson, N. R.

2013-06-01

At a single atom thick, it is challenging to distinguish graphene from its substrate using conventional techniques. In this paper we show that friction force microscopy (FFM) is a simple and quick technique for identifying graphene on a range of samples, from growth substrates to rough insulators. We show that FFM is particularly effective for characterizing graphene grown on copper where it can correlate the graphene growth to the three-dimensional surface topography. Atomic lattice stick-slip friction is readily resolved and enables the crystallographic orientation of the graphene to be mapped nondestructively, reproducibly and at high resolution. We expect FFM to be similarly effective for studying graphene growth on other metal/locally crystalline substrates, including SiC, and for studying growth of other two-dimensional materials such as molybdenum disulfide and hexagonal boron nitride.

A Simple low-cost device enables four epi-illumination techniques on standard light microscopes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Wheeler, Richard J.; Nord, Ashley L.; Berry, Richard M.

2016-02-01

Back-scattering darkfield (BSDF), epi-fluorescence (EF), interference reflection contrast (IRC), and darkfield surface reflection (DFSR) are advanced but expensive light microscopy techniques with limited availability. Here we show a simple optical design that combines these four techniques in a simple low-cost miniature epi-illuminator, which inserts into the differential interference-contrast (DIC) slider bay of a commercial microscope, without further additions required. We demonstrate with this device: 1) BSDF-based detection of Malarial parasites inside unstained human erythrocytes; 2) EF imaging with and without dichroic components, including detection of DAPI-stained Leishmania parasite without using excitation or emission filters; 3) RIC of black lipid membranes and other thin films, and 4) DFSR of patterned opaque and transparent surfaces. We believe that our design can expand the functionality of commercial bright field microscopes, provide easy field detection of parasites and be of interest to many users of light microscopy.

Preparation of herpes simplex virus-infected primary neurons for transmission electron microscopy.

PubMed

Miranda-Saksena, Monica; Boadle, Ross; Cunningham, Anthony L

2014-01-01

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction. Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus-cell interaction.

Rapid, High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy

PubMed Central

Cheong, Fook Chiong; Wong, Chui Ching; Gao, YunFeng; Nai, Mui Hoon; Cui, Yidan; Park, Sungsu; Kenney, Linda J.; Lim, Chwee Teck

2015-01-01

Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility. PMID:25762336

Physical characterization of uranium oxide pellets and powder applied in the Nuclear Forensics International Technical Working Group Collaborative Materials Exercise 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffiths, Grant; Keegan, E.; Young, E.

Physical characterization is one of the most broad and important categories of techniques to apply in a nuclear forensic examination. Physical characterization techniques vary from simple weighing and dimensional measurements to complex sample preparation and scanning electron microscopy-electron backscatter diffraction analysis. This paper reports on the physical characterization conducted by several international laboratories participating in the fourth Collaborative Materials Exercise, organized by the Nuclear Forensics International Technical Working Group. Methods include a range of physical measurements, microscopy-based observations, and profilometry. In conclusion, the value of these results for addressing key investigative questions concerning two uranium dioxide pellets and a uraniummore » dioxide powder is discussed.« less

Physical characterization of uranium oxide pellets and powder applied in the Nuclear Forensics International Technical Working Group Collaborative Materials Exercise 4

DOE PAGES

Griffiths, Grant; Keegan, E.; Young, E.; ...

2018-01-06

Physical characterization is one of the most broad and important categories of techniques to apply in a nuclear forensic examination. Physical characterization techniques vary from simple weighing and dimensional measurements to complex sample preparation and scanning electron microscopy-electron backscatter diffraction analysis. This paper reports on the physical characterization conducted by several international laboratories participating in the fourth Collaborative Materials Exercise, organized by the Nuclear Forensics International Technical Working Group. Methods include a range of physical measurements, microscopy-based observations, and profilometry. In conclusion, the value of these results for addressing key investigative questions concerning two uranium dioxide pellets and a uraniummore » dioxide powder is discussed.« less

Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging.

PubMed

Winterflood, Christian M; Platonova, Evgenia; Albrecht, David; Ewers, Helge

2015-07-07

Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants.

PubMed

Moore, Katie L; Lombi, Enzo; Zhao, Fang-Jie; Grovenor, Chris R M

2012-04-01

The ability to locate and quantify elemental distributions in plants is crucial to understanding plant metabolisms, the mechanisms of uptake and transport of minerals and how plants cope with toxic elements or elemental deficiencies. High-resolution secondary ion mass spectrometry (SIMS) is emerging as an important technique for the analysis of biological material at the subcellular scale. This article reviews recent work using the CAMECA NanoSIMS to determine elemental distributions in plants. The NanoSIMS is able to map elemental distributions at high resolution, down to 50 nm, and can detect very low concentrations (milligrams per kilogram) for some elements. It is also capable of mapping almost all elements in the periodic table (from hydrogen to uranium) and can distinguish between stable isotopes, which allows the design of tracer experiments. In this review, particular focus is placed upon studying the same or similar specimens with both the NanoSIMS and a wide range of complementary techniques, showing how the advantages of each technique can be combined to provide a fuller data set to address complex scientific questions. Techniques covered include optical microscopy, synchrotron techniques, including X-ray fluorescence and X-ray absorption spectroscopy, transmission electron microscopy, electron probe microanalysis, particle-induced X-ray emission and inductively coupled plasma mass spectrometry. Some of the challenges associated with sample preparation of plant material for SIMS analysis, the artefacts and limitations of the technique and future trends are also discussed.

Stress corrosion in titanium alloys and other metallic materials

NASA Technical Reports Server (NTRS)

Harkins, C. G. (Editor); Brotzen, F. R.; Hightower, J. W.; Mclellan, R. B.; Roberts, J. M.; Rudee, M. L.; Leith, I. R.; Basu, P. K.; Salama, K.; Parris, D. P.

1971-01-01

Multiple physical and chemical techniques including mass spectroscopy, atomic absorption spectroscopy, gas chromatography, electron microscopy, optical microscopy, electronic spectroscopy for chemical analysis (ESCA), infrared spectroscopy, nuclear magnetic resonance (NMR), X-ray analysis, conductivity, and isotopic labeling were used in investigating the atomic interactions between organic environments and titanium and titanium oxide surfaces. Key anhydrous environments studied included alcohols, which contain hydrogen; carbon tetrachloride, which does not contain hydrogen; and mixtures of alcohols and halocarbons. Effects of dissolved salts in alcohols were also studied. This program emphasized experiments designed to delineate the conditions necessary rather than sufficient for initiation processes and for propagation processes in Ti SCC.

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques.

PubMed

Plascencia-Villa, Germán; Starr, Clarise R; Armstrong, Linda S; Ponce, Arturo; José-Yacamán, Miguel

2012-11-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO(2), TiO(2) and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO(2) and TiO(2), whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy

PubMed Central

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin

2016-01-01

Objectives We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. Methods We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. Results An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. Conclusions The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis. PMID:27525165

All-optical optoacoustic microscopy based on probe beam deflection technique.

PubMed

Maswadi, Saher M; Ibey, Bennett L; Roth, Caleb C; Tsyboulski, Dmitri A; Beier, Hope T; Glickman, Randolph D; Oraevsky, Alexander A

2016-09-01

Optoacoustic (OA) microscopy using an all-optical system based on the probe beam deflection technique (PBDT) for detection of laser-induced acoustic signals was investigated as an alternative to conventional piezoelectric transducers. PBDT provides a number of advantages for OA microscopy including (i) efficient coupling of laser excitation energy to the samples being imaged through the probing laser beam, (ii) undistorted coupling of acoustic waves to the detector without the need for separation of the optical and acoustic paths, (iii) high sensitivity and (iv) ultrawide bandwidth. Because of the unimpeded optical path in PBDT, diffraction-limited lateral resolution can be readily achieved. The sensitivity of the current PBDT sensor of 22 μV/Pa and its noise equivalent pressure (NEP) of 11.4 Pa are comparable with these parameters of the optical micro-ring resonator and commercial piezoelectric ultrasonic transducers. Benefits of the present prototype OA microscope were demonstrated by successfully resolving micron-size details in histological sections of cardiac muscle.

High-resolution light-sheet microscopy: a simulation of an optical illumination system for oil immersion

NASA Astrophysics Data System (ADS)

Lu, Xiang; Heintzmann, Rainer; Leischner, Ulrich

2015-09-01

Light sheet microscopy is a microscopy technique characterized by an illumination from the side, perpendicular to the direction of observation. While this is often used and easy to implement for imaging samples with water-immersion, the application in combination with oil-immersion is less often used and requires a specific optimization. In this paper we present our design of a light-sheet illumination optical system with a ~1μm illumination thickness, a long working distance through the immersion oil, and including a focusing system allowing for moving the focus-spot of the lightsheet laterally through the field of view. This optical design allows for the acquisition of fluorescence images in 3D with isotropic resolution of below 1 micrometer of whole-mount samples with a size of ~1mm diameter. This technique enables high-resolution insights in the 3D structure of biological samples, e.g. for research of insect anatomy or for imaging of biopsies in medical diagnostics.

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

PubMed

Evans, Conor L; Potma, Eric O; Puoris'haag, Mehron; Côté, Daniel; Lin, Charles P; Xie, X Sunney

2005-11-15

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH(2) stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

Methods of Hematoxylin and Erosin Image Information Acquisition and Optimization in Confocal Microscopy.

PubMed

Yoon, Woong Bae; Kim, Hyunjin; Kim, Kwang Gi; Choi, Yongdoo; Chang, Hee Jin; Sohn, Dae Kyung

2016-07-01

We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.

Cocrystal screening of hydroxybenzamides with benzoic acid derivatives: a comparative study of thermal and solution-based methods.

PubMed

Manin, Alex N; Voronin, Alexander P; Drozd, Ksenia V; Manin, Nikolay G; Bauer-Brandl, Annette; Perlovich, German L

2014-12-18

The main problem occurring at the early stages of cocrystal search is the choice of an effective screening technique. Among the most popular techniques of obtaining cocrystals are crystallization from solution, crystallization from melt and solvent-drop grinding. This paper represents a comparative analysis of the following screening techniques: DSC cocrystal screening method, thermal microscopy and saturation temperature method. The efficiency of different techniques of cocrystal screening was checked in 18 systems. Benzamide and benzoic acid derivatives were chosen as model systems due to their ability to form acid-amide supramolecular heterosynthon. The screening has confirmed the formation of 6 new cocrystals. The screening by the saturation temperature method has the highest screen-out rate but the smallest range of application. DSC screening has a satisfactory accuracy and allows screening over a short time. Thermal microscopy is most efficient as an additional technique used to interpret ambiguous DSC screening results. The study also included an analysis of the influence of solvent type and component solubility on cocrystal formation. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by fluorescence microscopy.

PubMed

Alves, Wilber S; Manoel, Evelin A; Santos, Noemi S; Nunes, Rosane O; Domiciano, Giselli C; Soares, Marcia R

2017-04-01

Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microscopic Characterization of the Brazilian Giant Samba Virus.

PubMed

Schrad, Jason R; Young, Eric J; Abrahão, Jônatas S; Cortines, Juliana R; Parent, Kristin N

2017-02-14

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses .

Multiple speckle illumination for optical-resolution photoacoustic imaging

NASA Astrophysics Data System (ADS)

Poisson, Florian; Stasio, Nicolino; Moser, Christophe; Psaltis, Demetri; Bossy, Emmanuel

2017-03-01

Optical-resolution photoacoustic microscopy offers exquisite and specific contrast to optical absorption. Conventional approaches generally involves raster scanning a focused spot over the sample. Here, we demonstrate that a full-field illumination approach with multiple speckle illumination can also provide diffraction-limited optical-resolution photoacoustic images. Two different proof-of-concepts are demonstrated with micro-structured test samples. The first approach follows the principle of correlation/ghost imaging,1, 2 and is based on cross-correlating photoacoustic signals under multiple speckle illumination with known speckle patterns measured during a calibration step. The second approach is a speckle scanning microscopy technique, which adapts the technique proposed in fluorescence microscopy by Bertolotti and al.:3 in our work, spatially unresolved photoacoustic measurements are performed for various translations of unknown speckle patterns. A phase-retrieval algorithm is used to reconstruct the object from the knowledge of the modulus of its Fourier Transform yielded by the measurements. Because speckle patterns naturally appear in many various situations, including propagation through biological tissue or multi-mode fibers (for which focusing light is either very demanding if not impossible), speckle-illumination-based photoacoustic microscopy provides a powerful framework for the development of novel reconstruction approaches, well-suited to compressed sensing approaches.2

Microscopic Characterization of the Brazilian Giant Samba Virus

PubMed Central

Schrad, Jason R.; Young, Eric J.; Abrahão, Jônatas S.; Cortines, Juliana R.; Parent, Kristin N.

2017-01-01

Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses. PMID:28216551

Application of environmental scanning electron microscopy to determine biological surface structure.

PubMed

Kirk, S E; Skepper, J N; Donald, A M

2009-02-01

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Analytical Model of the Nonlinear Dynamics of Cantilever Tip-Sample Surface Interactions for Various Acoustic-Atomic Force Microscopies

NASA Technical Reports Server (NTRS)

Cantrell, John H., Jr.; Cantrell, Sean A.

2008-01-01

A comprehensive analytical model of the interaction of the cantilever tip of the atomic force microscope (AFM) with the sample surface is developed that accounts for the nonlinearity of the tip-surface interaction force. The interaction is modeled as a nonlinear spring coupled at opposite ends to linear springs representing cantilever and sample surface oscillators. The model leads to a pair of coupled nonlinear differential equations that are solved analytically using a standard iteration procedure. Solutions are obtained for the phase and amplitude signals generated by various acoustic-atomic force microscope (A-AFM) techniques including force modulation microscopy, atomic force acoustic microscopy, ultrasonic force microscopy, heterodyne force microscopy, resonant difference-frequency atomic force ultrasonic microscopy (RDF-AFUM), and the commonly used intermittent contact mode (TappingMode) generally available on AFMs. The solutions are used to obtain a quantitative measure of image contrast resulting from variations in the Young modulus of the sample for the amplitude and phase images generated by the A-AFM techniques. Application of the model to RDF-AFUM and intermittent soft contact phase images of LaRC-cp2 polyimide polymer is discussed. The model predicts variations in the Young modulus of the material of 24 percent from the RDF-AFUM image and 18 percent from the intermittent soft contact image. Both predictions are in good agreement with the literature value of 21 percent obtained from independent, macroscopic measurements of sheet polymer material.

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Superresolution Microscopy of the Nuclear Envelope and Associated Proteins.

PubMed

Xie, Wei; Horn, Henning F; Wright, Graham D

2016-01-01

Superresolution microscopy is undoubtedly one of the most exciting technologies since the invention of the optical microscope. Capable of nanometer-scale resolution to surpass the diffraction limit and coupled with the versatile labeling techniques available, it is revolutionizing the study of cell biology. Our understanding of the nucleus, the genetic and architectural center of the cell, has gained great advancements through the application of various superresolution microscopy techniques. This chapter describes detailed procedures of multichannel superresolution imaging of the mammalian nucleus, using structured illumination microscopy and single-molecule localization microscopy.

Live-cell stimulated Raman scattering imaging of alkyne-tagged biomolecules.

PubMed

Hong, Senlian; Chen, Tao; Zhu, Yuntao; Li, Ang; Huang, Yanyi; Chen, Xing

2014-06-02

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Circumventing photodamage in live-cell microscopy

PubMed Central

Magidson, Valentin; Khodjakov, Alexey

2013-01-01

Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

Noninvasive label-free monitoring of cosmetics and pharmaceuticals in human skin using nonlinear optical microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Osseiran, Sam; Wang, Hequn; Evans, Conor L.

2017-02-01

Over the past decade, nonlinear optical microscopy has seen a dramatic rise in its use in research settings due to its noninvasiveness, enhanced penetration depth, intrinsic optical sectioning, and the ability to probe chemical compounds with molecular specificity without exogenous contrast agents. Nonlinear optical techniques including two-photon excitation fluorescence (2PEF), fluorescence lifetime imaging microscopy (FLIM), second harmonic generation (SHG), coherent anti-Stokes and stimulated Raman scattering (CARS and SRS, respectively), as well as transient and sum frequency absorption (TA and SFA, respectively), have been widely used to explore the physiology and microanatomy of skin. Recently, these modalities have shed light on dermal processes that could not have otherwise been observed, including the spatiotemporal monitoring of cosmetics and pharmaceuticals. However, a challenge quickly arises when studying such chemicals in a dermatological context: many exogenous compounds have optical signatures that can interfere with the signals that would otherwise be acquired from intact skin. For example, oily solvents exhibit strong signals when probing CH2 vibrations with CARS/SRS; chemical sun filters appear bright in 2PEF microscopy; and darkly colored compounds readily absorb light across a broad spectrum, producing strong TA/SFA signals. Thus, this discussion will first focus on the molecular contrast in skin that can be probed using the aforementioned nonlinear optical techniques. This will be followed by an overview of strategies that take advantage of the exogenous compounds' optical signatures to probe spatiotemporal dynamics while preserving endogenous information from skin.

In vivo confocal microscopy of the cornea: New developments in image acquisition, reconstruction and analysis using the HRT-Rostock Corneal Module

PubMed Central

Petroll, W. Matthew; Robertson, Danielle M.

2015-01-01

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike. PMID:25998608

Chapter 14: Electron Microscopy on Thin Films for Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romero, Manuel; Abou-Ras, Daniel; Nichterwitz, Melanie

2016-07-22

This chapter overviews the various techniques applied in scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and highlights their possibilities and also limitations. It gives the various imaging and analysis techniques applied on a scanning electron microscope. The chapter shows that imaging is divided into that making use of secondary electrons (SEs) and of backscattered electrons (BSEs), resulting in different contrasts in the images and thus providing information on compositions, microstructures, and surface potentials. Whenever aiming for imaging and analyses at scales of down to the angstroms range, TEM and its related techniques are appropriate tools. In many cases,more » also SEM techniques provide the access to various material properties of the individual layers, not requiring specimen preparation as time consuming as TEM techniques. Finally, the chapter dedicates to cross-sectional specimen preparation for electron microscopy. The preparation decides indeed on the quality of imaging and analyses.« less

Microscopy basics and the study of actin-actin-binding protein interactions.

PubMed

Thomasson, Maggie S; Macnaughtan, Megan A

2013-12-15

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Reflectance Confocal Microscopy in Lentigo Maligna.

PubMed

Gamo, R; Pampín, A; Floristán, U

2016-12-01

Lentigo maligna is the most common type of facial melanoma. Diagnosis is complicated, however, as it shares clinical and dermoscopic characteristics with other cutaneous lesions of the face. Reflectance confocal microscopy is an imaging technique that permits the visualization of characteristic features of lentigo maligna. These include a disrupted honeycomb pattern and pagetoid cells with a tendency to show folliculotropism. These cells typically have a dendritic morphology, although they may also appear as round cells measuring over 20μm with atypical nuclei. Poorly defined dermal papillae and atypical cells may be seen at the dermal-epidermal junction and can form bridges resembling mitochondrial structures. Other characteristic findings include junctional swelling with atypical cells located around the follicles, resembling caput medusae. Reflectance confocal microscopy is a very useful tool for diagnosing lentigo maligna. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

Biomedical Applications of Nanodiamonds: An Overview.

PubMed

Passeri, D; Rinaldi, F; Ingallina, C; Carafa, M; Rossi, M; Terranova, M L; Marianecci, C

2015-02-01

Nanodiamonds are a novel class of nanomaterials which have raised much attention for application in biomedical field, as they combine the possibility of being produced on large scale using relatively inexpensive synthetic processes, of being fluorescent as a consequence of the presence of nitrogen vacancies, of having their surfaces functionalized, and of having good biocompatibility. Among other applications, we mainly focus on drug delivery, including cell interaction, targeting, cancer therapy, gene and protein delivery. In addition, nanodiamonds for bone and dental implants and for antibacterial use is discussed. Techniques for detection and imaging of nanodiamonds in biological tissues are also reviewed, including electron microscopy, fluorescence microscopy, Raman mapping, atomic force microscopy, thermal imaging, magnetic resonance imaging, and positron emission tomography, either in vitro, in vivo, or ex vivo. Toxicological aspects related to the use of nanodiamonds are also discussed. Finally, patents, preclinical and clinical trials based on the use of nanodiamonds for biomedical applications are reviewed.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Emerging optical nanoscopy techniques

PubMed Central

Montgomery, Paul C; Leong-Hoi, Audrey

2015-01-01

To face the challenges of modern health care, new imaging techniques with subcellular resolution or detection over wide fields are required. Far field optical nanoscopy presents many new solutions, providing high resolution or detection at high speed. We present a new classification scheme to help appreciate the growing number of optical nanoscopy techniques. We underline an important distinction between superresolution techniques that provide improved resolving power and nanodetection techniques for characterizing unresolved nanostructures. Some of the emerging techniques within these two categories are highlighted with applications in biophysics and medicine. Recent techniques employing wider angle imaging by digital holography and scattering lens microscopy allow superresolution to be achieved for subcellular and even in vivo, imaging without labeling. Nanodetection techniques are divided into four subcategories using contrast, phase, deconvolution, and nanomarkers. Contrast enhancement is illustrated by means of a polarized light-based technique and with strobed phase-contrast microscopy to reveal nanostructures. Very high sensitivity phase measurement using interference microscopy is shown to provide nanometric surface roughness measurement or to reveal internal nanometric structures. Finally, the use of nanomarkers is illustrated with stochastic fluorescence microscopy for mapping intracellular structures. We also present some of the future perspectives of optical nanoscopy. PMID:26491270

Atomic-scale mapping of electronic structures across heterointerfaces by cross-sectional scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Chiu, Ya-Ping; Huang, Bo-Chao; Shih, Min-Chuan; Huang, Po-Cheng; Chen, Chun-Wei

2015-09-01

Interfacial science has received much attention recently based on the development of state-of-the-art analytical tools that can create and manipulate the charge, spin, orbital, and lattice degrees of freedom at interfaces. Motivated by the importance of nanoscale interfacial science that governs device operation, we present a technique to probe the electronic characteristics of heterointerfaces with atomic resolution. In this work, the interfacial characteristics of heteroepitaxial structures are investigated and the fundamental mechanisms that pertain in these systems are elucidated through cross-sectional scanning tunneling microscopy (XSTM). The XSTM technique is employed here to directly observe epitaxial interfacial structures and probe local electronic properties with atomic-level capability. Scanning tunneling microscopy and spectroscopy experiments with atomic precision provide insight into the origin and spatial distribution of electronic properties across heterointerfaces. The first part of this report provides a brief description of the cleavage technique and spectroscopy analysis in XSTM measurements. The second part addresses interfacial electronic structures of several model heterostructures in current condensed matter research using XSTM. Topics to be discussed include high-κ‘s/III-V’s semiconductors, polymer heterojunctions, and complex oxide heterostructures, which are all material systems whose investigation using this technique is expected to benefit the research community. Finally, practical aspects and perspectives of using XSTM in interface science are presented.

Impact of virtual microscopy with conventional microscopy on student learning in dental histology.

PubMed

Hande, Alka Harish; Lohe, Vidya K; Chaudhary, Minal S; Gawande, Madhuri N; Patil, Swati K; Zade, Prajakta R

2017-01-01

In dental histology, the assimilation of histological features of different dental hard and soft tissues is done by conventional microscopy. This traditional method of learning prevents the students from screening the entire slide and change of magnification. To address these drawbacks, modification in conventional microscopy has evolved and become motivation for changing the learning tool. Virtual microscopy is the technique in which there is complete digitization of the microscopic glass slide, which can be analyzed on a computer. This research is designed to evaluate the effectiveness of virtual microscopy with conventional microscopy on student learning in dental histology. A cohort of 105 students were included and randomized into three groups: A, B, and C. Group A students studied the microscopic features of oral histologic lesions by conventional microscopy, Group B by virtual microscopy, and Group C by both conventional and virtual microscopy. The students' understanding of the subject was evaluated by a prepared questionnaire. The effectiveness of the study designs on knowledge gains and satisfaction levels was assessed by statistical assessment of differences in mean test scores. The difference in score between Groups A, B, and C at pre- and post-test was highly significant. This enhanced understanding of the subject may be due to benefits of using virtual microscopy in teaching histology. The augmentation of conventional microscopy with virtual microscopy shows enhancement of the understanding of the subject as compared to the use of conventional microscopy and virtual microscopy alone.

Fluorescence microscopy: A tool to study autophagy

NASA Astrophysics Data System (ADS)

Rai, Shashank; Manjithaya, Ravi

2015-08-01

Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

Unlocking the spatial inversion of large scanning magnetic microscopy datasets

NASA Astrophysics Data System (ADS)

Myre, J. M.; Lascu, I.; Andrade Lima, E.; Feinberg, J. M.; Saar, M. O.; Weiss, B. P.

2013-12-01

Modern scanning magnetic microscopy provides the ability to perform high-resolution, ultra-high sensitivity moment magnetometry, with spatial resolutions better than 10^-4 m and magnetic moments as weak as 10^-16 Am^2. These microscopy capabilities have enhanced numerous magnetic studies, including investigations of the paleointensity of the Earth's magnetic field, shock magnetization and demagnetization of impacts, magnetostratigraphy, the magnetic record in speleothems, and the records of ancient core dynamos of planetary bodies. A common component among many studies utilizing scanning magnetic microscopy is solving an inverse problem to determine the non-negative magnitude of the magnetic moments that produce the measured component of the magnetic field. The two most frequently used methods to solve this inverse problem are classic fast Fourier techniques in the frequency domain and non-negative least squares (NNLS) methods in the spatial domain. Although Fourier techniques are extremely fast, they typically violate non-negativity and it is difficult to implement constraints associated with the space domain. NNLS methods do not violate non-negativity, but have typically been computation time prohibitive for samples of practical size or resolution. Existing NNLS methods use multiple techniques to attain tractable computation. To reduce computation time in the past, typically sample size or scan resolution would have to be reduced. Similarly, multiple inversions of smaller sample subdivisions can be performed, although this frequently results in undesirable artifacts at subdivision boundaries. Dipole interactions can also be filtered to only compute interactions above a threshold which enables the use of sparse methods through artificial sparsity. To improve upon existing spatial domain techniques, we present the application of the TNT algorithm, named TNT as it is a "dynamite" non-negative least squares algorithm which enhances the performance and accuracy of spatial domain inversions. We show that the TNT algorithm reduces the execution time of spatial domain inversions from months to hours and that inverse solution accuracy is improved as the TNT algorithm naturally produces solutions with small norms. Using sIRM and NRM measures of multiple synthetic and natural samples we show that the capabilities of the TNT algorithm allow very large samples to be inverted without the need for alternative techniques to make the problems tractable. Ultimately, the TNT algorithm enables accurate spatial domain analysis of scanning magnetic microscopy data on an accelerated time scale that renders spatial domain analyses tractable for numerous studies, including searches for the best fit of unidirectional magnetization direction and high-resolution step-wise magnetization and demagnetization.

Microchemical Analysis Of Space Operation Debris

NASA Technical Reports Server (NTRS)

Cummings, Virginia J.; Kim, Hae Soo

1995-01-01

Report discusses techniques used in analyzing debris relative to space shuttle operations. Debris collected from space shuttle, expendable launch vehicles, payloads carried by space shuttle, and payloads carried by expendable launch vehicles. Optical microscopy, scanning electron microscopy with energy-dispersive spectrometry, analytical electron microscopy with wavelength-dispersive spectrometry, and X-ray diffraction chosen as techniques used in examining samples of debris.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Atomic oxygen effects on thin film space coatings studied by spectroscopic ellipsometry, atomic force microscopy, and laser light scattering

NASA Technical Reports Server (NTRS)

Synowicki, R. A.; Hale, Jeffrey S.; Woollam, John A.

1992-01-01

The University of Nebraska is currently evaluating Low Earth Orbit (LEO) simulation techniques as well as a variety of thin film protective coatings to withstand atomic oxygen (AO) degradation. Both oxygen plasma ashers and an electron cyclotron resonance (ECR) source are being used for LEO simulation. Thin film coatings are characterized by optical techniques including Variable Angle Spectroscopic Ellipsometry, Optical spectrophotometry, and laser light scatterometry. Atomic Force Microscopy (AFM) is also used to characterize surface morphology. Results on diamondlike carbon (DLC) films show that DLC degrades with simulated AO exposure at a rate comparable to Kapton polyimide. Since DLC is not as susceptible to environmental factors such as moisture absorption, it could potentially provide more accurate measurements of AO fluence on short space flights.

PVT Degradation Studies: Acoustic Diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dib, Gerges; Tucker, Brian J.; Kouzes, Richard T.

Under certain environmental conditions, polyvinyl toluene (PVT) plastic scintillator has been observed to undergo internal fogging. This document reports on a study of acoustic techniques to determine whether they can provide a diagnostic for the fogging of PVT. Different ultrasound techniques were employed for detecting the level of internal fogging in PVT, including wave velocity measurements, attenuation, nonlinear acoustics, and acoustic microscopy. The results indicate that there are linear relations between the wave velocity and wave attenuation with the level of internal fogging. The effects of fogging on ultrasound wave attenuation is further verified by acoustic microscopy imaging, where regionsmore » with fog in the specimen demonstration higher levels of attenuation compared to clear regions. Results from the nonlinear ultrasound measurements were inconclusive due to high sensitivities to transducer coupling and fixture variabilities.« less

Picoliter Drop-On-Demand Dispensing for Multiplex Liquid Cell Transmission Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patterson, Joseph P.; Parent, Lucas R.; Cantlon, Joshua

2016-05-03

Abstract Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights themore » great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.« less

Direct labeling and visualization of blood vessels with lipophilic carbocyanine dye DiI

PubMed Central

Li, Yiwen; Song, Ying; Zhao, Lian; Gaidosh, Gabriel; Laties, Alan M; Wen, Rong

2009-01-01

We describe a protocol to rapidly and reliably visualize blood vessels in experimental animals. Blood vessels are directly labeled by cardiac perfusion using a specially formulated aqueous solution containing 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), a lipophilic carbocyanine dye, which incorporates into endothelial cell membranes upon contact. By lateral diffusion, DiI also stains membrane structures, including angiogenic sprouts and pseudopodial processes that are not in direct contact. Tissues can be immediately examined by conventional and confocal fluorescence microscopy. High-quality serial optical sections using confocal microscopy are obtainable from thick tissue sections, especially at low magnification, for three-dimensional reconstruction. It takes less than 1 h to stain the vasculature in a whole animal. Compared with alternative techniques to visualize blood vessels, including space-occupying materials such as India ink or fluorescent dye-conjugated dextran, the corrosion casting technique, endothelial cell-specific markers and lectins, the present method simplifies the visualization of blood vessels and data analysis. PMID:18846097

Simple technique for high-throughput marking of distinguishable micro-areas for microscopy.

PubMed

Henrichs, Leonard F; Chen, L I; Bell, Andrew J

2016-04-01

Today's (nano)-functional materials, usually exhibiting complex physical properties require local investigation with different microscopy techniques covering different physical aspects such as dipolar and magnetic structure. However, often these must be employed on the very same sample position to be able to truly correlate those different information and corresponding properties. This can be very challenging if not impossible especially when samples lack prominent features for orientation. Here, we present a simple but effective method to mark hundreds of approximately 15×15 μm sample areas at one time by using a commercial transmission electron microscopy grid as shadow mask in combination with thin-film deposition. Areas can be easily distinguished when using a reference or finder grid structure as shadow mask. We show that the method is suitable to combine many techniques such as light microscopy, scanning probe microscopy and scanning electron microscopy. Furthermore, we find that best results are achieved when depositing aluminium on a flat sample surface using electron-beam evaporation which ensures good line-of-sight deposition. This inexpensive high-throughput method has several advantageous over other marking techniques such as focused ion-beam processing especially when batch processing or marking of many areas is required. Nevertheless, the technique could be particularly valuable, when used in junction with, for example focused ion-beam sectioning to obtain a thin lamellar of a particular pre-selected area. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Backscattered Electron Microscopy as an Advanced Technique in Petrography.

ERIC Educational Resources Information Center

Krinsley, David Henry; Manley, Curtis Robert

1989-01-01

Three uses of this method with sandstone, desert varnish, and granite weathering are described. Background information on this technique is provided. Advantages of this type of microscopy are stressed. (CW)

Photocarcinogenesis and Skin Cancer Prevention Strategies.

PubMed

Seebode, Christina; Lehmann, Janin; Emmert, Steffen

2016-03-01

In this review the basic principles of UV-induced carcinogenesis are summarized and the state of the art diagnosis and therapeutic strategies are discussed. The prevalent keratinocyte-derived neoplasms of the skin are basal cell and squamous cell carcinomas. Cutaneous melanoma is less frequent but associated with high mortality. Common risk factors for all three tumor entities include sun exposure and DNA-repair deficiencies. Photocarcinogenesis follows a multistep model of cancer development in which ultraviolet-induced DNA damage leads to mutations resulting in activation of oncogenes or silencing of tumor-suppressor genes. This ends in a cellular mutator phenotype even more prone to mutation acquisition. DNA repair, especially the nucleotide excision repair (NER) pathway, counteracts mutation formation and skin cancer development. This is vividly demonstrated by the NER-defective disorder xeroderma pigmentosum. Primary skin cancer preventative strategies, therefore, include reduction of DNA photodamage by protection from the sun. Secondary preventative strategies include skin cancer screening. This implies standard examination techniques with the naked eye, an epiluminescence microscope, or digital epiluminescence microscopy. More advanced techniques include confocal laser scan microscopy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Digital differential confocal microscopy based on spatial shift transformation.

PubMed

Liu, J; Wang, Y; Liu, C; Wilson, T; Wang, H; Tan, J

2014-11-01

Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Methods for characterizing plant fibers.

PubMed

Cruthers, Natasha; Carr, Debra; Niven, Brian; Girvan, Elizabeth; Laing, Raechel

2005-08-01

The effectiveness of different microscopy techniques for measuring the dimensions of ultimate fibers from harakeke (Phormium tenax, New Zealand flax) was investigated using a factorial experimental design. Constant variables were geographical location, location of specimens along the leaf, season (winter), individual plant, a fourth leaf from a north-facing fan, age of plant, and cultivars (two). Experimental variables were microscopy techniques and measurement axis. Measurements of width and length of harakeke ultimate fibers depended on the microscopic preparation/technique used as well as the cultivar examined. The best methods were (i) transverse sections of leaf specimens 4 microm thick, embedded in Paraplast and observed using light microscopy, and (ii) nonfixed ultimate fibers observed using scanning electron microscopy. (c) 2005 Wiley-Liss, Inc.

Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

PubMed Central

Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

2009-01-01

We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

Virtual Microscopy in Histopathology Training: Changing Student Attitudes in 3 Successive Academic Years.

PubMed

Bertram, Christof A; Firsching, Theresa; Klopfleisch, Robert

2018-01-01

Several veterinary faculties have integrated virtual microscopy into their curricula in recent years to improve and refine their teaching techniques. The many advantages of this recent technology are described in the literature, including remote access and an equal and constant slide quality for all students. However, no study has analyzed the change of perception toward virtual microscopy at different time points of students' academic educations. In the present study, veterinary students in 3 academic years were asked for their perspectives and attitudes toward virtual microscopy and conventional light microscopy. Third-, fourth-, and fifth-year veterinary students filled out a questionnaire with 12 questions. The answers revealed that virtual microscopy was overall well accepted by students of all academic years. Most students even suggested that virtual microscopy be implemented more extensively as the modality for final histopathology examinations. Nevertheless, training in the use of light microscopy and associated skills was surprisingly well appreciated. Regardless of their academic year, most students considered these skills important and necessary, and they felt that light microscopy should not be completely replaced. The reasons for this view differed depending on academic year, as the perceived main disadvantage of virtual microscopy varied. Third-year students feared that they would not acquire sufficient light microscopy skills. Fifth-year students considered technical difficulties (i.e., insufficient transmission speed) to be the main disadvantage of this newer teaching modality.

An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy.

PubMed

Warner, Cherish A; Biedrzycki, Meredith L; Jacobs, Samuel S; Wisser, Randall J; Caplan, Jeffrey L; Sherrier, D Janine

2014-12-01

We report on a nondestructive clearing technique that enhances transmission of light through specimens from diverse plant species, opening unique opportunities for microscope-enabled plant research. After clearing, plant organs and thick tissue sections are amenable to deep imaging. The clearing method is compatible with immunocytochemistry techniques and can be used in concert with common fluorescent probes, including widely adopted protein tags such as GFP, which has fluorescence that is preserved during the clearing process. © 2014 American Society of Plant Biologists. All Rights Reserved.

Calibrating the ChemCam LIBS for Carbonate Minerals on Mars

DOE R&D Accomplishments Database

Wiens, Roger C.; Clegg, Samuel M.; Ollila, Ann M.; Barefield, James E.; Lanza, Nina; Newsom, Horton E.

2009-01-01

The ChemCam instrument suite on board the NASA Mars Science Laboratory (MSL) rover includes the first LIBS instrument for extraterrestrial applications. Here we examine carbonate minerals in a simulated martian environment using the LIDS technique in order to better understand the in situ signature of these materials on Mars. Both chemical composition and rock type are determined using multivariate analysis (MVA) techniques. Composition is confirmed using scanning electron microscopy (SEM) techniques. Our initial results suggest that ChemCam can recognize and differentiate between carbonate materials on Mars.

Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells.

PubMed

Day, Richard N; Davidson, Michael W

2012-05-01

The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. Copyright © 2012 WILEY Periodicals, Inc.

Labeling Human Mesenchymal Stem Cells with Gold Nanocages for in vitro and in vivo Tracking by Two-Photon Microscopy and Photoacoustic Microscopy

PubMed Central

Zhang, Yu Shrike; Wang, Yu; Wang, Lidai; Wang, Yucai; Cai, Xin; Zhang, Chi; Wang, Lihong V.; Xia, Younan

2013-01-01

Stem cell tracking is a highly important subject. Current techniques based on nanoparticle-labeling, such as magnetic resonance imaging, fluorescence microscopy, and micro-computed tomography, are plagued by limitations including relatively low sensitivity or penetration depth, involvement of ionizing irradiation, and potential cytotoxicity of the nanoparticles. Here we introduce a new class of contrast agents based on gold nanocages (AuNCs) with hollow interiors and porous walls to label human mesenchymal stem cells (hMSCs) for both in vitro and in vivo tracking using two-photon microscopy and photoacoustic microscopy. As demonstrated by the viability assay, the AuNCs showed negligible cytotoxicity under a reasonable dose, and did not alter the differentiation potential of the hMSCs into desired lineages. We were able to image the cells labeled with AuNCs in vitro for at least 28 days in culture, as well as to track the cells that homed to the tumor region in nude mice in vivo. PMID:23946820

Reflective small angle electron scattering to characterize nanostructures on opaque substrates

NASA Astrophysics Data System (ADS)

Friedman, Lawrence H.; Wu, Wen-Li; Fu, Wei-En; Chien, Yunsan

2017-09-01

Feature sizes in integrated circuits (ICs) are often at the scale of 10 nm and are ever shrinking. ICs appearing in today's computers and hand held devices are perhaps the most prominent examples. These smaller feature sizes demand equivalent advances in fast and accurate dimensional metrology for both development and manufacturing. Techniques in use and continuing to be developed include X-ray based techniques, optical scattering, and of course the electron and scanning probe microscopy techniques. Each of these techniques has their advantages and limitations. Here, the use of small angle electron beam scattering measurements in a reflection mode (RSAES) to characterize the dimensions and the shape of nanostructures on flat and opaque substrates is demonstrated using both experimental and theoretical evidence. In RSAES, focused electrons are scattered at angles smaller than 1 ° with the assistance of electron optics typically used in transmission electron microscopy. A proof-of-concept experiment is combined with rigorous electron reflection simulations to demonstrate the efficiency and accuracy of RSAES as a method of non-destructive measurement of shapes of features less than 10 nm in size on flat and opaque substrates.

Reflective Small Angle Electron Scattering to Characterize Nanostructures on Opaque Substrates.

PubMed

Friedman, Lawrence H; Wu, Wen-Li; Fu, Wei-En; Chien, Yunsan

2017-09-01

Features sizes in integrated circuits (ICs) are often at the scale of 10 nm and are ever shrinking. ICs appearing in today's computers and hand held devices are perhaps the most prominent examples. These smaller feature sizes demand equivalent advances in fast and accurate dimensional metrology for both development and manufacturing. Techniques in use and continuing to be developed include X-ray based techniques, optical scattering and of course the electron and scanning probe microscopy techniques. Each of these techniques have their advantages and limitations. Here the use of small angle electron beam scattering measurements in a reflection mode (RSAES) to characterize the dimensions and the shape of nanostructures on flat and opaque substrates is demonstrated using both experimental and theoretical evidence. In RSAES, focused electrons are scattered at angles smaller than 1° with the assistance of electron optics typically used in transmission electron microscopy. A proof-of-concept experiment is combined with rigorous electron reflection simulations to demonstrate the efficiency and accuracy of RSAES as a method of non-destructive measurement of shapes of features less than 10 nm in size on flat and opaque substrates.

Large scale superres 3D imaging: light-sheet single-molecule localization microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Lu, Chieh Han; Chen, Peilin; Chen, Bi-Chang

2017-02-01

Optical imaging techniques provide much important information in understanding life science especially cellular structure and morphology because "seeing is believing". However, the resolution of optical imaging is limited by the diffraction limit, which is discovered by Ernst Abbe, i.e. λ/2(NA) (NA is the numerical aperture of the objective lens). Fluorescence super-resolution microscopic techniques such as Stimulated emission depletion microscopy (STED), Photoactivated localization microscopy (PALM), and Stochastic optical reconstruction microscopy (STORM) are invented to have the capability of seeing biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral resolution). These techniques do not physically violate the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their applications to the 2D imaging of fixed or dead samples due to the high laser power needed or slow speed for the localization process. Extended from 2D imaging, light sheet microscopy has been proven to have a lot of applications on 3D imaging at much better spatiotemporal resolutions due to its intrinsic optical sectioning and high imaging speed. Herein, we combine the advantage of localization microscopy and light-sheet microscopy to have super-resolved cellular imaging in 3D across large field of view. With high-density labeled spontaneous blinking fluorophore and wide-field detection of light-sheet microscopy, these allow us to construct 3D super-resolution multi-cellular imaging at high speed ( minutes) by light-sheet single-molecule localization microscopy.

Static and dynamic structural characterization of nanomaterial catalysts

NASA Astrophysics Data System (ADS)

Masiel, Daniel Joseph

Heterogeneous catalysts systems are pervasive in industry, technology and academia. These systems often involve nanostructured transition metal particles that have crucial interfaces with either their supports or solid products. Understanding the nature of these interfaces as well as the structure of the catalysts and support materials themselves is crucial for the advancement of catalysis in general. Recent developments in the field of transmission electron microscopy (TEM) including dynamic transmission electron microscopy (DTEM), electron tomography, and in situ techniques stand poised to provide fresh insight into nanostructured catalyst systems. Several electron microscopy techniques are applied in this study to elucidate the mechanism of silica nanocoil growth and to discern the role of the support material and catalyst size in carbon dioxide and steam reforming of methane. The growth of silica nanocoils by faceted cobalt nanoparticles is a process that was initially believed to take place via a vapor-liquid-solid growth mechanism similar to other nanowire growth techniques. The extensive TEM work described here suggests that the process may instead occur via transport of silicate and silica species over the nanoparticle surface. Electron tomography studies of the interface between the catalyst particles and the wire indicate that they grow from edges between facets. Studies on reduction of the Co 3O4 nanoparticle precursors to the faceted pure cobalt catalysts were carried out using DTEM and in situ heating. Supported catalyst systems for methane reforming were studied using dark field scanning TEM to better understand sintering effects and the increased activity of Ni/Co catalysts supported by carbon nanotubes. Several novel electron microscopy techniques are described including annular dark field DTEM and a metaheuristic algorithm for solving the phase problem of coherent diffractive imaging. By inserting an annular dark field aperture into the back focal plane of the objective lens in a DTEM, time-resolved dark field images can be produced that have vastly improved contrast for supported catalyst materials compared to bright field DTEM imaging. A new algorithm called swarm optimized phase retrieval is described that uses a population-based approach to solve for the missing phases of diffraction data from discrete particles.

Multifarious applications of atomic force microscopy in forensic science investigations.

PubMed

Pandey, Gaurav; Tharmavaram, Maithri; Rawtani, Deepak; Kumar, Sumit; Agrawal, Y

2017-04-01

Forensic science is a wide field comprising of several subspecialties and uses methods derived from natural sciences for finding criminals and other evidence valid in a legal court. A relatively new area; Nano-forensics brings a new era of investigation in forensic science in which instantaneous results can be produced that determine various agents such as explosive gasses, biological agents and residues in different crime scenes and terrorist activity investigations. This can be achieved by applying Nanotechnology and its associated characterization techniques in forensic sciences. Several characterization techniques exist in Nanotechnology and nano-analysis is one such technique that is used in forensic science which includes Electron microscopes (EM) like Transmission (TEM) and Scanning (SEM), Raman microscopy (Micro -Raman) and Scanning Probe Microscopes (SPMs) like Atomic Force Microscope (AFM). Atomic force microscopy enables surface characterization of different materials by examining their morphology and mechanical properties. Materials that are immeasurable such as hair, body fluids, textile fibers, documents, polymers, pressure sensitive adhesives (PSAs), etc. are often encountered during forensic investigations. This review article will mainly focus on the use of AFM in the examination of different evidence such as blood stains, forged documents, human hair samples, ammunitions, explosives, and other such applications in the field of Forensic Science. Copyright © 2017 Elsevier B.V. All rights reserved.

Synchrotron-based X-ray microscopic studies for bioeffects of nanomaterials.

PubMed

Zhu, Ying; Cai, Xiaoqing; Li, Jiang; Zhong, Zengtao; Huang, Qing; Fan, Chunhai

2014-04-01

There have been increasing interests in studying biological effects of nanomaterials, which are nevertheless faced up with many challenges due to the nanoscale dimensions and unique chemical properties of nanomaterials. Synchrotron-based X-ray microscopy, an advanced imaging technology with high spatial resolution and excellent elemental specificity, provides a new platform for studying interactions between nanomaterials and living systems. In this article, we review the recent progress of X-ray microscopic studies on bioeffects of nanomaterials in several living systems including cells, model organisms, animals and plants. We aim to provide an overview of the state of the art, and the advantages of using synchrotron-based X-ray microscopy for characterizing in vitro and in vivo behaviors and biodistribution of nanomaterials. We also expect that the use of a combination of new synchrotron techniques should offer unprecedented opportunities for better understanding complex interactions at the nano-biological interface and accounting for unique bioeffects of nanomaterials. Synchrotron-based X-ray microscopy is a non-destructive imaging technique that enables high resolution spatial mapping of metals with elemental level detection methods. This review summarizes the current use and perspectives of this novel technique in studying the biology and tissue interactions of nanomaterials. Copyright © 2014 Elsevier Inc. All rights reserved.

Single-molecule fluorescence microscopy review: shedding new light on old problems

PubMed Central

Shashkova, Sviatlana

2017-01-01

Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques. PMID:28694303

Intravital microscopy

PubMed Central

Masedunskas, Andrius; Milberg, Oleg; Porat-Shliom, Natalie; Sramkova, Monika; Wigand, Tim; Amornphimoltham, Panomwat; Weigert, Roberto

2012-01-01

Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs. PMID:22992750

A scanning transmission electron microscopy approach to analyzing large volumes of tissue to detect nanoparticles.

PubMed

Kempen, Paul J; Thakor, Avnesh S; Zavaleta, Cristina; Gambhir, Sanjiv S; Sinclair, Robert

2013-10-01

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 mm³ of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

A Scanning Transmission Electron Microscopy (STEM) Approach to Analyzing Large Volumes of Tissue to Detect Nanoparticles

PubMed Central

Kempen, Paul J.; Thakor, Avnesh S.; Zavaleta, Cristina; Gambhir, Sanjiv S.; Sinclair, Robert

2013-01-01

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time consuming analytical characterization. We utilized this technique to analyze 243,000 µm3 of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail-vein accumulated in the liver while those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation. PMID:23803218

Self-referenced axial chromatic dispersion measurement in multiphoton microscopy through 2-color THG imaging.

PubMed

Du, Yu; Zhuang, Ziwei; He, Jiexing; Liu, Hongji; Qiu, Ping; Wang, Ke

2018-05-16

With tunable excitation light, multiphoton microscopy (MPM) is widely used for imaging biological structures at subcellular resolution. Axial chromatic dispersion, present in virtually every transmissive optical system including the multiphoton microscope, leads to focal (and the resultant image) plane separation. Here we demonstrate experimentally a technique to measure the axial chromatic dispersion in a multiphoton microscope, using simultaneous 2-color third-harmonic generation (THG) imaging excited by a 2-color soliton source with tunable wavelength separation. Our technique is self-referenced, eliminating potential measurement error when 1-color tunable excitation light is used which necessitates reciprocating motion of the mechanical translation stage. Using this technique, we demonstrate measured axial chromatic dispersion with 2 different objective lenses in a multiphoton microscope. Further measurement in a biological sample also indicates that this axial chromatic dispersion, in combination with 2-color imaging, may open up opportunity for simultaneous imaging of two different axial planes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Preparing Al-Mg Substrate for Thermal Spraying: Evaluation of Surface State After Different Pretreatments

NASA Astrophysics Data System (ADS)

Lukauskaitė, R.; Valiulis, A. V.; Černašėjus, O.; Škamat, J.; Rębiś, J. A.

2016-08-01

The article deals with the pretreatment technique for preparing the surface of aluminum alloy EN AW 5754 before thermal spray. The surface after different pretreatments, including degreasing with acetone, chemical etching with acidic and alkali solutions, grit-blasting, cathodic cleaning, and some combinations of these techniques, has been studied. The investigation of pre-treated surfaces covered the topographical study (using scanning electron microscopy, atomic force microscopy, and 3D profilometry), the chemical analysis by x-ray photoelectron spectroscopy, the evaluation of surface wettability (sessile drop method), and the assessment of surface free energy. Compared with all the techniques used in present work, the cathodic cleaning and its combination with grit-blasting provide the most preferable chemistry of the surface. Due to the absence of hydroxides at the surface and, possible, due to the diffusion of magnesium to the surface of substrate, the surface wettability and the surface free energy have been significantly improved. No direct correlation between the surface topography and the surface wettability has been established.

Controllable self-induced passivation of hybrid lead iodide perovskites toward high performance solar cells.

PubMed

Chen, Qi; Zhou, Huanping; Song, Tze-Bin; Luo, Song; Hong, Ziruo; Duan, Hsin-Sheng; Dou, Letian; Liu, Yongsheng; Yang, Yang

2014-07-09

To improve the performance of the polycrystalline thin film devices, it requires a delicate control of its grain structures. As one of the most promising candidates among current thin film photovoltaic techniques, the organic/inorganic hybrid perovskites generally inherit polycrystalline nature and exhibit compositional/structural dependence in regard to their optoelectronic properties. Here, we demonstrate a controllable passivation technique for perovskite films, which enables their compositional change, and allows substantial enhancement in corresponding device performance. By releasing the organic species during annealing, PbI2 phase is presented in perovskite grain boundaries and at the relevant interfaces. The consequent passivation effects and underlying mechanisms are investigated with complementary characterizations, including scanning electron microscopy (SEM), X-ray diffraction (XRD), time-resolved photoluminescence decay (TRPL), scanning Kelvin probe microscopy (SKPM), and ultraviolet photoemission spectroscopy (UPS). This controllable self-induced passivation technique represents an important step to understand the polycrystalline nature of hybrid perovskite thin films and contributes to the development of perovskite solar cells judiciously.

Nanoscale infrared spectroscopy as a non-destructive probe of extraterrestrial samples.

PubMed

Dominguez, Gerardo; Mcleod, A S; Gainsforth, Zack; Kelly, P; Bechtel, Hans A; Keilmann, Fritz; Westphal, Andrew; Thiemens, Mark; Basov, D N

2014-12-09

Advances in the spatial resolution of modern analytical techniques have tremendously augmented the scientific insight gained from the analysis of natural samples. Yet, while techniques for the elemental and structural characterization of samples have achieved sub-nanometre spatial resolution, infrared spectral mapping of geochemical samples at vibrational 'fingerprint' wavelengths has remained restricted to spatial scales >10 μm. Nevertheless, infrared spectroscopy remains an invaluable contactless probe of chemical structure, details of which offer clues to the formation history of minerals. Here we report on the successful implementation of infrared near-field imaging, spectroscopy and analysis techniques capable of sub-micron scale mineral identification within natural samples, including a chondrule from the Murchison meteorite and a cometary dust grain (Iris) from NASA's Stardust mission. Complementary to scanning electron microscopy, energy-dispersive X-ray spectroscopy and transmission electron microscopy probes, this work evidences a similarity between chondritic and cometary materials, and inaugurates a new era of infrared nano-spectroscopy applied to small and invaluable extraterrestrial samples.

Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

PubMed

Johnson, Sam A

2015-01-01

Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches. © 2014 Wiley Periodicals, Inc.

Stochastic Optical Reconstruction Microscopy (STORM).

PubMed

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Measuring Roughnesses Of Optical Surfaces

NASA Technical Reports Server (NTRS)

Coulter, Daniel R.; Al-Jumaily, Gahnim A.; Raouf, Nasrat A.; Anderson, Mark S.

1994-01-01

Report discusses use of scanning tunneling microscopy and atomic force microscopy to measure roughnesses of optical surfaces. These techniques offer greater spatial resolution than other techniques. Report notes scanning tunneling microscopes and atomic force microscopes resolve down to 1 nm.

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping.

PubMed

Roth, Gary A; Sosa Peña, Maria del Pilar; Neu-Baker, Nicole M; Tahiliani, Sahil; Brenner, Sara A

2015-12-08

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Decalin-assisted light emitting porous Si formation and its optical, surface and morphological properties

NASA Astrophysics Data System (ADS)

Karatutlu, Ali; Istengir, Sumeyra; Cosgun, Sedat; Seker, Isa; Unal, Bayram

2017-11-01

In this research paper, light emitting porous silicon (Lep-Si) samples were fabricated by a surfactant-mediated chemical stain etching solution in order to form homogenous luminescent nanostructures at room temperature. As an industrially important solvent, decalin (decahydronaphtalene) was used as a surfactant in the HF/HNO3 solutions in order to control the etching process. Morphological, surface and optical properties of the Lep-Si samples were examined using atomic force microscopy, X-ray photoelectron spectroscopy, photoluminescence (PL) spectroscopy, and laser scanning confocal microscopy (LSCM) techniques. These characterization techniques were correlated with the various etching times including depth dependent luminescence profiles for the first time. We report the optimum conditions for production of the most efficient Lep-Si using decalin (decahydronaphtalene) and possible structural origins of light emission using the depth dependent luminescence measurements.

Gold nanoparticles for cancer detection and treatment: The role of adhesion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oni, Y.; Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey 08544; Hao, K.

2014-02-28

This paper presents the results of an experimental study of the effects of adhesion between gold nanoparticles and surfaces that are relevant to the potential applications in cancer detection and treatment. Adhesion is measured using a dip coating/atomic force microscopy (DC/AFM) technique. The adhesion forces are obtained for dip-coated gold nanoparticles that interact with peptide or antibody-based molecular recognition units (MRUs) that attach specifically to breast cancer cells. They include MRUs that attach specifically to receptors on breast cancer cells. Adhesion forces between anti-cancer drugs such as paclitaxel, and the constituents of MRU-conjugated Au nanoparticle clusters, are measured using forcemore » microscopy techniques. The implications of the results are then discussed for the design of robust gold nanoparticle clusters and for potential applications in localized drug delivery and hyperthermia.« less

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique.

PubMed

Snyman, Celia; Elliott, Edith

2011-12-15

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components. Copyright © 2011 Elsevier Inc. All rights reserved.

Surface-specific additive manufacturing test artefacts

NASA Astrophysics Data System (ADS)

Townsend, Andrew; Racasan, Radu; Blunt, Liam

2018-06-01

Many test artefact designs have been proposed for use with additive manufacturing (AM) systems. These test artefacts have primarily been designed for the evaluation of AM form and dimensional performance. A series of surface-specific measurement test artefacts designed for use in the verification of AM manufacturing processes are proposed here. Surface-specific test artefacts can be made more compact because they do not require the large dimensions needed for accurate dimensional and form measurements. The series of three test artefacts are designed to provide comprehensive information pertaining to the manufactured surface. Measurement possibilities include deviation analysis, surface texture parameter data generation, sub-surface analysis, layer step analysis and build resolution comparison. The test artefacts are designed to provide easy access for measurement using conventional surface measurement techniques, for example, focus variation microscopy, stylus profilometry, confocal microscopy and scanning electron microscopy. Additionally, the test artefacts may be simply visually inspected as a comparative tool, giving a fast indication of process variation between builds. The three test artefacts are small enough to be included in every build and include built-in manufacturing traceability information, making them a convenient physical record of the build.

Techniques for super-resolution microscopy using NV-diamond

NASA Astrophysics Data System (ADS)

Trifonov, Alexei; Glenn, David; Bar-Gill, Nir; Le Sage, David; Walsworth, Ronald

2011-05-01

We discuss the development and application of techniques for super-resolution microscopy using NV centers in diamond: stimulated emission depletion (STED), metastable ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM). NV centers do not bleach under optical excitation, are not biotoxic, and have long-lived electronic spin coherence and spin-state-dependent fluorescence. Thus NV-diamond has great potential as a fluorescent biomarker and as a magnetic biosensor.

Application of Contact Mode AFM to Manufacturing Processes

NASA Astrophysics Data System (ADS)

Giordano, Michael A.; Schmid, Steven R.

A review of the application of contact mode atomic force microscopy (AFM) to manufacturing processes is presented. A brief introduction to common experimental techniques including hardness, scratch, and wear testing is presented, with a discussion of challenges in the extension of manufacturing scale investigations to the AFM. Differences between the macro- and nanoscales tests are discussed, including indentation size effects and their importance in the simulation of processes such as grinding. The basics of lubrication theory are presented and friction force microscopy is introduced as a method of investigating metal forming lubrication on the nano- and microscales that directly simulates tooling/workpiece asperity interactions. These concepts are followed by a discussion of their application to macroscale industrial manufacturing processes and direct correlations are made.

Detection, mapping, and quantification of single walled carbon nanotubes in histological specimens with photoacoustic microscopy.

PubMed

Avti, Pramod K; Hu, Song; Favazza, Christopher; Mikos, Antonios G; Jansen, John A; Shroyer, Kenneth R; Wang, Lihong V; Sitharaman, Balaji

2012-01-01

In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Optical-resolution (OR) and acoustic-resolution (AR)--Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs.

3D Image Analysis of Geomaterials using Confocal Microscopy

NASA Astrophysics Data System (ADS)

Mulukutla, G.; Proussevitch, A.; Sahagian, D.

2009-05-01

Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the shapes of the segmented vesicles, vapor bubbles, and void spaces due to the optical measurements, so corrective actions are being explored. This will establish a practical and reliable framework for an adaptive 3D image processing technique for the analysis of geomaterials using confocal microscopy.

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Single-molecule techniques in biophysics: a review of the progress in methods and applications.

PubMed

Miller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J M; Leake, Mark C

2018-02-01

Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in 'force spectroscopy' techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

Single-molecule techniques in biophysics: a review of the progress in methods and applications

NASA Astrophysics Data System (ADS)

Miller, Helen; Zhou, Zhaokun; Shepherd, Jack; Wollman, Adam J. M.; Leake, Mark C.

2018-02-01

Single-molecule biophysics has transformed our understanding of biology, but also of the physics of life. More exotic than simple soft matter, biomatter lives far from thermal equilibrium, covering multiple lengths from the nanoscale of single molecules to up to several orders of magnitude higher in cells, tissues and organisms. Biomolecules are often characterized by underlying instability: multiple metastable free energy states exist, separated by levels of just a few multiples of the thermal energy scale k B T, where k B is the Boltzmann constant and T absolute temperature, implying complex inter-conversion kinetics in the relatively hot, wet environment of active biological matter. A key benefit of single-molecule biophysics techniques is their ability to probe heterogeneity of free energy states across a molecular population, too challenging in general for conventional ensemble average approaches. Parallel developments in experimental and computational techniques have catalysed the birth of multiplexed, correlative techniques to tackle previously intractable biological questions. Experimentally, progress has been driven by improvements in sensitivity and speed of detectors, and the stability and efficiency of light sources, probes and microfluidics. We discuss the motivation and requirements for these recent experiments, including the underpinning mathematics. These methods are broadly divided into tools which detect molecules and those which manipulate them. For the former we discuss the progress of super-resolution microscopy, transformative for addressing many longstanding questions in the life sciences, and for the latter we include progress in ‘force spectroscopy’ techniques that mechanically perturb molecules. We also consider in silico progress of single-molecule computational physics, and how simulation and experimentation may be drawn together to give a more complete understanding. Increasingly, combinatorial techniques are now used, including correlative atomic force microscopy and fluorescence imaging, to probe questions closer to native physiological behaviour. We identify the trade-offs, limitations and applications of these techniques, and discuss exciting new directions.

Fluorescence microscopy for the characterization of structural integrity

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Leonhardt, Todd A.

1991-01-01

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

Optimal model-based sensorless adaptive optics for epifluorescence microscopy.

PubMed

Pozzi, Paolo; Soloviev, Oleg; Wilding, Dean; Vdovin, Gleb; Verhaegen, Michel

2018-01-01

We report on a universal sample-independent sensorless adaptive optics method, based on modal optimization of the second moment of the fluorescence emission from a point-like excitation. Our method employs a sample-independent precalibration, performed only once for the particular system, to establish the direct relation between the image quality and the aberration. The method is potentially applicable to any form of microscopy with epifluorescence detection, including the practically important case of incoherent fluorescence emission from a three dimensional object, through minor hardware modifications. We have applied the technique successfully to a widefield epifluorescence microscope and to a multiaperture confocal microscope.

Nomarski differential interference contrast microscopy for surface slope measurements: an examination of techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartman, J.S.; Gordon, R.L.; Lessor, D.L.

1981-08-01

Alternate measurement and data analysis procedures are discussed and compared for the application of reflective Nomarski differential interference contrast microscopy for the determination of surface slopes. The discussion includes the interpretation of a previously reported iterative procedure using the results of a detailed optical model and the presentation of a new procedure based on measured image intensity extrema. Surface slope determinations from these procedures are presented and compared with results from a previously reported curve fit analysis of image intensity data. The accuracy and advantages of the different procedures are discussed.

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Cement paste surface roughness analysis using coherence scanning interferometry and confocal microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Apedo, K.L., E-mail: apedo@unistra.fr; Munzer, C.; He, H.

2015-02-15

Scanning electron microscopy and scanning probe microscopy have been used for several decades to better understand the microstructure of cementitious materials. Very limited work has been performed to date to study the roughness of cementitious materials by optical microscopy such as coherence scanning interferometry (CSI) and chromatic confocal sensing (CCS). The objective of this paper is to better understand how CSI can be used as a tool to analyze surface roughness and topography of cement pastes. Observations from a series of images acquired using this technique on both polished and unpolished samples are described. The results from CSI are comparedmore » with those from a STIL confocal microscopy technique (SCM). Comparison between both optical techniques demonstrates the ability of CSI to measure both polished and unpolished cement pastes. - Highlights: • Coherence scanning interferometry (CSI) was used to analyze cement paste surfaces. • The results from the CSI were compared with those from a confocal microscopy. • 3D roughness parameters were obtained using the window resizing method. • Polished and unpolished cement pastes were studied.« less

PREFACE: Ultrafast biophotonics Ultrafast biophotonics

NASA Astrophysics Data System (ADS)

Gu, Min; Reid, Derryck; Ben-Yakar, Adela

2010-08-01

The use of light to explore biology can be traced to the first observations of tissue made with early microscopes in the mid-seventeenth century, and has today evolved into the discipline which we now know as biophotonics. This field encompasses a diverse range of activities, each of which shares the common theme of exploiting the interaction of light with biological material. With the rapid advancement of ultrafast optical technologies over the last few decades, ultrafast lasers have increasingly found applications in biophotonics, to the extent that the distinctive new field of ultrafast biophotonics has now emerged, where robust turnkey ultrafast laser systems are facilitating cutting-edge studies in the life sciences to take place in everyday laboratories. The broad spectral bandwidths, precision timing resolution, low coherence and high peak powers of ultrafast optical pulses provide unique opportunities for imaging and manipulating biological systems. Time-resolved studies of bio-molecular dynamics exploit the short pulse durations from such lasers, while other applications such as optical coherence tomography benefit from the broad optical bandwidths possible by using super-continuum generation and additionally allowing for high speed imaging with speeds as high as 47 000 scans per second. Continuing progress in laser-system technology is accelerating the adoption of ultrafast techniques across the life sciences, both in research laboratories and in clinical applications, such as laser-assisted in situ keratomileusis (LASIK) eye surgery. Revolutionizing the field of optical microscopy, two-photon excitation fluorescence (TPEF) microscopy has enabled higher spatial resolution with improved depth penetration into biological specimens. Advantages of this nonlinear optical process include: reduced photo-interactions, allowing for extensive imaging time periods; simultaneously exciting multiple fluorescent molecules with only one excitation wavelength; and reduced chromatic aberration effects. These extensive advantages have led to further exploration of nonlinear processes including second-harmonic generation (SHG) microscopy and third-harmonic generation (THG) microscopy. Second-harmonic generation has provided biologists with an extremely powerful tool for generating contrast in biological imaging, with the additional benefit of non-invasive three-dimensional imaging. The recent popularity of THG microscopy is largely due to the fact that three-dimensional imaging is achievable without the need for any labels, but rather relying on the intrinsic properties of the biological specimen itself. This optical nonlinear technique has attracted much attention recently from the biological community due to its non-invasive capabilities. Users of ultrafast lasers in the biological and medical fields are becoming a fast-growing community, employing pulse-shaping microscopy, resolution-enhancing microscopy techniques, linear and nonlinear micro-spectroscopy, functional deep-tissue imaging, optical coherence tomography, nonlinear fluorescence microscopy, molecular imaging and control, harmonic microscopy and femtosecond lifetime imaging, for cutting-edge research concerning the interaction of light with biological dynamics. The adaptability of ultrafast lasers to interact with a large array of materials through nonlinear excitation has enabled precise control of laser fluence allowing for highly localized material interactions, permitting micro-structured fabricated surfaces. The resultant multi-dimensional fabricated micro-structures are capable of replicating and/or manipulating microenvironments for controlled cell biology. In this special issue of Journal of Optics readers have a chance to view a collection of new contributions to the growing research field of ultrafast biophotonics. They are presented with recent advances in ultrafast technology applied to biological and medical investigations, where topics include advances in the visualization and identification of photo-reaction dynamics of biological functions under relevant physiological conditions, theoretically proposed imaging designs for obtaining super-resolved optical sectioned images in single exposures and fabricated micro-structured surfaces for biological micro-environments. We hope the collection will stimulate innovative new research in this growing field by showcasing new techniques for the visualization and manipulation of complex biological systems using linear and and nonlinear optical processes. Professor Min Gu would like to acknowledge Dr Betty Kouskousis for her contribution and support towards this editorial.

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Ophthalmic applications of confocal microscopy: diagnostics, refractive surgery, and eye banking

NASA Astrophysics Data System (ADS)

Masters, Barry R.

1990-11-01

Confocal microscopy of ocular tissue provides two advantages over traditional imaging techniques: increased range and transverse resolution and increased contrast. The semitransparent cornea and ocular lens in the living eye can be optically sectioned and observed by reflected light confocal microscopy. Within the cornea we observed various cell components nerve fibers nerve cell bodies and fibrous networks. The confocal microscopic images from the in-situ ocular lens show the lens capsule the lens epithelium and the individual lens fibrils. All of the reflected light confocal microscopic images have high contrast and high resolution. Some of the applications of confocal imaging in ophthalmology include: diagnostics of the cornea and the ocular lens examination prior to and after refractive surgery examination of intraocular lenses (IOL) and examination of eye bank material. Other ophthalmic uses of confocal imaging include: studies of wound healing therapeutics and the effects of contact lenses on the cornea. The proposed features of a clinical confocal microscope are reviewed. 2.

Applicability of confocal laser scanning microscopy for evaluation and monitoring of cutaneous wound healing

NASA Astrophysics Data System (ADS)

Lange-Asschenfeldt, Susanne; Bob, Adrienne; Terhorst, Dorothea; Ulrich, Martina; Fluhr, Joachim; Mendez, Gil; Roewert-Huber, Hans-Joachim; Stockfleth, Eggert; Lange-Asschenfeldt, Bernhard

2012-07-01

There is a high demand for noninvasive imaging techniques for wound assessment. In vivo reflectance confocal laser scanning microscopy (CLSM) represents an innovative optical technique for noninvasive evaluation of normal and diseased skin in vivo at near cellular resolution. This study was designed to test the feasibility of CLSM for noninvasive analysis of cutaneous wound healing in 15 patients (7 male/8 female), including acute and chronic, superficial and deep dermal skin wounds. A commercially available CLSM system was used for the assessment of wound bed and wound margins in order to obtain descriptive cellular and morphological parameters of cutaneous wound repair noninvasively and over time. CLSM was able to visualize features of cutaneous wound repair in epidermal and superficial dermal wounds, including aspects of inflammation, neovascularisation, and tissue remodelling in vivo. Limitations include the lack of mechanic fixation of the optical system on moist surfaces restricting the analysis of chronic skin wounds to the wound margins, as well as a limited optical resolution in areas of significant slough formation. By describing CLSM features of cutaneous inflammation, vascularisation, and epithelialisation, the findings of this study support the role of CLSM in modern wound research and management.

Creep fatigue life prediction for engine hot section materials (isotropic)

NASA Technical Reports Server (NTRS)

Nelson, R. S.; Levan, G. W.; Harvey, P. R.

1992-01-01

This Final Report covers the activities completed under the optional program of the NASA HOST Contract, NAS3-23288. The initial effort of the optional program was report-in NASA CR189221, which consisted of high temperature strain controlled fatigue tests to study the effects of thermomechanical fatigue, multiaxial loading, reactive environments, and imposed stresses. The baseline alloy used in the tests included B1900+Hf (with or without coating) and wrought INCO 718. Tests conducted on B1900+Hf included environmental tests using various atmospheres (75 psig oxygen, purified argon, or block exposures) and specimen tests of wrought INCO 718 included tensile, creep, stress rupture, TMF, multiaxial, and mean stress tests. Results of these testings were used to calibrate a CDA model for INCO 718 alloy and to develop modifications or corrections to the CDA model to handle additional failure mechanisms. The Socie parameter was found to provide the best correlation for INCO multiaxial loading. Microstructural evaluations consisting of optical, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) techniques, and surface replication techniques to determine crack initiation lives provided data which were used to develop life prediction models.

SRRF: Universal live-cell super-resolution microscopy.

PubMed

Culley, Siân; Tosheva, Kalina L; Matos Pereira, Pedro; Henriques, Ricardo

2018-08-01

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

A review of cellphone microscopy for disease detection.

PubMed

Dendere, R; Myburg, N; Douglas, T S

2015-12-01

The expansion in global cellphone network coverage coupled with advances in cellphone imaging capabilities present an opportunity for the advancement of cellphone microscopy as a low-cost alternative to conventional microscopy for disease detection in resource-limited regions. The development of cellphone microscopy has also benefitted from the availability of low-cost miniature microscope components such as low-power light-emitting diodes and ball lenses. As a result, researchers are developing hardware and software techniques that would enable such microscopes to produce high-resolution, diagnostic-quality images. This approach may lead to more widespread delivery of diagnostic services in resource-limited areas where there is a shortage of the skilled labour required for conventional microscopy and where prevalence of infectious and other diseases is still high. In this paper, we review current techniques, clinical applications and challenges faced in the field of cellphone microscopy. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Infrared spectroscopy of molecular submonolayers on surfaces by infrared scanning tunneling microscopy: tetramantane on Au111.

PubMed

Pechenezhskiy, Ivan V; Hong, Xiaoping; Nguyen, Giang D; Dahl, Jeremy E P; Carlson, Robert M K; Wang, Feng; Crommie, Michael F

2013-09-20

We have developed a new scanning-tunneling-microscopy-based spectroscopy technique to characterize infrared (IR) absorption of submonolayers of molecules on conducting crystals. The technique employs a scanning tunneling microscope as a precise detector to measure the expansion of a molecule-decorated crystal that is irradiated by IR light from a tunable laser source. Using this technique, we obtain the IR absorption spectra of [121]tetramantane and [123]tetramantane on Au(111). Significant differences between the IR spectra for these two isomers show the power of this new technique to differentiate chemical structures even when single-molecule-resolved scanning tunneling microscopy (STM) images look quite similar. Furthermore, the new technique was found to yield significantly better spectral resolution than STM-based inelastic electron tunneling spectroscopy, and to allow determination of optical absorption cross sections. Compared to IR spectroscopy of bulk tetramantane powders, infrared scanning tunneling microscopy (IRSTM) spectra reveal narrower and blueshifted vibrational peaks for an ordered tetramantane adlayer. Differences between bulk and surface tetramantane vibrational spectra are explained via molecule-molecule interactions.

Cornea and anterior eye assessment with slit lamp biomicroscopy, specular microscopy, confocal microscopy, and ultrasound biomicroscopy

PubMed Central

Martin, Raul

2018-01-01

Current corneal assessment technologies make the process of corneal evaluation extremely fast and simple, and several devices and technologies show signs that help in identification of different diseases thereby, helping in diagnosis, management, and follow-up of patients. The purpose of this review is to present and update readers on the evaluation of cornea and ocular surface. This first part reviews a description of slit lamp biomicroscopy (SLB), endothelial specular microscopy, confocal microscopy, and ultrasound biomicroscopy examination techniques and the second part describes the corneal topography and tomography, providing up-to-date information on the clinical recommendations of these techniques in eye care practice. Although the SLB is a traditional technique, it is of paramount importance in clinical diagnosis and compulsory when an eye test is conducted in primary or specialist eye care practice. Different techniques allow the early diagnosis of many diseases, especially when clinical signs have not yet become apparent and visible with SLB. These techniques also allow for patient follow-up in several clinical conditions or diseases, facilitating clinical decisions and improving knowledge regarding the corneal anatomy. PMID:29380757

Deciphering the physics and chemistry of perovskites with transmission electron microscopy.

PubMed

Polking, Mark J

2016-03-28

Perovskite oxides exhibit rich structural complexity and a broad range of functional properties, including ferroelectricity, ferromagnetism, and superconductivity. The development of aberration correction for the transmission electron microscope and concurrent progress in electron spectroscopy, electron holography, and other techniques has fueled rapid progress in the understanding of the physics and chemistry of these materials. New techniques based on the transmission electron microscope are first surveyed, and the applications of these techniques for the study of the structure, chemistry, electrostatics, and dynamics of perovskite oxides are then explored in detail, with a particular focus on ferroelectric materials.

Developing new optical imaging techniques for single particle and molecule tracking in live cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Wei

Differential interference contrast (DIC) microscopy is a far-field as well as wide-field optical imaging technique. Since it is non-invasive and requires no sample staining, DIC microscopy is suitable for tracking the motion of target molecules in live cells without interfering their functions. In addition, high numerical aperture objectives and condensers can be used in DIC microscopy. The depth of focus of DIC is shallow, which gives DIC much better optical sectioning ability than those of phase contrast and dark field microscopies. In this work, DIC was utilized to study dynamic biological processes including endocytosis and intracellular transport in live cells.more » The suitability of DIC microscopy for single particle tracking in live cells was first demonstrated by using DIC to monitor the entire endocytosis process of one mesoporous silica nanoparticle (MSN) into a live mammalian cell. By taking advantage of the optical sectioning ability of DIC, we recorded the depth profile of the MSN during the endocytosis process. The shape change around the nanoparticle due to the formation of a vesicle was also captured. DIC microscopy was further modified that the sample can be illuminated and imaged at two wavelengths simultaneously. By using the new technique, noble metal nanoparticles with different shapes and sizes were selectively imaged. Among all the examined metal nanoparticles, gold nanoparticles in rod shapes were found to be especially useful. Due to their anisotropic optical properties, gold nanorods showed as diffraction-limited spots with disproportionate bright and dark parts that are strongly dependent on their orientation in the 3D space. Gold nanorods were developed as orientation nanoprobes and were successfully used to report the self-rotation of gliding microtubules on kinesin coated substrates. Gold nanorods were further used to study the rotational motions of cargoes during the endocytosis and intracellular transport processes in live mammalian cells. New rotational information was obtained: (1) during endocytosis, cargoes lost their rotation freedom at the late stage of internalization; (2) cargoes performed train-like motion when they were transported along the microtubule network by motor proteins inside live cells; (3) During the pause stage of fast axonal transport, cargoes were still bound to the microtubule tracks by motor proteins. Total internal reflection fluorescence microscopy (TIRFM) is another non-invasive and far-field optical imaging technique. Because of its near-field illumination mechanism, TIRFM has better axial resolution than epi-fluorescence microscopy and confocal microscopy. In this work, an auto-calibrated, prism type, angle-scanning TIRFM instrument was built. The incident angle can range from subcritical angles to nearly 90°, with an angle interval less than 0.2°. The angle precision of the new instrument was demonstrated through the finding of the surface plasmon resonance (SPR) angle of metal film coated glass slide. The new instrument improved significantly the precision in determining the axial position. As a result, the best obtained axial resolution was ~ 8 nm, which is better than current existing instruments similar in function. The instrument was further modified to function as a pseudo TIRF microscope. The illumination depth can be controlled by changing the incident angle of the excitation laser beam or adjusting the horizontal position of the illumination laser spot on the prism top surface. With the new technique, i.e., variable-illumination-depth pseudo TIRF microscopy, the whole cell body from bottom to top was scanned.« less

Spectroscopic photon localization microscopy: breaking the resolution limit of single molecule localization microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Dong, Biqin; Almassalha, Luay Matthew; Urban, Ben E.; Nguyen, The-Quyen; Khuon, Satya; Chew, Teng-Leong; Backman, Vadim; Sun, Cheng; Zhang, Hao F.

2017-02-01

Distinguishing minute differences in spectroscopic signatures is crucial for revealing the fluorescence heterogeneity among fluorophores to achieve a high molecular specificity. Here we report spectroscopic photon localization microscopy (SPLM), a newly developed far-field spectroscopic imaging technique, to achieve nanoscopic resolution based on the principle of single-molecule localization microscopy while simultaneously uncovering the inherent molecular spectroscopic information associated with each stochastic event (Dong et al., Nature Communications 2016, in press). In SPLM, by using a slit-less monochromator, both the zero-order and the first-order diffractions from a grating were recorded simultaneously by an electron multiplying charge-coupled device to reveal the spatial distribution and the associated emission spectra of individual stochastic radiation events, respectively. As a result, the origins of photon emissions from different molecules can be identified according to their spectral differences with sub-nm spectral resolution, even when the molecules are within close proximity. With the newly developed algorithms including background subtraction and spectral overlap unmixing, we established and tested a method which can significantly extend the fundamental spatial resolution limit of single molecule localization microscopy by molecular discrimination through spectral regression. Taking advantage of this unique capability, we demonstrated improvement in spatial resolution of PALM/STORM up to ten fold with selected fluorophores. This technique can be readily adopted by other research groups to greatly enhance the optical resolution of single molecule localization microscopy without the need to modify their existing staining methods and protocols. This new resolving capability can potentially provide new insights into biological phenomena and enable significant research progress to be made in the life sciences.

Nuclear microscopy in biomedical analysis with special emphasis on clinical metal biology

NASA Astrophysics Data System (ADS)

Lindh, Ulf; Frisk, Peter; Nyström, Joakim; Danersund, Antero; Hudecek, Romuald; Lindvall, Anders; Thunell, Stig

1997-07-01

Nuclear microscopy based upon developments in high energy ion beam techniques is by now an accepted technique in many fields of research. The advancements into the biomedical field have, however, been slower than expected. A major factor explaining this tendency is the availability of nuclear microscopy. This paper reviews briefly the biomedical work using nuclear microscopy that has been carried out since the 4 th International Conference on Nuclear Microprobe Technology and Applications held in Shanghai. Nuclear microscopy of isolated individual blood cells from patients adversely affected by metal exposure from dental amalgam has been performed both before and after removal of the metallic fillings. The elemental profile of blood cells was more or less normalised after treatment. Some of these results will be presented to illustrate a medical application. Results from bulk analysis by ICP-MS of erythrocytes and plasma before and after treatment will also be presented to illustrate the difference in information content between these two approaches as well as the need for complementary information in solving biomedical problems. As part of a larger study of acute porphyria, nuclear microscopy of blood cells was included among the 78 laboratory tests. The approach in this study was unbiased in the sense that no hypothesis was formulated as to which laboratory parameters would be the most explanatory for health or disease. Multivariate discriminant analysis was applied to the large amounts of data acquired. This approach led to the hypothesis that oxidative stress increased the synthesis of manganese-dependent superoxide dismutase in the mitochondria of polymorphonuclear leukocytes, explaining the increase of manganese in these cells. Antioxidant therapy was therefore applied to a couple of patients with porphyria, however, without clinical success.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy.

PubMed

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

Multiple excitation nano-spot generation and confocal detection for far-field microscopy

NASA Astrophysics Data System (ADS)

Mondal, Partha Pratim

2010-03-01

An imaging technique is developed for the controlled generation of multiple excitation nano-spots for far-field microscopy. The system point spread function (PSF) is obtained by interfering two counter-propagating extended depth-of-focus PSF (DoF-PSF), resulting in highly localized multiple excitation spots along the optical axis. The technique permits (1) simultaneous excitation of multiple planes in the specimen; (2) control of the number of spots by confocal detection; and (3) overcoming the point-by-point based excitation. Fluorescence detection from the excitation spots can be efficiently achieved by Z-scanning the detector/pinhole assembly. The technique complements most of the bioimaging techniques and may find potential application in high resolution fluorescence microscopy and nanoscale imaging.

PIZZA: a phase-induced zonal Zernike apodization designed for stellar coronagraphy

NASA Astrophysics Data System (ADS)

Martinache, Frantz

2004-08-01

I explore here the possibilities offered by the general formalism of coronagraphy for the very special case of phase contrast. This technique, invented by Zernike, is commonly used in microscopy, to see phase objects such as micro-organisms, and in strioscopy, to control the quality of optics polishing. It may find application in telescope pupil apodization with significant advantages over classical pupil apodization techniques, including high throughput and no off-axis resolution loss, which is essential for exoplanet imaging.

Superresolution microscopy for microbiology

PubMed Central

Coltharp, Carla; Xiao, Jie

2014-01-01

Summary This review provides a practical introduction to superresolution microscopy from the perspective of microbiological research. Because of the small sizes of bacterial cells, superresolution methods are particularly powerful and suitable for revealing details of cellular structures that are not resolvable under conventional fluorescence light microscopy. Here we describe the methodological concepts behind three major categories of super-resolution light microscopy: photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) and stimulated emission-depletion (STED) microscopy. We then present recent applications of each of these techniques to microbial systems, which have revealed novel conformations of cellular structures and described new properties of in vivo protein function and interactions. Finally, we discuss the unique issues related to implementing each of these superresolution techniques with bacterial specimens and suggest avenues for future development. The goal of this review is to provide the necessary technical background for interested microbiologists to choose the appropriate super-resolution method for their biological systems, and to introduce the practical considerations required for designing and analysing superresolution imaging experiments. PMID:22947061

Nano-Optics for Chemical and Materials Characterization

NASA Astrophysics Data System (ADS)

Beversluis, Michael; Stranick, Stephan

2007-03-01

Light microscopy can provide non-destructive, real-time, three-dimensional imaging with chemically-specific contrast, but diffraction frequently limits the resolution to roughly 200 nm. Recently, structured illumination techniques have allowed fluorescence imaging to reach 50 nm resolution [1]. Since these fluorescence techniques were developed for use in microbiology, a key challenge is to take the resolution-enhancing features and apply them to contrast mechanisms like vibrational spectroscopy (e.g., Raman and CARS microscopy) that provide morphological and chemically specific imaging.. We are developing a new hybrid technique that combines the resolution enhancement of structured illumination microscopy with scanning techniques that can record hyperspectral images with 100 nm spatial resolution. We will show such superresolving images of semiconductor nanostructures and discuss the advantages and requirements for this technique. Referenence: 1. M. G. L. Gustafsson, P. Natl. Acad. Sci. USA 102, 13081-13086 (2005).

Integrating Elemental Analysis and Chromatography Techniques by Analyzing Metal Oxide and Organic UV Absorbers in Commercial Sunscreens

ERIC Educational Resources Information Center

Quin~ones, Rosalynn; Bayline, Jennifer Logan; Polvani, Deborah A.; Neff, David; Westfall, Tamara D.; Hijazi, Abdullah

2016-01-01

A series of undergraduate laboratory experiments that utilize reversed-phase HPLC separation, inductively coupled plasma spectroscopy (ICP), and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) are described for the analysis of commercial sunscreens. The active ingredients of many sunscreen brands include zinc or titanium…

Trachymolgus purpureus sp. nov., an armored snout mite (Acari: Bdellidae) from the Ozark highlands: morphology, development, and key to Trachymolgus Berlese

USDA-ARS?s Scientific Manuscript database

Trachymolgus purpureus Fisher & Dowling sp. nov. is described from the Ozark highlands of North America. A diversity of imaging techniques are used to illustrate the species including field emission low-temperature scanning electron microscopy (FE-LTSEM), stereomicrography, compound light micrograph...

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

PubMed

Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Birk, Udo J; Dobrucki, Jurek W; Cremer, Christoph

2016-05-01

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. Copyright © 2016. Published by Elsevier Inc.

Identifying Nanoscale Structure-Function Relationships Using Multimodal Atomic Force Microscopy, Dimensionality Reduction, and Regression Techniques.

PubMed

Kong, Jessica; Giridharagopal, Rajiv; Harrison, Jeffrey S; Ginger, David S

2018-05-31

Correlating nanoscale chemical specificity with operational physics is a long-standing goal of functional scanning probe microscopy (SPM). We employ a data analytic approach combining multiple microscopy modes, using compositional information in infrared vibrational excitation maps acquired via photoinduced force microscopy (PiFM) with electrical information from conductive atomic force microscopy. We study a model polymer blend comprising insulating poly(methyl methacrylate) (PMMA) and semiconducting poly(3-hexylthiophene) (P3HT). We show that PiFM spectra are different from FTIR spectra, but can still be used to identify local composition. We use principal component analysis to extract statistically significant principal components and principal component regression to predict local current and identify local polymer composition. In doing so, we observe evidence of semiconducting P3HT within PMMA aggregates. These methods are generalizable to correlated SPM data and provide a meaningful technique for extracting complex compositional information that are impossible to measure from any one technique.

Environmental scanning electron microscopy in cell biology.

PubMed

McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

2013-01-01

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy.

PubMed

Keevil, C W

2003-01-01

Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.

Individual human cell responses to low doses of chemicals studied by synchrotron infrared spectromicroscopy

NASA Astrophysics Data System (ADS)

Holman, Hoi-Ying N.; Goth-Goldstein, Regine; Blakely, Elanor A.; Bjornstad, Kathy; Martin, Michael C.; McKinney, Wayne R.

2000-05-01

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in the individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR-FTIR microscopy probes intact living cells providing a composite view of all of the molecular response and the ability to monitor the response over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low- doses of chemicals. In this study we used the high spatial - resolution SR-FTIR vibrational spectromicroscopy as a sensitive analytical tool to detect chemical- and radiation- induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of dioxin. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio- compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

Resinless section electron microscopy reveals the yeast cytoskeleton.

PubMed

Penman, J; Penman, S

1997-04-15

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Three-dimensional imaging of porous media using confocal laser scanning microscopy.

PubMed

Shah, S M; Crawshaw, J P; Boek, E S

2017-02-01

In the last decade, imaging techniques capable of reconstructing three-dimensional (3-D) pore-scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO 2 storage potential. CLSM has a unique capability of producing 3-D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z-dimension) that can be imaged in porous materials. In this study, we introduce a 'grind and slice' technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3-D confocal volumetric data into pores and grains. Finally, we use the resulting 3-D pore-scale binarized confocal data obtained to quantitatively determine petrophysical pore-scale properties such as total porosity, macro- and microporosity and single-phase permeability using lattice Boltzmann (LB) simulations, validated by experiments. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Resinless section electron microscopy reveals the yeast cytoskeleton

PubMed Central

Penman, Joshua; Penman, Sheldon

1997-01-01

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture. PMID:9108046

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

PubMed

Matsuda, Tomoki; Nagai, Takeharu

2014-12-01

Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

The Neuro-Oncology Branch (NOB), Center for Cancer Research (CCR), National Cancer Institute (NCI) of the National Institutes of Health (NIH) is seeking outstanding postdoctoral candidates interested in studying the metabolic changes in brain tumors such as glioblastoma multiforme (GBMs).  NOB’s Metabolomics program is interested in revealing the metabolic alterations of isocitrate dehydrogenase (IDH1)-mutated GBMs and in exploiting these deregulations for therapeutic applications.  A combination of methods such as molecular biology, animal models, as well as in vitro and in vivo metabolomics using Raman Imaging Microscopy, Nuclear Magnetic Resonance spectroscopy (NMR), Mass Spectrometry (MS) and Magnetic Resonance Imaging (MRI) techniques are employed.  The position will specifically focus on molecular biology and Raman Imaging Microscopy, which includes work in Western Blotting, mammalian cell culture and other common biomedical techniques used in cancer bio  logy labs such as handling tissue samples, preparing tissue slides, staining, and extracting proteins from brain tissue.

Comparison of nanoparticle diffusion using fluorescence correlation spectroscopy and differential dynamic microscopy within concentrated polymer solutions

NASA Astrophysics Data System (ADS)

Shokeen, Namita; Issa, Christopher; Mukhopadhyay, Ashis

2017-12-01

We studied the diffusion of nanoparticles (NPs) within aqueous entangled solutions of polyethylene oxide (PEO) by using two different optical techniques. Fluorescence correlation spectroscopy, a method widely used to investigate nanoparticle dynamics in polymer solution, was used to measure the long-time diffusion coefficient (D) of 25 nm radius particles within high molecular weight, Mw = 600 kg/mol PEO in water solutions. Differential dynamic microscopy (DDM) was used to determine the wave-vector dependent dynamics of NPs within the same polymer solutions. Our results showed good agreement between the two methods, including demonstration of normal diffusion and almost identical diffusion coefficients obtained by both techniques. The research extends the scope of DDM to study the dynamics and rheological properties of soft matter at a nanoscale. The measured diffusion coefficients followed a scaling theory, which can be explained by the coupling between polymer dynamics and NP motion.

First oxygen from lunar basalt

NASA Technical Reports Server (NTRS)

Gibson, M. A.; Knudsen, C. W.; Brueneman, D. J.; Kanamori, H.; Ness, R. O.; Sharp, L. L.; Brekke, D. W.; Allen, C. C.; Morris, R. V.; Keller, L. P.

1993-01-01

The Carbotek/Shimizu process to produce oxygen from lunar soils has been successfully demonstrated on actual lunar samples in laboratory facilities at Carbotek with Shimizu funding and support. Apollo sample 70035 containing approximately 25 percent ilmenite (FeTiO3) was used in seven separate reactions with hydrogen varying temperature and pressure: FeTiO3 + H2 yields Fe + TiO2 + H2O. The experiments gave extremely encouraging results as all ilmenite was reduced in every experiment. The lunar ilmenite was found to be about twice as reactive as terrestrial ilmenite samples. Analytical techniques of the lunar and terrestrial ilmenite experiments performed by NASA Johnson Space Center include iron Mossbauer spectroscopy (FeMS), optical microscopy, SEM, TEM, and XRD. The Energy and Environmental Research Center at the University of North Dakota performed three SEM techniques (point count method, morphology determination, elemental mapping), XRD, and optical microscopy.

Application of Advanced Atomic Force Microscopy Techniques to Study Quantum Dots and Bio-materials

NASA Astrophysics Data System (ADS)

Guz, Nataliia

In recent years, there has been an increase in research towards micro- and nanoscale devices as they have proliferated into diverse areas of scientific exploration. Many of the general fields of study that have greatly affected the advancement of these devices includes the investigation of their properties. The sensitivity of Atomic Force Microscopy (AFM) allows detecting charges up to the single electron value in quantum dots in ambient conditions, the measurement of steric forces on the surface of the human cell brush, determination of cell mechanics, magnetic forces, and other important properties. Utilizing AFM methods, the fast screening of quantum dot efficiency and the differences between cancer, normal (healthy) and precancer (immortalized) human cells has been investigated. The current research using AFM techniques can help to identify biophysical differences of cancer cells to advance our understanding of the resistance of the cells against the existing medicine.

DOE Office of Scientific and Technical Information (OSTI.GOV)

García, Abraham; Cotto, María; Duconge, José

The use of hydrogen as replacement for fossil fuels, on which we depend today, is a matter of great relevance. The sustainable generation of hydrogen as fuel is relevant from an environmental and economic point of view. In this study we have explored new synthetic routes for developing new photocatalysts to be used in water splitting, for hydrogen production. Different techniques have been used to produce hydrogen, such as electrolysis, even though these processes have been found to be energetically non suitable. In this research various photocatalytic materials were presented as possible alternatives for using in water splitting processes. Characterizationmore » of the new synthesized materials has been done by using different experimental techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), surface area BET, and X-ray Diffraction (XRD). The efficiency of the synthesized photocatalysts was determined by evaluating the hydrogen evolution by the photocatalytic water splitting reaction.« less

Aberrations and adaptive optics in super-resolution microscopy.

PubMed

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-08-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy - or rather nanoscopy - to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy.

Polarized Light Microscopy

NASA Technical Reports Server (NTRS)

Frandsen, Athela F.

2016-01-01

Polarized light microscopy (PLM) is a technique which employs the use of polarizing filters to obtain substantial optical property information about the material which is being observed. This information can be combined with other microscopy techniques to confirm or elucidate the identity of an unknown material, determine whether a particular contaminant is present (as with asbestos analysis), or to provide important information that can be used to refine a manufacturing or chemical process. PLM was the major microscopy technique in use for identification of materials for nearly a century since its introduction in 1834 by William Fox Talbot, as other techniques such as SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared spectroscopy), XPD (X-ray Powder Diffraction), and TEM (Transmission Electron Microscopy) had not yet been developed. Today, it is still the only technique approved by the Environmental Protection Agency (EPA) for asbestos analysis, and is often the technique first applied for identification of unknown materials. PLM uses different configurations in order to determine different material properties. With each configuration additional clues can be gathered, leading to a conclusion of material identity. With no polarizing filter, the microscope can be used just as a stereo optical microscope, and view qualities such as morphology, size, and number of phases. With a single polarizing filter (single polars), additional properties can be established, such as pleochroism, individual refractive indices, and dispersion staining. With two polarizing filters (crossed polars), even more can be deduced: isotropy vs. anisotropy, extinction angle, birefringence/degree of birefringence, sign of elongation, and anomalous polarization colors, among others. With the use of PLM many of these properties can be determined in a matter of seconds, even for those who are not highly trained. McCrone, a leader in the field of polarized light microscopy, often advised, If you cant determine a specific optical property of a particle after two minutes, move onto another configuration. Since optical properties can be seen so very quickly and easily under polarized light, it is only necessary to spend a maximum of two minutes on a technique to determine a particular property, though often only a few seconds are required.

Persistent measles virus infection of the intestine: confirmation by immunogold electron microscopy.

PubMed Central

Lewin, J; Dhillon, A P; Sim, R; Mazure, G; Pounder, R E; Wakefield, A J

1995-01-01

This study sought to investigate persistent measles virus infection of the intestine: a novel protocol for immunogold electron microscopy was developed using a polyclonal anti-measles nucleoprotein antibody on reprocessed, formalin fixed paraffin wax embedded tissue sections. Antibody binding was detected using both immunoperoxidase and light microscopy on tissue sections, and 10 nm gold conjugated secondary antibody and electron microscopy on ultrathin sections. The techniques were validated using both measles infected vero cells and human tissues with established measles infection: these included brain affected by subacute sclerosing panencephalitis and acute measles appendicitis. The technique was applied subsequently to six untreated cases of granulomatous Crohn's disease, and two cases of ileocaecal tuberculosis, a granulomatous control. Mumps primary antibody--applied to both mumps infected vero cells, and measles infected vero cells and tissues studied by immunoperoxidase, and measles antibody on mumps infected cells studied by immunoperoxidase and immunogold--were used as specificity controls: the primary antibodies identified their respective target antigen and there was no antibody cross reactivity. Measles virus nucleocapsids labelled with gold conjugated antibody in both infected cells and tissues, including foci of granulomatous inflammation in five of six cases of Crohn's disease: in the fifth case, the granuloma could not be identified in ultrathin section. In one of the tuberculosis cases, a low level of signal was noted while the second case was negative. Labelling adopted a characteristic pattern in all infected tissues, strengthening the specificity of these findings. This study provides the first direct confirmation of persistent measles virus infection of the intestine. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7737565

Directly Observing Micelle Fusion and Growth in Solution by Liquid-Cell Transmission Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parent, Lucas R.; Bakalis, Evangelos; Ramírez-Hernández, Abelardo

Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporalmore » and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution.« less

Nanotechnology solutions for Alzheimer's disease: advances in research tools, diagnostic methods and therapeutic agents.

PubMed

Nazem, Amir; Mansoori, G Ali

2008-03-01

A century of research has passed since the discovery and definition of Alzheimer's disease (AD), the primary common dementing disorder worldwide. However, AD lacks definite diagnostic approaches and effective cure at the present. Moreover, the currently available diagnostic tools are not sufficient for an early screening of AD in order to start preventive approaches. Recently the emerging field of nanotechnology has promised new techniques to solve some of the AD challenges. Nanotechnology refers to the techniques of designing and manufacturing nanosize (1-100 nm) structures through controlled positional and/or self-assembly of atoms and molecules. In this report, we present the promises that nanotechnology brings in research on the AD diagnosis and therapy. They include its potential for the better understanding of the AD root cause molecular mechanisms, AD's early diagnoses, and effective treatment. The advances in AD research offered by the atomic force microscopy, single molecule fluorescence microscopy and NanoSIMS microscopy are examined here. In addition, the recently proposed applications of nanotechnology for the early diagnosis of AD including bio-barcode assay, localized surface plasmon resonance nanosensor, quantum dot and nanomechanical cantilever arrays are analyzed. Applications of nanotechnology in AD therapy including neuroprotections against oxidative stress and anti-amyloid therapeutics, neuroregeneration and drug delivery beyond the blood brain barrier (BBB) are discussed and analyzed. All of these applications could improve the treatment approach of AD and other neurodegenerative diseases. The complete cure of AD may become feasible by a combination of nanotechnology and some other novel approaches, like stem cell technology.

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

PubMed

Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

2016-06-06

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

In-situ Isotopic Analysis at Nanoscale using Parallel Ion Electron Spectrometry: A Powerful New Paradigm for Correlative Microscopy

NASA Astrophysics Data System (ADS)

Yedra, Lluís; Eswara, Santhana; Dowsett, David; Wirtz, Tom

2016-06-01

Isotopic analysis is of paramount importance across the entire gamut of scientific research. To advance the frontiers of knowledge, a technique for nanoscale isotopic analysis is indispensable. Secondary Ion Mass Spectrometry (SIMS) is a well-established technique for analyzing isotopes, but its spatial-resolution is fundamentally limited. Transmission Electron Microscopy (TEM) is a well-known method for high-resolution imaging down to the atomic scale. However, isotopic analysis in TEM is not possible. Here, we introduce a powerful new paradigm for in-situ correlative microscopy called the Parallel Ion Electron Spectrometry by synergizing SIMS with TEM. We demonstrate this technique by distinguishing lithium carbonate nanoparticles according to the isotopic label of lithium, viz. 6Li and 7Li and imaging them at high-resolution by TEM, adding a new dimension to correlative microscopy.

Axial range of conjugate adaptive optics in two-photon microscopy

PubMed Central

Paudel, Hari P.; Taranto, John; Mertz, Jerome; Bifano, Thomas

2015-01-01

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy. PMID:26367938

Axial range of conjugate adaptive optics in two-photon microscopy.

PubMed

Paudel, Hari P; Taranto, John; Mertz, Jerome; Bifano, Thomas

2015-08-10

We describe an adaptive optics technique for two-photon microscopy in which the deformable mirror used for aberration compensation is positioned in a plane conjugate to the plane of the aberration. We demonstrate in a proof-of-principle experiment that this technique yields a large field of view advantage in comparison to standard pupil-conjugate adaptive optics. Further, we show that the extended field of view in conjugate AO is maintained over a relatively large axial translation of the deformable mirror with respect to the conjugate plane. We conclude with a discussion of limitations and prospects for the conjugate AO technique in two-photon biological microscopy.

Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy.

PubMed

Chemes, Hector E

2013-01-01

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

Localization of single biological molecules out of the focal plane

NASA Astrophysics Data System (ADS)

Gardini, L.; Capitanio, M.; Pavone, F. S.

2014-03-01

Since the behaviour of proteins and biological molecules is tightly related to the cell's environment, more and more microscopy techniques are moving from in vitro to in living cells experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). We developed an apparatus that combines different microscopy techniques to satisfy all the technical requirements for 3D tracking of single fluorescent molecules inside living cells with nanometer accuracy. To account for the optical sectioning of thick samples we built up a HILO (Highly Inclined and Laminated Optical sheet) microscopy system through which we can excite the sample in a widefield (WF) configuration by a thin sheet of light that can follow the molecule up and down along the z axis spanning the entire thickness of the cell with a SNR much higher than traditional WF microscopy. Since protein dynamics inside a cell involve all three dimensions, we included a method to measure the x, y, and z coordinates with nanometer accuracy, exploiting the properties of the point-spread-function of out-of-focus quantum dots bound to the protein of interest. Finally, a feedback system stabilizes the microscope from thermal drifts, assuring accurate localization during the entire duration of the experiment.

Correlation of two-photon in vivo imaging and FIB/SEM microscopy

PubMed Central

Blazquez-Llorca, L; Hummel, E; Zimmerman, H; Zou, C; Burgold, S; Rietdorf, J; Herms, J

2015-01-01

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two-photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long-term two-photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer’s disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three-dimensional high-resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system. Lay Description Neuroscience and the understanding of brain functions are closely linked to the technical advances in microscopy. In this study we performed a correlative microscopy technique that offers the possibility to combine 2 photon in vivo imaging and FIB/SEM microscopy. Long term 2 photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool to study the dynamics of neurodegenerative diseases, such as Alzheimer’s disease. However, light microscopy has important limitations in revealing synapses that are the connections between neurons, and for this purpose, the electron microscopy is necessary. FIB/SEM microscopy is a novel tool for three-dimensional (3D) high resolution reconstructions since it acquires automated serial images at ultrastructural level. This correlative technique will open up new horizons and opportunities for unravelling the complexity of the nervous system. PMID:25786682

Integral refractive index imaging of flowing cell nuclei using quantitative phase microscopy combined with fluorescence microscopy.

PubMed

Dardikman, Gili; Nygate, Yoav N; Barnea, Itay; Turko, Nir A; Singh, Gyanendra; Javidi, Barham; Shaked, Natan T

2018-03-01

We suggest a new multimodal imaging technique for quantitatively measuring the integral (thickness-average) refractive index of the nuclei of live biological cells in suspension. For this aim, we combined quantitative phase microscopy with simultaneous 2-D fluorescence microscopy. We used 2-D fluorescence microscopy to localize the nucleus inside the quantitative phase map of the cell, as well as for measuring the nucleus radii. As verified offline by both 3-D confocal fluorescence microscopy and 2-D fluorescence microscopy while rotating the cells during flow, the nucleus of cells in suspension that are not during division can be assumed to be an ellipsoid. The entire shape of a cell in suspension can be assumed to be a sphere. Then, the cell and nucleus 3-D shapes can be evaluated based on their in-plain radii available from the 2-D phase and fluorescent measurements, respectively. Finally, the nucleus integral refractive index profile is calculated. We demonstrate the new technique on cancer cells, obtaining nucleus refractive index values that are lower than those of the cytoplasm, coinciding with recent findings. We believe that the proposed technique has the potential to be used for flow cytometry, where full 3-D refractive index tomography is too slow to be implemented during flow.

Detection, Mapping, and Quantification of Single Walled Carbon Nanotubes in Histological Specimens with Photoacoustic Microscopy

PubMed Central

Mikos, Antonios G.; Jansen, John A.; Shroyer, Kenneth R.; Wang, Lihong V.; Sitharaman, Balaji

2012-01-01

Aims In the present study, the efficacy of multi-scale photoacoustic microscopy (PAM) was investigated to detect, map, and quantify trace amounts [nanograms (ng) to micrograms (µg)] of SWCNTs in a variety of histological tissue specimens consisting of cancer and benign tissue biopsies (histological specimens from implanted tissue engineering scaffolds). Materials and Methods Optical-resolution (OR) and acoustic-resolution (AR) - Photoacoustic microscopy (PAM) was employed to detect, map and quantify the SWCNTs in a variety of tissue histological specimens and compared with other optical techniques (bright-field optical microscopy, Raman microscopy, near infrared (NIR) fluorescence microscopy). Results Both optical-resolution and acoustic-resolution PAM, allow the detection and quantification of SWCNTs in histological specimens with scalable spatial resolution and depth penetration. The noise-equivalent detection sensitivity to SWCNTs in the specimens was calculated to be as low as ∼7 pg. Image processing analysis further allowed the mapping, distribution, and quantification of the SWCNTs in the histological sections. Conclusions The results demonstrate the potential of PAM as a promising imaging technique to detect, map, and quantify SWCNTs in histological specimens, and could complement the capabilities of current optical and electron microscopy techniques in the analysis of histological specimens containing SWCNTs. PMID:22496892

Widefield fluorescence sectioning with HiLo microscopy.

PubMed

Mertz, Jerome; Lim, Daryl; Chu, Kengyeh K; Bozinovic, Nenad; Ford, Timothy

2009-01-01

HiLo microscopy is a widefield fluorescence imaging technique that provides depth discrimination by combining two images, one with non-uniform illumination and one with uniform illumination. We discuss the theory of this technique and a variety of practical implementations in brain-tissue imaging and fluorescence endomicroscopy.

Holographic techniques for cellular fluorescence microscopy

NASA Astrophysics Data System (ADS)

Kim, Myung K.

2017-04-01

We have constructed a prototype instrument for holographic fluorescence microscopy (HFM) based on self-interference incoherent digital holography (SIDH) and demonstrate novel imaging capabilities such as differential 3D fluorescence microscopy and optical sectioning by compressive sensing.

Imaging of single retinal ganglion cell with differential interference contrast microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Oh, Juyeong; Kim, Yu Jeong; Kim, Chul-Ki; Lee, Taik Jin; Seo, Mina; Lee, Seok; Woo, Deok Ha; Jun, Seong Chan; Park, Ki-Ho; Kim, Seok Hwan; Kim, Jae Hun

2017-02-01

Glaucoma is a progressive optic neuropathy, characterized by the selective loss of retinal ganglion cells (RGCs). Therefore, monitoring the change of number or morphology of RGC is essential for the early detection as well as investigation of pathophysiology of glaucoma. Since RGC layer is transparent and hyporeflective, the direct optical visualization of RGCs has not been successful so far. Therefore, glaucoma evaluation mostly depends on indirect diagnostic methods such as the evaluation of optic disc morphology or retinal nerve fiber layer thickness measurement by optical coherence tomography. We have previously demonstrated single photoreceptor cell imaging with differential interference contrast (DIC) microscopy. Herein, we successfully visualized single RGC using DIC microscopy. Since RGC layer is much less reflective than photoreceptor layer, various techniques including the control of light wavelength and bandwidth using a tunable band pass filter were adopted to reduce the chromatic aberration in z-axis for higher and clearer resolution. To verify that the imaged cells were the RGCs, the flat-mounted retina of Sprague-Dawley rat, in which the RGCs were retrogradely labeled with fluorescence, was observed by both fluorescence and DIC microscopies for direct comparison. We have confirmed that the cell images obtained by fluorescence microscopy were perfectly matched with cell images by DIC microscopy. As conclusion, we have visualized single RGC with DIC microscopy, and confirmed with fluorescence microscopy.

Lens-free microscopy of cerebrospinal fluid for the laboratory diagnosis of meningitis

NASA Astrophysics Data System (ADS)

Delacroix, Robin; Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Blandin, Pierre; Dinten, Jean-Marc; Drancourt, Michel; Allier, Cédric

2018-02-01

The cytology of the cerebrospinal fluid is traditionally performed by an operator (physician, biologist) by means of a conventional light microscope. The operator visually counts the leukocytes (white blood cells) present in a sample of cerebrospinal fluid (10 μl). It is a tedious job and the result is operator-dependent. Here in order to circumvent the limitations of manual counting, we approach the question of numeration of erythrocytes and leukocytes for the cytological diagnosis of meningitis by means of lens-free microscopy. In a first step, a prospective counts of leukocytes was performed by five different operators using conventional optical microscopy. The visual counting yielded an overall 16.7% misclassification of 72 cerebrospinal fluid specimens in meningitis/non-meningitis categories using a 10 leukocyte/μL cut-off. In a second step, the lens-free microscopy algorithm was adapted step-by-step for counting cerebrospinal fluid cells and discriminating leukocytes from erythrocytes. The optimization of the automatic lens-free counting was based on the prospective analysis of 215 cerebrospinal fluid specimens. The optimized algorithm yielded a 100% sensitivity and a 86% specificity compared to confirmed diagnostics. In a third step, a blind lens-free microscopic analysis of 116 cerebrospinal fluid specimens, including six cases of microbiology confirmed infectious meningitis, yielded a 100% sensitivity and a 79% specificity. Adapted lens-free microscopy is thus emerging as an operator-independent technique for the rapid numeration of leukocytes and erythrocytes in cerebrospinal fluid. In particular, this technique is well suited to the rapid diagnosis of meningitis at point-of-care laboratories.

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE PAGES

Ducic, Tanja; Paunesku, Tatjana; Chen, Si; ...

2016-12-09

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

Structural and elemental changes in glioblastoma cells in situ: complementary imaging with high resolution visible light- and X-ray microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ducic, Tanja; Paunesku, Tatjana; Chen, Si

The glioblastoma (GBM) is characterized by a short median survival and an almost 100% tumor related mortality. GBM cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores application of X-ray and visible light microscopy to display the elemental and structural images of cells from 3 patient derived GMB samples and an established GMB cell line. Slight differences in elemental concentrations, in actin cytoskeleton organization and cell morphology were noted between all cells types by X-ray fluorescence and full field soft X-ray microscopy, as well as the Structured Illumination Super-resolution Microscope (SIM). Different samplemore » preparation approaches were used to match each imaging technique. While preparation for SIM included cell fixation and staining, intact frozen hydrated cells were used for the trace element imaging by hard X-ray fluorescence and exploration of the structural features by soft X-ray absorption tomography. In conclusion, each technique documented differences between samples with regard to morphology and elemental composition and underscored the importance of use of multiple patient derived samples for detailed GBM study.« less

The actin cytoskeleton in whole mount preparations and sections.

PubMed

Resch, Guenter P; Urban, Edit; Jacob, Sonja

2010-01-01

In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

Segmentation of fluorescence microscopy cell images using unsupervised mining.

PubMed

Du, Xian; Dua, Sumeet

2010-05-28

The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

Optical toolkits for in vivo deep tissue laser scanning microscopy: a primer

NASA Astrophysics Data System (ADS)

Lee, Woei Ming; McMenamin, Thomas; Li, Yongxiao

2018-06-01

Life at the microscale is animated and multifaceted. The impact of dynamic in vivo microscopy in small animals has opened up opportunities to peer into a multitude of biological processes at the cellular scale in their native microenvironments. Laser scanning microscopy (LSM) coupled with targeted fluorescent proteins has become an indispensable tool to enable dynamic imaging in vivo at high temporal and spatial resolutions. In the last few decades, the technique has been translated from imaging cells in thin samples to mapping cells in the thick biological tissue of living organisms. Here, we sought to provide a concise overview of the design considerations of a LSM that enables cellular and subcellular imaging in deep tissue. Individual components under review include: long working distance microscope objectives, laser scanning technologies, adaptive optics devices, beam shaping technologies and photon detectors, with an emphasis on more recent advances. The review will conclude with the latest innovations in automated optical microscopy, which would impact tracking and quantification of heterogeneous populations of cells in vivo.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching

PubMed Central

Gerbich, Therese M.; Rana, Kishan; Suzuki, Aussie; Schaefer, Kristina N.; Heppert, Jennifer K.; Boothby, Thomas C.; Allbritton, Nancy L.; Gladfelter, Amy S.; Maddox, Amy S.

2018-01-01

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution. PMID:29490939

Non-invasive current and voltage imaging techniques for integrated circuits using scanning probe microscopy. Final report, LDRD Project FY93 and FY94

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campbell, A.N.; Cole, E.I. Jr.; Tangyunyong, Paiboon

This report describes the first practical, non-invasive technique for detecting and imaging currents internal to operating integrated circuits (ICs). This technique is based on magnetic force microscopy and was developed under Sandia National Laboratories` LDRD (Laboratory Directed Research and Development) program during FY 93 and FY 94. LDRD funds were also used to explore a related technique, charge force microscopy, for voltage probing of ICs. This report describes the technical work performed under this LDRD as well as the outcomes of the project in terms of publications and awards, intellectual property and licensing, synergistic work, potential future work, hiring ofmore » additional permanent staff, and benefits to DOE`s defense programs (DP).« less

Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

NASA Astrophysics Data System (ADS)

Esposito, Alessandro

2006-05-01

This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

Diagnostic performance of direct wet mount microscopy in detecting intestinal helminths among pregnant women attending ante-natal care (ANC) in East Wollega, Oromia, Ethiopia.

PubMed

Mengist, Hylemariam Mihiretie; Demeke, Gebreselassie; Zewdie, Olifan; Belew, Adugna

2018-05-04

The aim of this study was to evaluate the diagnostic performance of direct wet mount microscopy compared to formalin ether concentration (FEC) technique in detecting intestinal helminths in pregnant women. The total prevalence of intestinal helminths was 18.8% (70/372) by direct wet mount microscopy and 24.7% (92/372) by FEC technique (P < 0.001). The sensitivity, negative predictive value (NPV) and test efficiency (TE) of direct wet mount microscopy in diagnosing intestinal helminths was 76, 92.7 and 94%, respectively. The sensitivity of direct w et mount microscopy was very low in detecting ova of Hymenolepis nana. The two methods showed excellent agreement in detecting ova of Hook worm and Ascaris lumbricoides (Kappa > 0.81) but they fairly agreed in detecting ova of Hymenolepis nana (Kappa = 0.39). Intestinal helminths were underdiagnosed and the total diagnostic performance of direct wet mount microscopy was significantly poor in detecting intestinal helminths as compared to FEC technique. Routine use of FEC method is recommended for the diagnosis of intestinal helminths in pregnant women.

The external morphology of adult female Egrasilus labracis as shown using hexamethyldisilazane treated, uncoated specimens for scanning electron microscopy.

PubMed

Murray, Harry M; Hill, Stephen J; Ang, Keng P

2016-07-01

The description and application of a modified Scanning Electron Microscope preparation technique using hexamethyldisilazane for small parasitic copepods was demonstrated though a high resolution depiction of individuals of Ergasilus labracis sampled from three spined stickleback (Gasterosteus aculeatus) in Bay D'Espoir, Newfoundland during summer 2015 and from archival samples retrieved from Atlantic salmon par (Salmo salar) stored at the Atlantic reference centre, St. Andrews, New Brunswick. The specimens were very well preserved showing high quality detail of important features and verifying those previously described using light microscopy by Hogans. Additionally the technique allowed excellent in situ demonstrations of mouth parts, swimming legs, and unusual and previously undescribed features of the second antenna including prominent striations and pore-like structures found to define the claw. It is thought that this technique will become a quick and efficient tool for describing important taxonomic features of small parasitic copepods like E. labracis or other similar small aquatic organisms. Microsc. Res. Tech. 79:657-663, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Advanced microscopy of star-shaped gold nanoparticles and their adsorption-uptake by macrophages

PubMed Central

Plascencia-Villa, Germán; Bahena, Daniel; Rodríguez, Annette R.; Ponce, Arturo; José-Yacamán, Miguel

2013-01-01

Metallic nanoparticles have diverse applications in biomedicine, as diagnostics, image contrast agents, nanosensors and drug delivery systems. Anisotropic metallic nanoparticles possess potential applications in cell imaging and therapy+diagnostics (theranostics), but controlled synthesis and growth of these anisotropic or branched nanostructures has been challenging and usually require use of high concentrations of surfactants. Star-shaped gold nanoparticles were synthesized in high yield through a seed mediated route using HEPES as a precise shape-directing capping agent. Characterization was performed using advanced electron microscopy techniques including atomic resolution TEM, obtaining a detailed characterization of nanostructure and atomic arrangement. Spectroscopy techniques showed that particles have narrow size distribution, monodispersity and high colloidal stability, with absorbance into NIR region and high efficiency for SERS applications. Gold nanostars showed to be biocompatible and efficiently adsorbed and internalized by macrophages, as revealed by advanced FE-SEM and backscattered electron imaging techniques of complete unstained uncoated cells. Additionally, low voltage STEM and X-ray microanalysis revealed the ultra-structural location and confirmed stability of nanoparticles after endocytosis with high spatial resolution. PMID:23443314

Characterization of Metal Powders Used for Additive Manufacturing.

PubMed

Slotwinski, J A; Garboczi, E J; Stutzman, P E; Ferraris, C F; Watson, S S; Peltz, M A

2014-01-01

Additive manufacturing (AM) techniques can produce complex, high-value metal parts, with potential applications as critical parts, such as those found in aerospace components. The production of AM parts with consistent and predictable properties requires input materials (e.g., metal powders) with known and repeatable characteristics, which in turn requires standardized measurement methods for powder properties. First, based on our previous work, we assess the applicability of current standardized methods for powder characterization for metal AM powders. Then we present the results of systematic studies carried out on two different powder materials used for additive manufacturing: stainless steel and cobalt-chrome. The characterization of these powders is important in NIST efforts to develop appropriate measurements and standards for additive materials and to document the property of powders used in a NIST-led additive manufacturing material round robin. An extensive array of characterization techniques was applied to these two powders, in both virgin and recycled states. The physical techniques included laser diffraction particle size analysis, X-ray computed tomography for size and shape analysis, and optical and scanning electron microscopy. Techniques sensitive to structure and chemistry, including X-ray diffraction, energy dispersive analytical X-ray analysis using the X-rays generated during scanning electron microscopy, and X-Ray photoelectron spectroscopy were also employed. The results of these analyses show how virgin powder changes after being exposed to and recycled from one or more Direct Metal Laser Sintering (DMLS) additive manufacturing build cycles. In addition, these findings can give insight into the actual additive manufacturing process.

Leakage radiation interference microscopy.

PubMed

Descrovi, Emiliano; Barakat, Elsie; Angelini, Angelo; Munzert, Peter; De Leo, Natascia; Boarino, Luca; Giorgis, Fabrizio; Herzig, Hans Peter

2013-09-01

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

Combined time-lapse cinematography and immuno-electron microscopy.

PubMed

Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

1990-04-01

A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

Molecular expressions: exploring the world of optics and microscopy. http://microscopy.fsu.edu.

PubMed

Eliceiri, Kevin W

2004-08-01

Our knowledge of the structure, dynamics and physiology of a cell has increased significantly in the last ten years through the emergence of new optical imaging modalities such as optical sectioning microscopy, computer- enhanced video microscopy and laser-scanning microscopy. These techniques together with the use of genetically engineered fluorophores have helped scientists visualize the 3-dimensional dynamic processes of living cells. However as powerful as these imaging tools are, they can often be difficult to understand and fully utilize. Below I will discuss my favorite website: The Molecular Expressions Web Site that endeavors to present the power of microscopy to its visitors. The Molecular Expressions group does a remarkable job of not only clearly presenting the principles behind these techniques in a manner approachable by lay and scientific audiences alike but also provides representative data from each as well.

Scanning Tunneling Microscopy analysis of space-exposed polymer films

NASA Technical Reports Server (NTRS)

Kalil, Carol R.; Young, Philip R.

1993-01-01

The characterization of the surface of selected space-exposed polymer films by Scanning Tunneling Microscopy (STM) is reported. Principles of STM, an emerging new technique for materials analysis, are reviewed. The analysis of several films which received up to 5.8 years of low Earth orbital (LEO) exposure onboard the NASA Long Duration Exposure Facility (LDEF) is discussed. Specimens included FEP Teflon thermal blanket material, Kapton film, and several experimental polymer films. Ultraviolet and atomic oxygen-induced crazing and erosion are described. The intent of this paper is to demonstrate how STM is enhancing the understanding of LEO space environmental effects on polymer films.

Particle image diffusometry: Resolving diffusion coefficient field from microscopy movie data without particle tracking

NASA Astrophysics Data System (ADS)

Hanasaki, Itsuo; Ooi, Yuto

2018-06-01

We propose a technique to evaluate the field of diffusion coefficient for particle dispersion where the Brownian motion is heterogeneous in space and single particle tracking (SPT) analysis is hindered by high concentration of the particles and/or their small size. We realize this "particle image diffusometry" by the principle of the differential dynamic microscopy (DDM). We extend the DDM by introducing the automated objective decision of the scaling regime itself. Label-free evaluation of spatially non-uniform diffusion coefficients without SPT is useful in the diverse applications including crystal nucleation and glass transition where non-invasive observation is desired.

Looking at tardigrades in a new light: using epifluorescence to interpret structure.

PubMed

Perry, E S; Miller, W R; Lindsay, S

2015-02-01

The use of epifluorescence microscopy coupled with ultraviolet (UV) autofluorescence is suggested as a means to view and interpret tardigrade structures. Endogenous fluorochromes are a known component of tardigrade cuticle, claws and bucco-pharyngeal apparatus. By imaging the autofluorescence from tardigrades, it is possible to document these structures in detail, including the subdivisions and boundaries of echiniscid (heterotardigrade) plates and the nature and spatial relationships of the texture (pores, granules, papillae and tubercles) on the various plates. This allows the determination of taxonomic features not easily seen with other microscopic techniques. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

The use of analytical surface tools in the fundamental study of wear. [atomic nature of wear

NASA Technical Reports Server (NTRS)

Buckley, D. H.

1977-01-01

Various techniques and surface tools available for the study of the atomic nature of the wear of materials are reviewed These include chemical etching, x-ray diffraction, electron diffraction, scanning electron microscopy, low-energy electron diffraction, Auger emission spectroscopy analysis, electron spectroscopy for chemical analysis, field ion microscopy, and the atom probe. Properties of the surface and wear surface regions which affect wear, such as surface energy, crystal structure, crystallographic orientation, mode of dislocation behavior, and cohesive binding, are discussed. A number of mechanisms involved in the generation of wear particles are identified with the aid of the aforementioned tools.

Cytological Analysis of Meiosis in Caenorhabditis elegans

PubMed Central

Phillips, Carolyn M.; McDonald, Kent L.; Dernburg, Abby F.

2011-01-01

The nematode Caenorhabditis elegans has emerged as an informative experimental system for analysis of meiosis, in large part because of the advantageous physical organization of meiotic nuclei as a gradient of stages within the germline. Here we provide tools for detailed observational studies of cells within the worm gonad, including techniques for light and electron microscopy. PMID:19685325

Single-shot full resolution region-of-interest (ROI) reconstruction in image plane digital holographic microscopy

NASA Astrophysics Data System (ADS)

Singh, Mandeep; Khare, Kedar

2018-05-01

We describe a numerical processing technique that allows single-shot region-of-interest (ROI) reconstruction in image plane digital holographic microscopy with full pixel resolution. The ROI reconstruction is modelled as an optimization problem where the cost function to be minimized consists of an L2-norm squared data fitting term and a modified Huber penalty term that are minimized alternately in an adaptive fashion. The technique can provide full pixel resolution complex-valued images of the selected ROI which is not possible to achieve with the commonly used Fourier transform method. The technique can facilitate holographic reconstruction of individual cells of interest from a large field-of-view digital holographic microscopy data. The complementary phase information in addition to the usual absorption information already available in the form of bright field microscopy can make the methodology attractive to the biomedical user community.

Nonlinear dynamic phase contrast microscopy for microfluidic and microbiological applications

NASA Astrophysics Data System (ADS)

Denz, C.; Holtmann, F.; Woerdemann, M.; Oevermann, M.

2008-08-01

In live sciences, the observation and analysis of moving living cells, molecular motors or motion of micro- and nano-objects is a current field of research. At the same time, microfluidic innovations are needed for biological and medical applications on a micro- and nano-scale. Conventional microscopy techniques are reaching considerable limits with respect to these issues. A promising approach for this challenge is nonlinear dynamic phase contrast microscopy. It is an alternative full field approach that allows to detect motion as well as phase changes of living unstained micro-objects in real-time, thereby being marker free, without contact and non destructive, i.e. fully biocompatible. The generality of this system allows it to be combined with several other microscope techniques such as conventional bright field or fluorescence microscopy. In this article we will present the dynamic phase contrast technique and its applications in analysis of micro organismic dynamics, micro flow velocimetry and micro-mixing analysis.

Aberrations and adaptive optics in super-resolution microscopy

PubMed Central

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 1; A Study Employing Neural Cell Adhesion Molecules (N-CAM) Immunocytochemistry and Conventional Morphological Techniques (Light and Electron Microscopy)

NASA Technical Reports Server (NTRS)

Daunton, N. G.; DAmelio, F.; Wu, L.; Ilyina-Kakueva, E. I.; Krasnov, I. B.; Hyde, T. M.; Sigworth, S. K.

1994-01-01

The effects of spaceflight upon the 'slow' muscle adductor longus was examined in rats flown in the Soviet Biosatellite COSMOS 2044. Three groups - synchronous, vivarium and basal served as controls. The techniques employed included standard methods for light microscopy, N-CAM immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy, contraction bands and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leucocytes and mononuclear cells. N-CAM immunoreactivity was seen (N-CAM-IR) on the myofiber surface, satellite cells and in regenerating myofibers reminiscent of myotubes. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments that displayed varied distributive patterns. The principal electron microscopic changes of the neuromuscular junctions consisted of a decrease or absence of synaptic vesicles, degeneration of axon terminals, increased number of microtubules, vacant axonal spaces and axonal sprouting. The present observations indicate that major alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

PubMed Central

Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

2007-01-01

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

PubMed

Shebanova, A S; Bogdanov, A G; Ismagulova, T T; Feofanov, A V; Semenyuk, P I; Muronets, V I; Erokhina, M V; Onishchenko, G E; Kirpichnikov, M P; Shaitan, K V

2014-01-01

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Detection of stiff nanoparticles within cellular structures by contact resonance atomic force microscopy subsurface nanomechanical imaging.

PubMed

Reggente, Melania; Passeri, Daniele; Angeloni, Livia; Scaramuzzo, Francesca Anna; Barteri, Mario; De Angelis, Francesca; Persiconi, Irene; De Stefano, Maria Egle; Rossi, Marco

2017-05-04

Detecting stiff nanoparticles buried in soft biological matrices by atomic force microscopy (AFM) based techniques represents a new frontier in the field of scanning probe microscopies, originally developed as surface characterization methods. Here we report the detection of stiff (magnetic) nanoparticles (NPs) internalized in cells by using contact resonance AFM (CR-AFM) employed as a potentially non-destructive subsurface characterization tool. Magnetite (Fe 3 O 4 ) NPs were internalized in microglial cells from cerebral cortices of mouse embryos of 18 days by phagocytosis. Nanomechanical imaging of cells was performed by detecting the contact resonance frequencies (CRFs) of an AFM cantilever held in contact with the sample. Agglomerates of NPs internalized in cells were visualized on the basis of the local increase in the contact stiffness with respect to the surrounding biological matrix. A second AFM-based technique for nanomechanical imaging, i.e., HarmoniX™, as well as magnetic force microscopy and light microscopy were used to confirm the CR-AFM results. Thus, CR-AFM was demonstrated as a promising technique for subsurface imaging of nanomaterials in biological samples.

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

Gamete competence assessment by polarizing optics in assisted reproduction.

PubMed

Montag, Markus; Köster, Maria; van der Ven, Katrin; van der Ven, Hans

2011-01-01

The purpose of this study was first to give an overview of the historical development of polarization microscopy, second to describe the various applications of this technique in assisted reproduction techniques (ART) and third to discuss the potential benefit of polarization microscopy as a predictor for IVF success. The history of polarization microscopy was undertaken by performing a backward search in the scientific literature using Google and internet sites of several Societies for Microscopy and Cell Biology. Studies of polarization microscopy in ART were identified by using a systematic literature search in PubMed and Scopus. A total of 62 articles were identified by the direct search and further relevant articles were found by screening the cited literature in these articles. The topics relevant for assisted reproduction were spindle and zona imaging in combination with IVF success, meiotic cell cycle progression, pharmaceutical studies and cryopreservation. A separate topic was the use of sperm birefringence in ART. The majority of studies are observational studies and were not performed in a randomized manner and there is no direct comparison of techniques using other gamete selection markers. Despite this, most studies show that polarization microscopy may help us to further increase our knowledge on gametes and meiosis. Whether certain applications such as spindle or zona imaging may lead to an increase in IVF success is unclear at present. Publications on the use of polarization microscopy on sperm are still very limited.

Condenser-free contrast methods for transmitted-light microscopy

PubMed Central

WEBB, K F

2015-01-01

Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared. PMID:25226859

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques.

PubMed

Marcos-Garcés, V; Harvat, M; Molina Aguilar, P; Ferrández Izquierdo, A; Ruiz-Saurí, A

2017-08-01

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Fluorescence lifetime in cardiovascular diagnostics

NASA Astrophysics Data System (ADS)

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Fluorescence lifetime in cardiovascular diagnostics.

PubMed

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications.

Demonstration of transmission high energy electron microscopy

DOE PAGES

Merrill, F. E.; Goett, J.; Gibbs, J. W.; ...

2018-04-06

High energy electrons have been used to investigate an extension of transmission electron microscopy. This technique, transmission high energy electron microscopy (THEEM), provides two additional capabilities to electron microscopy. First, high energy electrons are more penetrating than low energy electrons, and thus, they are able to image through thicker samples. Second, the accelerating mode of a radio-frequency linear accelerator provides fast exposures, down to 1 ps, which are ideal for flash radiography, making THEEM well suited to study the evolution of fast material processes under dynamic conditions. Lastly, initial investigations with static objects and during material processing have been performedmore » to investigate the capabilities of this technique.« less

Open-source do-it-yourself multi-color fluorescence smartphone microscopy

PubMed Central

Sung, Yulung; Campa, Fernando; Shih, Wei-Chuan

2017-01-01

Fluorescence microscopy is an important technique for cellular and microbiological investigations. Translating this technique onto a smartphone can enable particularly powerful applications such as on-site analysis, on-demand monitoring, and point-of-care diagnostics. Current fluorescence smartphone microscope setups require precise illumination and imaging alignment which altogether limit its broad adoption. We report a multi-color fluorescence smartphone microscope with a single contact lens-like add-on lens and slide-launched total-internal-reflection guided illumination for three common tasks in investigative fluorescence microscopy: autofluorescence, fluorescent stains, and immunofluorescence. The open-source, simple and cost-effective design has the potential for do-it-yourself fluorescence smartphone microscopy. PMID:29188104

Use of Kelvin probe force microscopy for identification of CVD grown graphene flakes on copper foil

NASA Astrophysics Data System (ADS)

Kumar, Rakesh; Mehta, B. R.; Kanjilal, D.

2017-05-01

Graphene flakes have been grown by chemical vapour deposition (CVD) method on Cu foils. The obtained graphene flakes have been characterized by optical microscopy, field emission scanning electron microscopy, Kelvin probe force microscopy (KPFM) and Raman spectroscopy. The graphene flakes grown on Cu foil comprise mainly single layer graphene and confirm that the nucleation for graphene growth starts very quickly. Moreover, KPFM has been found to be a valuable technique to differentiate between covered and uncovered portion of Cu foil by graphene flakes deposited for shorter duration. The results show that KPFM can be a very useful technique in understanding the mechanism of graphene growth.

Airborne asbestos in Colorado public schools.

PubMed

Chadwick, D A; Buchan, R M; Beaulieu, H J

1985-02-01

Levels of airborne asbestos for six Colorado public school facilities with sprayed-on asbestos materials were documented using three analytical techniques. Phase contrast microscopy showed levels up to the thousandths of a fiber per cubic centimeter (f/cc), scanning electron microscopy (SEM) up to the hundredths of a f/cc, and transmission electron microscopy coupled to selected area electron diffraction and energy dispersive X-ray analysis (TEM-SAED-EDXA) up to the tenths of an asbestos f/cc. Phase contrast microscopy was found to be an inadequate analytical technique for documenting the levels of airborne asbestos fibers in the schools: only large fibers which were not embedded in the filter were counted, and asbestos fibers were not distinguished from nonasbestos.

EDITORIAL: Probing the nanoworld Probing the nanoworld

NASA Astrophysics Data System (ADS)

Miles, Mervyn

2009-10-01

In nanotechnology, it is the unique properties arising from nanometre-scale structures that lead not only to their technological importance but also to a better understanding of the underlying science. Over the last twenty years, material properties at the nanoscale have been dominated by the properties of carbon in the form of the C60 molecule, single- and multi-wall carbon nanotubes, nanodiamonds, and recently graphene. During this period, research published in the journal Nanotechnology has revealed the amazing mechanical properties of such materials as well as their remarkable electronic properties with the promise of new devices. Furthermore, nanoparticles, nanotubes, nanorods, and nanowires from metals and dielectrics have been characterized for their electronic, mechanical, optical, chemical and catalytic properties. Scanning probe microscopy (SPM) has become the main characterization technique and atomic force microscopy (AFM) the most frequently used SPM. Over the past twenty years, SPM techniques that were previously experimental in nature have become routine. At the same time, investigations using AFM continue to yield impressive results that demonstrate the great potential of this powerful imaging tool, particularly in close to physiological conditions. In this special issue a collaboration of researchers in Europe report the use of AFM to provide high-resolution topographical images of individual carbon nanotubes immobilized on various biological membranes, including a nuclear membrane for the first time (Lamprecht C et al 2009 Nanotechnology 20 434001). Other SPM developments such as high-speed AFM appear to be making a transition from specialist laboratories to the mainstream, and perhaps the same may be said for non-contact AFM. Looking to the future, characterisation techniques involving SPM and spectroscopy, such as tip-enhanced Raman spectroscopy, could emerge as everyday methods. In all these advanced techniques, routinely available probes will be needed to make them mainstream methods, as was indeed the case for establishing AFM. At the same time, both transmission electron microscopy and scanning electron microscopy have undergone major developments in resolution, spectroscopic techniques, and new techniques such as tomography. It is exciting to speculate in which areas new properties of materials at the nanoscale will be discovered over the next twenty years, and how characterization methods will evolve, but it is the unimaginable and unpredictable that will bring the most dramatic changes to nanotechnology.

Optical Biomarkers of Serous and Mucinous Human Ovarian Tumor Assessed with Nonlinear Optics Microscopies

PubMed Central

Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; Baratti, Mariana O.; Almeida, Diogo B.; Andrade, L. A. L. A.; Bottcher-Luiz, Fátima; Carvalho, Hernandes F.; Cesar, Carlos L.

2012-01-01

Background Nonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression. Methodology/Principal Findings We obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells. Conclusions/Significance NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions. PMID:23056557

Application of high-angle annular dark field scanning transmission electron microscopy, scanning transmission electron microscopy-energy dispersive X-ray spectrometry, and energy-filtered transmission electron microscopy to the characterization of nanoparticles in the environment.

PubMed

Utsunomiya, Satoshi; Ewing, Rodney C

2003-02-15

A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.

Applications of microscopy in Salmonella research.

PubMed

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Broadband quantitative phase microscopy with extended field of view using off-axis interferometric multiplexing.

PubMed

Girshovitz, Pinhas; Frenklach, Irena; Shaked, Natan T

2015-11-01

We propose a new portable imaging configuration that can double the field of view (FOV) of existing off-axis interferometric imaging setups, including broadband off-axis interferometers. This configuration is attached at the output port of the off-axis interferometer and optically creates a multiplexed interferogram on the digital camera, which is composed of two off-axis interferograms with straight fringes at orthogonal directions. Each of these interferograms contains a different FOV of the imaged sample. Due to the separation of these two FOVs in the spatial-frequency domain, they can be fully reconstructed separately, while obtaining two complex wavefronts from the sample at once. Since the optically multiplexed off-axis interferogram is recorded by the camera in a single exposure, fast dynamics can be recorded with a doubled imaging area. We used this technique for quantitative phase microscopy of biological samples with extended FOV. We demonstrate attaching the proposed module to a diffractive phase microscopy interferometer, illuminated by a broadband light source. The biological samples used for the experimental demonstrations include microscopic diatom shells, cancer cells, and flowing blood cells.

Microstructure, Morphology, and Nanomechanical Properties Near Fine Holes Produced by Electro-Discharge Machining

NASA Astrophysics Data System (ADS)

Blau, P. J.; Howe, J. Y.; Coffey, D. W.; Trejo, R. M.; Kenik, E. D.; Jolly, B. C.; Yang, N.

2012-08-01

Fine holes in metal alloys are employed for many important technological purposes, including cooling and the precise atomization of liquids. For example, they play an important role in the metering and delivery of fuel to the combustion chambers in energy-efficient, low-emission diesel engines. Electro-discharge machining (EDM) is one process employed to produce such holes. Since the hole shape and bore morphology can affect fluid flow, and holes also represent structural discontinuities in the tips of the spray nozzles, it is important to understand the microstructures adjacent to these holes, the features of the hole walls, and the nanomechanical properties of the material that was in some manner altered by the EDM hole-making process. Several techniques were used to characterize the structure and properties of spray-holes in a commercial injector nozzle. These include scanning electron microscopy, cross sectioning and metallographic etching, bore surface roughness measurements by optical interferometry, scanning electron microscopy, and transmission electron microscopy of recast EDM layers extracted with the help of a focused ion beam.

A STED-FLIM microscope applied to imaging the natural killer cell immune synapse

NASA Astrophysics Data System (ADS)

Lenz, M. O.; Brown, A. C. N.; Auksorius, E.; Davis, D. M.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.

2011-03-01

We present a stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, excited by a microstructured optical fibre supercontinuum source that is pumped by a femtosecond Ti:Sapphire-laser, which is also used for depletion. Implemented using a piezo-scanning stage on a laser scanning confocal fluorescence microscope system with FLIM realised using time correlated single photon counting (TCSPC), this provides convenient switching between confocal and STED-FLIM with spatial resolution down to below 60 nm. We will present our design considerations to make a robust instrument for biological applications including a comparison between fixed phase plate and spatial light modulator (SLM) approaches to shape the STED beam and the correlation of STED and confocal FLIM microscopy. Following our previous application of FLIM-FRET to study intercellular signalling at the immunological synapse (IS), we are employing STED microscopy to characterize the spatial distribution of cellular molecules with subdiffraction resolution at the IS. In particular, we are imaging cytoskeletal structure at the Natural Killer cell activated immune synapse. We will also present our progress towards multilabel STED microscopy to determine how relative spatial molecular organization, previously undetectable by conventional microscopy techniques, is important for NK cell cytotoxic function. Keywords: STED, Stimulated Emission Depletion Microscopy, Natural Killer (NK) cell, Fluorescence lifetime imaging, FLIM, Super resolution microscopy.

A comparison of honey bee-collected pollen from working agricultural lands using light microscopy and ITS metabarcoding

USGS Publications Warehouse

Smart, Matthew; Cornman, Robert S.; Iwanowicz, Deborah; McDermott-Kubeczko, Margaret; Pettis, Jeff S; Spivak, Marla S; Otto, Clint R.

2017-01-01

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010–2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

NASA Astrophysics Data System (ADS)

Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

2014-02-01

Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

The Review of Nuclear Microscopy Techniques: An Approach for Nondestructive Trace Elemental Analysis and Mapping of Biological Materials.

PubMed

Mulware, Stephen Juma

2015-01-01

The properties of many biological materials often depend on the spatial distribution and concentration of the trace elements present in a matrix. Scientists have over the years tried various techniques including classical physical and chemical analyzing techniques each with relative level of accuracy. However, with the development of spatially sensitive submicron beams, the nuclear microprobe techniques using focused proton beams for the elemental analysis of biological materials have yielded significant success. In this paper, the basic principles of the commonly used microprobe techniques of STIM, RBS, and PIXE for trace elemental analysis are discussed. The details for sample preparation, the detection, and data collection and analysis are discussed. Finally, an application of the techniques to analysis of corn roots for elemental distribution and concentration is presented.

Optical sectioning microscopes with no moving parts using a micro-stripe array light emitting diode.

PubMed

Poher, V; Zhang, H X; Kennedy, G T; Griffin, C; Oddos, S; Gu, E; Elson, D S; Girkin, M; French, P M W; Dawson, M D; Neil, M A

2007-09-03

We describe an optical sectioning microscopy system with no moving parts based on a micro-structured stripe-array light emitting diode (LED). By projecting arbitrary line or grid patterns onto the object, we are able to implement a variety of optical sectioning microscopy techniques such as grid-projection structured illumination and line scanning confocal microscopy, switching from one imaging technique to another without modifying the microscope setup. The micro-structured LED and driver are detailed and depth discrimination capabilities are measured and calculated.

Multilayer mounting for long-term light sheet microscopy of zebrafish.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-02-27

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.

PubMed

Takeuchi, Miyuki; Takabe, Keiji; Mineyuki, Yoshinobu

2016-01-01

Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.

Single-Shot Optical Sectioning Using Two-Color Probes in HiLo Fluorescence Microscopy

PubMed Central

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-01-01

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. PMID:21641327

Correlative light-electron fractography for fatigue striations characterization in metallic alloys.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-09-01

The correlative light-electron fractography technique combines correlative microscopy concepts to the extended depth-from-focus reconstruction method, associating the reliable topographic information of 3-D maps from light microscopy ordered Z-stacks to the finest lateral resolution and large focus depth from scanning electron microscopy. Fatigue striations spacing analysis can be precisely measured, by correcting the mean surface tilting with the knowledge of local elevation data from elevation maps. This new technique aims to improve the accuracy of quantitative fractography in fatigue fracture investigations. Copyright © 2013 Wiley Periodicals, Inc.

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

PubMed Central

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology. PMID:24637614

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

PubMed Central

Lavagnino, Zeno; Sancataldo, Giuseppe; d’Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca

2016-01-01

In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces. PMID:27033347

Advances in imaging and quantification of electrical properties at the nanoscale using Scanning Microwave Impedance Microscopy (sMIM)

NASA Astrophysics Data System (ADS)

Friedman, Stuart; Stanke, Fred; Yang, Yongliang; Amster, Oskar

Scanning Microwave Impedance Microscopy (sMIM) is a mode for Atomic Force Microscopy (AFM) enabling imaging of unique contrast mechanisms and measurement of local permittivity and conductivity at the 10's of nm length scale. sMIM has been applied to a variety of systems including nanotubes, nanowires, 2D materials, photovoltaics and semiconductor devices. Early results were largely semi-quantitative. This talk will focus on techniques for extracting quantitative physical parameters such as permittivity, conductivity, doping concentrations and thin film properties from sMIM data. Particular attention will be paid to non-linear materials where sMIM has been used to acquire nano-scale capacitance-voltage curves. These curves can be used to identify the dopant type (n vs p) and doping level in doped semiconductors, both bulk samples and devices. Supported in part by DOE-SBIR DE-SC0009856.

Making the Nanoworld Accessible: Nanoscience Education Using Scanning Probe Methods

NASA Astrophysics Data System (ADS)

Knorr, Daniel; Killgore, Jason; Gray, Tomoko; Ginger, David; Wei, Joseph; Chen, Yeechi; Sarikaya, Mehmet; Fong, Hanson; Griffith, Tom; Overney, Rene

2008-03-01

A partnership between researchers and educators at the University of Washington, North Seattle Community College and two companies, Nanosurf, AG and nanoScience Instruments has been forged to develop a nationally replicable model of a sustainable and up-to-date undergraduate teaching laboratory of scanning probe microscopy (SPM) methods applied to nanoscience and nanotechnology. Within this partnership a new paradigm of operating and maintaining a SPM laboratory has been developed that provides a truly hands-on experience in a classroom laboratory setting with a small student to instrument ratio involving a variety of SPM techniques and topics. To date, we have run a first successful undergraduate laboratory workshop, where students were able to have extensive hands-on experience on five SPM modes of operation including: electrostatic force microscopy involving photovoltaic polymeric materials, tunneling microscopy and the determination of the workfunction, and nanolithography using the dip-pen method. http://depts.washington.edu/nanolab/NUE/UNIQUE/NUE/UNIQUE.htm

Dynamic imaging with electron microscopy

ScienceCinema

Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

2018-02-13

Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

Diagnosing the Internal Architecture of Zeolite Ferrierite

PubMed Central

Schmidt, Joel E.; Hendriks, Frank C.; Lutz, Martin; Post, L. Christiaan; Fu, Donglong

2017-01-01

Abstract Large crystals of zeolite ferrierite (FER) are important model systems for spatially resolved catalysis and diffusion studies, though there is considerable variation in crystal habit depending on the chemical composition and employed synthesis conditions. A synergistic combination of techniques has been applied, including single crystal X‐ray diffraction, high‐temperature in situ confocal fluorescence microscopy, fluorescent probe molecules, wide‐field microscopy and atomic force microscopy to unravel the internal architecture of three distinct FER zeolites. Pyrolyzed template species can be used as markers for the 8‐membered ring direction as they are trapped in the terraced roof of the FER crystals. This happens as the materials grow in a layer‐by‐layer, defect‐free manner normal to the large crystal surface, and leads to a facile method to diagnose the pore system orientation, which avoids tedious single crystal X‐ray diffraction experiments. PMID:28809081

Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. A morphological study including immunoelectron microscopy.

PubMed

Felix, I A; Horvath, E; Kovacs, K; Smyth, H S; Killinger, D W; Vale, J

1986-01-01

A 29-year old giantess with growth hormone excess and hyperprolactinemia underwent transsphenoidal surgery to remove her pituitary tumor. Electron microscopy revealed a mammosomatotroph adenoma composed of one cell type. Immunoelectron microscopy, using the immunogold technique, demonstrated predominantly growth hormone or prolactin or a varying mixture of both growth hormone and prolactin in the adenoma cells. The presence of growth hormone and prolactin was found not only in the cytoplasm of the same adenoma cells but also in the same secretory granules. In the nontumorous adenohypophysis, somatotrophs and lactotrophs showed ultrastructural signs of hyperactivity. This finding is in contrast with the presence of suppressed somatotrophs and lactotrophs seen in nontumorous portions of adult pituitaries harboring growth hormone or prolactin-secreting adenomas. Our morphological study reinforces the view that growth hormone-producing pituitary tumors, originating in childhood, are different from those of the adult gland.

Quantification of transendothelial migration using three-dimensional confocal microscopy.

PubMed

Cain, Robert J; d'Água, Bárbara Borda; Ridley, Anne J

2011-01-01

Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy

PubMed Central

Neuman, Keir C.; Nagy, Attila

2012-01-01

Single-molecule force spectroscopy has emerged as a powerful tool to investigate the forces and motions associated with biological molecules and enzymatic activity. The most common force spectroscopy techniques are optical tweezers, magnetic tweezers and atomic force microscopy. These techniques are described and illustrated with examples highlighting current capabilities and limitations. PMID:18511917

Fourier ptychographic microscopy at telecommunication wavelengths using a femtosecond laser

NASA Astrophysics Data System (ADS)

Ahmed, Ishtiaque; Alotaibi, Maged; Skinner-Ramos, Sueli; Dominguez, Daniel; Bernussi, Ayrton A.; de Peralta, Luis Grave

2017-12-01

We report the implementation of the Fourier Ptychographic Microscopy (FPM) technique, a phase retrieval technique, at telecommunication wavelengths using a low-coherence ultrafast pulsed laser source. High quality images, near speckle-free, were obtained with the proposed approach. We demonstrate that FPM can also be used to image periodic features through a silicon wafer.

NDE of structural ceramics

NASA Technical Reports Server (NTRS)

Klima, S. J.; Vary, A.

1986-01-01

Radiographic, ultrasonic, scanning laser acoustic microscopy (SLAM), and thermo-acoustic microscopy techniques were used to characterize silicon nitride and silicon carbide modulus-of-rupture test specimens in various stages of fabrication. Conventional and microfocus X-ray techniques were found capable of detecting minute high density inclusions in as-received powders, green compacts, and fully densified specimens. Significant density gradients in sintered bars were observed by radiography, ultrasonic velocity, and SLAM. Ultrasonic attenuation was found sensitive to microstructural variations due to grain and void morphology and distribution. SLAM was also capable of detecting voids, inclusions and cracks in finished test bars. Consideration is given to the potential for applying thermo-acoustic microscopy techniques to green and densified ceramics. The detection probability statistics and some limitations of radiography and SLAM also are discussed.

Changes Found on Run-In and Scuffed Surfaces of Steel Chrome Plate, and Cast Iron

NASA Technical Reports Server (NTRS)

Good, J. N.; Godfrey, Douglas

1947-01-01

A study was made of run-in and scuffed steel, chrome-plate, and cast-iron surfaces. X-ray and electron diffraction techniques, micro-hardness determinations, and microscopy were used. Surface changes varied and were found to include three classes: chemical reaction, hardening, and crystallite-size alteration. The principal chemical reactions were oxidation and carburization.

Electron microscopy of lamin and the nuclear lamina in Caenorhabditis elegans.

PubMed

Cohen, Merav; Santarella, Rachel; Wiesel, Naama; Mattaj, Iain; Gruenbaum, Yosef

2008-01-01

The nuclear lamina is found between the inner nuclear membrane and the peripheral chromatin. Lamins are the main components of the nuclear lamina, where they form protein complexes with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. Lamins are required for mechanical stability, chromatin organization, Pol II transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in human lamins cause at least 13 distinct human diseases, collectively termed laminopathies, affecting muscle, adipose, bone, nerve and skin cells, and range from muscular dystrophies to accelerated aging. Caenorhabditis elegans has unique advantages in studying lamins and nuclear lamina genes including low complexity of lamina genes and the unique ability of bacterially expressed C. elegans lamin protein to form stable 10 nm fibers. In addition, transgenic techniques, simple application of RNA interference, sophisticated genetic analyses, and the production of a large collection of mutant lines, all make C. elegans especially attractive for studying the functions of its nuclear lamina genes. In this chapter we will include a short review of our current knowledge of nuclear lamina in C. elegans and will describe electron microscopy techniques used for their analyses.

Interference techniques in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Dogan, Mehmet

We developed a set of interference-based optical microscopy techniques to study biological structures through nanometer-scale axial localization of fluorescent biomarkers. Spectral self-interference fluorescence microscopy (SSFM) utilizes interference of direct and reflected waves emitted from fluorescent molecules in the vicinity of planar reflectors to reveal the axial position of the molecules. A comprehensive calculation algorithm based on Green's function formalism is presented to verify the validity of approximations used in a far-field approach that describes the emission of fluorescent markers near interfaces. Using the validated model, theoretical limits of axial localization were determined with emphasis given to numerical aperture (NA) dependence of localization uncertainty. SSFM was experimentally demonstrated in conformational analysis of nucleoproteins. In particular, interaction between surface-tethered 75-mer double strand DNA and integration host factor (IHF) protein was probed on Si-SiO2 substrates by determining the axial position of fluorescent labels attached to the free ends of DNA molecules. Despite its sub-nanometer precision axial localization capability, SSFM lacks high lateral resolution due to the low-NA requirement for planar reflectors. We developed a second technique, 4Pi-SSFM, which improves the lateral resolution of a conventional SSFM system by an order of magnitude while achieving nanometer-scale axial localization precision. Using two opposing high-NA objectives, fluorescence signal is interferometrically collected and spectral interference pattern is recorded. Axial position of emitters is found from analysis of the spectra. The 4Pi-SSFM technique was experimentally demonstrated by determining the surface profiles of fabricated glass surfaces and outer membranes of Shigella, a type of Gram-negative bacteria. A further discussion is presented to localize surface O antigen, which is an important oligosaccharide structure in the virulence mechanism of the Gram-negative bacteria, including E. coli and Shigella.

Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy

NASA Astrophysics Data System (ADS)

Gomez-Morilla, Inmaculada; Thoree, Vinay; Powell, Jonathan J.; Kirkby, Karen J.; Grime, Geoffrey W.

2006-08-01

Microscopic particles (0.5-2 μm diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles.

The development of optical microscopy techniques for the advancement of single-particle studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchuk, Kyle

2013-05-15

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-fieldmore » imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called “non-blinking” quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.« less

The development of optical microscopy techniques for the advancement of single-particle studies

NASA Astrophysics Data System (ADS)

Marchuk, Kyle

Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called "non-blinking" quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to find the 3D orientation of stationary metallic anisotropic nanoparticles utilizing only long-axis SPR enhancement. The polarization direction of the illuminating light was rotated causing the relative intensity of p-polarized and s-polarized light within the evanescent field to change. The interaction of the evanescent field with the particles is dependent on the orientation of the particle producing an intensity curve. This curve and the in-plane angle can be compared with simulations to accurately determine the 3D orientation. Differential interference contrast (DIC) microscopy is another non-invasive far-field technique based upon interferometry that does not rely on staining or other contrast enhancing techniques. In addition, high numerical aperture condensers and objectives can be used to give a very narrow depth of field allowing for the optical tomography of samples, which makes it an ideal candidate to study biological systems. DIC microscopy has also proven itself in determining the orientation of gold nanorods in both engineered environments and within cells. Many types of nanoparticles and nanostructures have been synthesized using lithographic techniques on silicon wafer substrates. Traditionally, reflective mode DIC microscopes have been developed and applied to the topographical study of reflective substrates and the imaging of chips on silicon wafers. Herein, a laser-illuminated reflected-mode DIC was developed for studying nanoparticles on reflective surfaces.

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balke, Nina; Jesse, Stephen; Yu, Pu

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

Quantification of surface displacements and electromechanical phenomena via dynamic atomic force microscopy

DOE PAGES

Balke, Nina; Jesse, Stephen; Yu, Pu; ...

2016-09-15

Detection of dynamic surface displacements associated with local changes in material strain provides access to a number of phenomena and material properties. Contact resonance-enhanced methods of atomic force microscopy (AFM) have been shown capable of detecting ~1–3 pm-level surface displacements, an approach used in techniques such as piezoresponse force microscopy, atomic force acoustic microscopy, and ultrasonic force microscopy. Here, based on an analytical model of AFM cantilever vibrations, we demonstrate a guideline to quantify surface displacements with high accuracy by taking into account the cantilever shape at the first resonant contact mode, depending on the tip–sample contact stiffness. The approachmore » has been experimentally verified and further developed for piezoresponse force microscopy (PFM) using well-defined ferroelectric materials. These results open up a way to accurate and precise measurements of surface displacement as well as piezoelectric constants at the pm-scale with nanometer spatial resolution and will allow avoiding erroneous data interpretations and measurement artifacts. Furthermore, this analysis is directly applicable to all cantilever-resonance-based scanning probe microscopy (SPM) techniques.« less

Characterization of the host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy, and immunological techniques

USGS Publications Warehouse

Bartholomew, J.L.; Smith, C.E.; Rohovec, J.S.; Fryer, J.L.

1989-01-01

The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta-susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.

Characterization of Metal Powders Used for Additive Manufacturing

PubMed Central

Slotwinski, JA; Garboczi, EJ; Stutzman, PE; Ferraris, CF; Watson, SS; Peltz, MA

2014-01-01

Additive manufacturing (AM) techniques1 can produce complex, high-value metal parts, with potential applications as critical parts, such as those found in aerospace components. The production of AM parts with consistent and predictable properties requires input materials (e.g., metal powders) with known and repeatable characteristics, which in turn requires standardized measurement methods for powder properties. First, based on our previous work, we assess the applicability of current standardized methods for powder characterization for metal AM powders. Then we present the results of systematic studies carried out on two different powder materials used for additive manufacturing: stainless steel and cobalt-chrome. The characterization of these powders is important in NIST efforts to develop appropriate measurements and standards for additive materials and to document the property of powders used in a NIST-led additive manufacturing material round robin. An extensive array of characterization techniques was applied to these two powders, in both virgin and recycled states. The physical techniques included laser diffraction particle size analysis, X-ray computed tomography for size and shape analysis, and optical and scanning electron microscopy. Techniques sensitive to structure and chemistry, including X-ray diffraction, energy dispersive analytical X-ray analysis using the X-rays generated during scanning electron microscopy, and X-Ray photoelectron spectroscopy were also employed. The results of these analyses show how virgin powder changes after being exposed to and recycled from one or more Direct Metal Laser Sintering (DMLS) additive manufacturing build cycles. In addition, these findings can give insight into the actual additive manufacturing process. PMID:26601040

Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM

PubMed Central

Ghorbal, Achraf; Grisotto, Federico; Charlier, Julienne; Palacin, Serge; Goyer, Cédric; Demaille, Christophe; Ben Brahim, Ammar

2013-01-01

This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM) electrode. The capabilities of the AFM-SECM-electrode were tested with the nano-electrografting of vinylic monomers initiated by aryl diazonium salts. Nano-electrochemical and technical processes were thoroughly described, so as to allow experiments reproducing. A plausible explanation of chemical and electrochemical mechanisms, leading to the nano-grafting process, was reported. This combined technique represents the first step towards improved nano-processes for the nano-electrografting. PMID:28348337

Three dimensional electron microscopy and in silico tools for macromolecular structure determination

PubMed Central

Borkotoky, Subhomoi; Meena, Chetan Kumar; Khan, Mohammad Wahab; Murali, Ayaluru

2013-01-01

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it. PMID:27092033

Application of Multiphoton Microscopy in Dermatological Studies: a Mini-Review

PubMed Central

Yew, Elijah; Rowlands, Christopher

2014-01-01

This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance. PMID:25075226

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization

PubMed Central

Bukharina, Natalia S.; Archakov, Alexander I.; Ivanov, Yuri D.

2018-01-01

This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed. PMID:29642632

Optical Coherence Microscopy

NASA Astrophysics Data System (ADS)

Aguirre, Aaron D.; Zhou, Chao; Lee, Hsiang-Chieh; Ahsen, Osman O.; Fujimoto, James G.

Cellular imaging of human tissues remains an important advance for many clinical applications of optical coherence tomography (OCT). Imaging cells with traditional OCT systems has not been possible due to the limited transverse resolution of such techniques. Optical coherence microscopy (OCM) refers to OCT methods that achieve high transverse resolution to visualize cells and subcellular features. This chapter provides a comprehensive discussion of the rationale for cellular imaging in human tissues as well as a review of the key technological advances required to achieve it. Time domain and Fourier domain OCM approaches are described with an emphasis on state of the art system designs, including miniaturized endoscopic imaging probes. Clinical applications are discussed and multiple examples of cellular imaging in human tissues are provided.

Simulation of Mirror Electron Microscopy Caustic Images in Three-Dimensions

NASA Astrophysics Data System (ADS)

Kennedy, S. M.; Zheng, C. X.; Jesson, D. E.

A full, three-dimensional (3D) ray tracing approach is developed to simulate the caustics visible in mirror electron microscopy (MEM). The method reproduces MEM image contrast resulting from 3D surface relief. To illustrate the potential of the simulation methods, we study the evolution of crater contrast associated with a movie of GaAs structures generated by the droplet epitaxy technique. Specifically, we simulate the image contrast resulting from both a precursor stage and the final crater morphology which is consistent with an inverted pyramid consisting of (111) facet walls. The method therefore facilities the study of how self-assembled quantum structures evolve with time and, in particular, the development of anisotropic features including faceting.

In vivo confocal microscopy, an inner vision of the cornea - a major review.

PubMed

Guthoff, Rudolf F; Zhivov, Andrey; Stachs, Oliver

2009-01-01

The demands of modern ophthalmology have evolved from descriptive findings from the slit lamp to in vivo assessment of cellular level changes. Nowadays, the latter can be provided by in vivo confocal microscopy. This article gives an overview of confocal principles using tandem scanning, scanning slit and laser scanning techniques used in ophthalmology. The main part of the paper describes the clinical applications emphasizing the anatomy of the normal and pathological cornea, and illustrates side-effects of topical medication, contact lens wear, cross-linking and refractive surgery. Finally, a summary about experimental applications, including animal studies, surface characterization and volume rendering as well as future developments, is given.

Synthesis and characterization of a narrow size distribution of zinc oxide nanoparticles.

PubMed

Zak, A Khorsand; Razali, R; Majid, W H Abd; Darroudi, Majid

2011-01-01

Zinc oxide nanoparticles (ZnO-NPs) were synthesized via a solvothermal method in triethanolamine (TEA) media. TEA was utilized as a polymer agent to terminate the growth of ZnO-NPs. The ZnO-NPs were characterized by a number of techniques, including X-ray diffraction analysis, transition electron microscopy, and field emission electron microscopy. The ZnO-NPs prepared by the solvothermal process at 150°C for 18 hours exhibited a hexagonal (wurtzite) structure, with a crystalline size of 33 ± 2 nm, and particle size of 48 ± 7 nm. The results confirm that TEA is a suitable polymer agent to prepare homogenous ZnO-NPs.

Effects of Polyethylene Glycol and Citric Acid on Preparation and Hydrodechlorination Activity of Molybdenum Phosphide

NASA Astrophysics Data System (ADS)

Liu, Xiaomeng; Lu, Shaoxiang; Xu, Hanghui; Ren, Lili

2018-07-01

Molybdenum phosphide (MoP), modified by polyethylene glycol (PEG) and citric acid (CA), exhibited 2 to 3 times superior activity than the MoP modified by CA alone. And the optimal activity temperature was reduced from 500 to 450oC. The catalyst was fully characterized by a variety of techniques including X-ray diffraction (XRD), N2 adsorption-desorption isotherm, scanning electron microscopy (SEM), transmission electron microscopy (TEM). The results showed that the addition of PEG and CA increased the surface area of MoP and decreased the particle size of MoP. Furthermore, the reaction mechanism also has been discussed by combining the activity data and characterization results.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy.

PubMed

Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

2016-06-01

We recently proposed a straightforward fluorescence microscopy technique to study adhesion of Giant Unilamellar Vesicles. This technique is based on dual observations which combine epi-fluorescence microscopy and total internal reflection fluorescence (TIRF) microscopy: TIRF images are normalized by epi-fluorescence ones. By this way, it is possible to map the membrane/substrate separation distance with a nanometric resolution, typically ~20 nm, with a maximal working range of 300-400 nm. The purpose of this paper is to demonstrate that this technique is useful to quantify vesicle adhesion from ultra-weak to strong membrane-surface interactions. Thus, we have examined unspecific and specific adhesion conditions. Concerning unspecific adhesion, we have controlled the strength of electrostatic forces between negatively charged vesicles and various functionalized surfaces which exhibit a positive or a negative effective charge. Specific adhesion was highlighted with lock-and-key forces mediated by the well defined biotin/streptavidin recognition. Copyright © 2016 Elsevier B.V. All rights reserved.

New Photocatalysts for Hydrogen Production; Nuevos Fotocatalizadores para la Producción de Hidrógeno

DOE PAGES

García, Abraham; Cotto, María; Duconge, José; ...

2014-06-10

The use of hydrogen as replacement for fossil fuels, on which we depend today, is a matter of great relevance. The sustainable generation of hydrogen as fuel is relevant from an environmental and economic point of view. In this study we have explored new synthetic routes for developing new photocatalysts to be used in water splitting, for hydrogen production. Different techniques have been used to produce hydrogen, such as electrolysis, even though these processes have been found to be energetically non suitable. In this research various photocatalytic materials were presented as possible alternatives for using in water splitting processes. Characterizationmore » of the new synthesized materials has been done by using different experimental techniques including Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), surface area BET, and X-ray Diffraction (XRD). The efficiency of the synthesized photocatalysts was determined by evaluating the hydrogen evolution by the photocatalytic water splitting reaction.« less

High-resolution analytical imaging and electron holography of magnetite particles in amyloid cores of Alzheimer’s disease

PubMed Central

Plascencia-Villa, Germán; Ponce, Arturo; Collingwood, Joanna F.; Arellano-Jiménez, M. Josefina; Zhu, Xiongwei; Rogers, Jack T.; Betancourt, Israel; José-Yacamán, Miguel; Perry, George

2016-01-01

Abnormal accumulation of brain metals is a key feature of Alzheimer’s disease (AD). Formation of amyloid-β plaque cores (APC) is related to interactions with biometals, especially Fe, Cu and Zn, but their particular structural associations and roles remain unclear. Using an integrative set of advanced transmission electron microscopy (TEM) techniques, including spherical aberration-corrected scanning transmission electron microscopy (Cs-STEM), nano-beam electron diffraction, electron holography and analytical spectroscopy techniques (EDX and EELS), we demonstrate that Fe in APC is present as iron oxide (Fe3O4) magnetite nanoparticles. Here we show that Fe was accumulated primarily as nanostructured particles within APC, whereas Cu and Zn were distributed through the amyloid fibers. Remarkably, these highly organized crystalline magnetite nanostructures directly bound into fibrillar Aβ showed characteristic superparamagnetic responses with saturated magnetization with circular contours, as observed for the first time by off-axis electron holography of nanometer scale particles. PMID:27121137

Fluorescence microscopy techniques for quantitative evaluation of organic biocide distribution in antifouling paint coatings: application to model antifouling coatings.

PubMed

Goodes, L R; Dennington, S P; Schuppe, H; Wharton, J A; Bakker, M; Klijnstra, J W; Stokes, K R

2012-01-01

A test matrix of antifouling (AF) coatings including pMMA, an erodible binder and a novel trityl copolymer incorporating Cu₂O and a furan derivative (FD) natural product, were subjected to pontoon immersion and accelerated rotor tests. Fluorescence and optical microscopy techniques were applied to these coatings for quantification of organic biocide and pigment distribution. Total leaching of the biocide from the novel copolymer binder was observed within 6 months of rotor immersion, compared to 35% from the pMMA coating. In pontoon immersions, 61% of the additive was lost from the pMMA coating, and 53% from the erodible binder. Profiles of FD content in the binders revealed an accelerated loss of additive from the surface of the CDP resulting from rosin degradation, compared to even depletion from pMMA. In all samples, release of the biocide was inhibited beyond the Cu₂O front, corresponding to the leached layer in samples where Cu₂O release occurred.

Toward quantitative fluorescence microscopy with DNA origami nanorulers.

PubMed

Beater, Susanne; Raab, Mario; Tinnefeld, Philip

2014-01-01

The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution. © 2014 Elsevier Inc. All rights reserved.

High-resolution analytical imaging and electron holography of magnetite particles in amyloid cores of Alzheimer’s disease

NASA Astrophysics Data System (ADS)

Plascencia-Villa, Germán; Ponce, Arturo; Collingwood, Joanna F.; Arellano-Jiménez, M. Josefina; Zhu, Xiongwei; Rogers, Jack T.; Betancourt, Israel; José-Yacamán, Miguel; Perry, George

2016-04-01

Abnormal accumulation of brain metals is a key feature of Alzheimer’s disease (AD). Formation of amyloid-β plaque cores (APC) is related to interactions with biometals, especially Fe, Cu and Zn, but their particular structural associations and roles remain unclear. Using an integrative set of advanced transmission electron microscopy (TEM) techniques, including spherical aberration-corrected scanning transmission electron microscopy (Cs-STEM), nano-beam electron diffraction, electron holography and analytical spectroscopy techniques (EDX and EELS), we demonstrate that Fe in APC is present as iron oxide (Fe3O4) magnetite nanoparticles. Here we show that Fe was accumulated primarily as nanostructured particles within APC, whereas Cu and Zn were distributed through the amyloid fibers. Remarkably, these highly organized crystalline magnetite nanostructures directly bound into fibrillar Aβ showed characteristic superparamagnetic responses with saturated magnetization with circular contours, as observed for the first time by off-axis electron holography of nanometer scale particles.

Enhanced materials from nature: nanocellulose from citrus waste.

PubMed

Mariño, Mayra; Lopes da Silva, Lucimara; Durán, Nelson; Tasic, Ljubica

2015-04-03

Nanocellulose is a relatively inexpensive, highly versatile bio-based renewable material with advantageous properties, including biodegradability and nontoxicity. Numerous potential applications of nanocellulose, such as its use for the preparation of high-performance composites, have attracted much attention from industry. Owing to the low energy consumption and the addition of significant value, nanocellulose extraction from agricultural waste is one of the best alternatives for waste treatment. Different techniques for the isolation and purification of nanocellulose have been reported, and combining these techniques influences the morphology of the resultant fibers. Herein, some of the extraction routes for obtaining nanocellulose from citrus waste are addressed. The morphology of nanocellulose was determined by Scanning Electron Microscopy (SEM) and Field Emission Scanning Electron Microscopy (FESEM), while cellulose crystallinity indexes (CI) from lyophilized samples were determined using solid-state Nuclear Magnetic Resonance (NMR) and X-Ray Diffraction (XRD) measurements. The resultant nanofibers had 55% crystallinity, an average diameter of 10 nm and a length of 458 nm.

Nanoscale surface characterization using laser interference microscopy

NASA Astrophysics Data System (ADS)

Ignatyev, Pavel S.; Skrynnik, Andrey A.; Melnik, Yury A.

2018-03-01

Nanoscale surface characterization is one of the most significant parts of modern materials development and application. The modern microscopes are expensive and complicated tools, and its use for industrial tasks is limited due to laborious sample preparation, measurement procedures, and low operation speed. The laser modulation interference microscopy method (MIM) for real-time quantitative and qualitative analysis of glass, metals, ceramics, and various coatings has a spatial resolution of 0.1 nm for vertical and up to 100 nm for lateral. It is proposed as an alternative to traditional scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods. It is demonstrated that in the cases of roughness metrology for super smooth (Ra >1 nm) surfaces the application of a laser interference microscopy techniques is more optimal than conventional SEM and AFM. The comparison of semiconductor test structure for lateral dimensions measurements obtained with SEM and AFM and white light interferometer also demonstrates the advantages of MIM technique.

Tomographic phase microscopy and its biological applications

NASA Astrophysics Data System (ADS)

Choi, Wonshik

2012-12-01

Conventional interferometric microscopy techniques such as digital holographic microscopy and quantitative phase microscopy are often classified as 3D imaging techniques because a recorded complex field image can be numerically propagated to a different depth. In a strict sense, however, a single complex field image contains only 2D information on a specimen. The measured 2D image is only a subset of the 3D structure. For the 3D mapping of an object, multiple independent 2D images are to be taken, for example at multiple incident angles or wavelengths, and then combined by the so-called optical diffraction tomography (ODT). In this Letter, tomographic phase microscopy (TPM) is reviewed that experimentally realizes the concept of the ODT for the 3D mapping of biological cells in their native state, and some of its interesting biological and biomedical applications are introduced. [Figure not available: see fulltext.

Determination of the five parameter grain boundary character distribution of nanocrystalline alpha-zirconium thin films using transmission electron microscopy

DOE PAGES

Ghamarian, I.; Samani, P.; Rohrer, G. S.; ...

2017-03-24

Grain boundary engineering and other fundamental materials science problems (e.g., phase transformations and physical properties) require an improvement in the understanding of the type and population of grain boundaries in a given system – yet, databases are limited in number and spare in detail, including for hcp crystals such as zirconium. One way to rapidly obtain databases to analyze is to use small-grained materials and high spatial resolution orientation microscopy techniques, such as ASTAR™/precession electron diffraction. To demonstrate this, a study of grain boundary character distributions was conducted for α-zirconium deposited at room temperature on fused silica substrates using physicalmore » vapor deposition. The orientation maps of the nanocrystalline thin films were acquired by the ASTARα/precession electron diffraction technique, a new transmission electron microscope based orientation microscopy method. The reconstructed grain boundaries were classified as pure tilt, pure twist, 180°-twist and 180°-tilt grain boundaries based on the distribution of grain boundary planes with respect to the angle/axis of misorientation associated with grain boundaries. The results of the current study were compared to the results of a similar study on α-titanium and the molecular dynamics results of grain boundary energy for α-titanium.« less

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques†

PubMed Central

Plascencia-Villa, Germán; Starr, Clarise R.; Armstrong, Linda S.; Ponce, Arturo

2016-01-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO2, TiO2 and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO2 and TiO2, whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution. PMID:23023106

Porous silicon - rare earth doped xerogel and glass composites

NASA Astrophysics Data System (ADS)

Balakrishnan, S.; Gun'ko, Yurii K.; Perova, T. S.; Rafferty, A.; Astrova, E. V.; Moore, R. A.

2005-06-01

The development of components for photonics applications is growing exponentially. The sol-gel method is now recognised as a convenient and flexible way to deposit oxide or glass films on a variety of hosts, including porous silicon. In the present work we incorporated erbium and europium doped xerogel into porous silicon and developed new porous silicon - rare earth doped glass composites. Various characteris-ation techniques including FTIR, Raman Spectroscopy, Thermal Gravimetric Analysis and Scanning Electron Microscopy were employed in this work.

Multi-scale Observation of Biological Interactions of Nanocarriers: from Nano to Macro

PubMed Central

Jin, Su-Eon; Bae, Jin Woo; Hong, Seungpyo

2010-01-01

Microscopic observations have played a key role in recent advancements in nanotechnology-based biomedical sciences. In particular, multi-scale observation is necessary to fully understand the nano-bio interfaces where a large amount of unprecedented phenomena have been reported. This review describes how to address the physicochemical and biological interactions of nanocarriers within the biological environments using microscopic tools. The imaging techniques are categorized based on the size scale of detection. For observation of the nano-scale biological interactions of nanocarriers, we discuss atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For the micro to macro-scale (in vitro and in vivo) observation, we focus on confocal laser scanning microscopy (CLSM) as well as in vivo imaging systems such as magnetic resonance imaging (MRI), superconducting quantum interference devices (SQUIDs), and IVIS®. Additionally, recently developed combined techniques such as AFM-CLSM, correlative Light and Electron Microscopy (CLEM), and SEM-spectroscopy are also discussed. In this review, we describe how each technique helps elucidate certain physicochemical and biological activities of nanocarriers such as dendrimers, polymers, liposomes, and polymeric/inorganic nanoparticles, thus providing a toolbox for bioengineers, pharmaceutical scientists, biologists, and research clinicians. PMID:20232368

Adequacy of surface analytical tools for studying the tribology of ceramics

NASA Technical Reports Server (NTRS)

Sliney, H. E.

1986-01-01

Surface analytical tools are very beneficial in tribological studies of ceramics. Traditional methods of optical microscopy, XRD, XRF, and SEM should be combined with newer surface sensitive techniques especially AES and XPS. ISS and SIMS can also be useful in providing additional compositon details. Tunneling microscopy and electron energy loss spectroscopy are less known techniques that may also prove useful.

Scalp imaging techniques

NASA Astrophysics Data System (ADS)

Otberg, Nina; Shapiro, Jerry; Lui, Harvey; Wu, Wen-Yu; Alzolibani, Abdullateef; Kang, Hoon; Richter, Heike; Lademann, Jürgen

2017-05-01

Scalp imaging techniques are necessary tools for the trichological practice and for visualization of permeation, penetration and absorption processes into and through the scalp and for the research on drug delivery and toxicology. The present letter reviews different scalp imaging techniques and discusses their utility. Moreover, two different studies on scalp imaging techniques are presented in this letter: (1) scalp imaging with phototrichograms in combination with laser scanning microscopy, and (2) follicular measurements with cyanoacrylate surface replicas and light microscopy in combination with laser scanning microscopy. The experiments compare different methods for the determination of hair density on the scalp and different follicular measures. An average terminal hair density of 132 hairs cm-2 was found in 6 Caucasian volunteers and 135 hairs cm-2 in 6 Asian volunteers. The area of the follicular orifices accounts to 16.3% of the skin surface on average measured with laser scanning microscopy images. The potential volume of the follicular infundibulum was calculated based on the laser scanning measurements and is found to be 4.63 mm3 per cm2 skin on average. The experiments show that hair follicles are quantitatively relevant pathways and potential reservoirs for topically applied drugs and cosmetics.

High-spatial-resolution sub-surface imaging using a laser-based acoustic microscopy technique.

PubMed

Balogun, Oluwaseyi; Cole, Garrett D; Huber, Robert; Chinn, Diane; Murray, Todd W; Spicer, James B

2011-01-01

Scanning acoustic microscopy techniques operating at frequencies in the gigahertz range are suitable for the elastic characterization and interior imaging of solid media with micrometer-scale spatial resolution. Acoustic wave propagation at these frequencies is strongly limited by energy losses, particularly from attenuation in the coupling media used to transmit ultrasound to a specimen, leading to a decrease in the depth in a specimen that can be interrogated. In this work, a laser-based acoustic microscopy technique is presented that uses a pulsed laser source for the generation of broadband acoustic waves and an optical interferometer for detection. The use of a 900-ps microchip pulsed laser facilitates the generation of acoustic waves with frequencies extending up to 1 GHz which allows for the resolution of micrometer-scale features in a specimen. Furthermore, the combination of optical generation and detection approaches eliminates the use of an ultrasonic coupling medium, and allows for elastic characterization and interior imaging at penetration depths on the order of several hundred micrometers. Experimental results illustrating the use of the laser-based acoustic microscopy technique for imaging micrometer-scale subsurface geometrical features in a 70-μm-thick single-crystal silicon wafer with a (100) orientation are presented.

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

PubMed Central

Sun, Yuliang; Juzenas, Kevin

2017-01-01

Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

NASA Astrophysics Data System (ADS)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Electron microscopy characterization of Ni-Cr-B-Si-C laser deposited coatings.

PubMed

Hemmati, I; Rao, J C; Ocelík, V; De Hosson, J Th M

2013-02-01

During laser deposition of Ni-Cr-B-Si-C alloys with high amounts of Cr and B, various microstructures and phases can be generated from the same chemical composition that results in heterogeneous properties in the clad layer. In this study, the microstructure and phase constitution of a high-alloy Ni-Cr-B-Si-C coating deposited by laser cladding were analyzed by a combination of several microscopy characterization techniques including scanning electron microscopy in secondary and backscatter imaging modes, energy dispersive spectroscopy (EDS), electron backscatter diffraction (EBSD), and transmission electron microscopy (TEM). The combination of EDS and EBSD allowed unequivocal identification of micron-sized precipitates as polycrystalline orthorhombic CrB, single crystal tetragonal Cr5B3, and single crystal hexagonal Cr7C3. In addition, TEM characterization showed various equilibrium and metastable Ni-B, Ni-Si, and Ni-Si-B eutectic products in the alloy matrix. The findings of this study can be used to explain the phase formation reactions and to tune the microstructure of Ni-Cr-B-Si-C coatings to obtain the desired properties.

Direct manipulation of metallic nanosheets by shear force microscopy.

PubMed

Bi, Z; Cai, W; Wang, Y; Shang, G

2018-05-15

Micro/nanomanipulation is a rapidly growing technology and holds promising applications in various fields, including photonic/electronic devices, chemical/biosensors etc. In this work, we present that shear force microscopy (ShFM) can be exploited to manipulate metallic nanosheets besides imaging. The manipulation is realized via controlling the shear force sensor probe position and shear force magnitude based on our homemade ShFM system under an optical microscopy for in situ observation. The main feature of the ShFM system is usage of a piezoelectric bimorph sensor, which has the ability of self-excitation and detection. Moreover, the shear force magnitude as a function of the spring constant of the sensor and setpoint is obtained, which indicates that operation modes can be switched between imaging and manipulation through designing the spring constant before experiment and changing the setpoint during manipulation process, respectively. We believe that this alternative manipulation technique could be used to assemble other nanostructures with different shapes, sizes and compositions for new properties and wider applications. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

The MIND PALACE: A Multi-Spectral Imaging and Spectroscopy Database for Planetary Science

NASA Astrophysics Data System (ADS)

Eshelman, E.; Doloboff, I.; Hara, E. K.; Uckert, K.; Sapers, H. M.; Abbey, W.; Beegle, L. W.; Bhartia, R.

2017-12-01

The Multi-Instrument Database (MIND) is the web-based home to a well-characterized set of analytical data collected by a suite of deep-UV fluorescence/Raman instruments built at the Jet Propulsion Laboratory (JPL). Samples derive from a growing body of planetary surface analogs, mineral and microbial standards, meteorites, spacecraft materials, and other astrobiologically relevant materials. In addition to deep-UV spectroscopy, datasets stored in MIND are obtained from a variety of analytical techniques obtained over multiple spatial and spectral scales including electron microscopy, optical microscopy, infrared spectroscopy, X-ray fluorescence, and direct fluorescence imaging. Multivariate statistical analysis techniques, primarily Principal Component Analysis (PCA), are used to guide interpretation of these large multi-analytical spectral datasets. Spatial co-referencing of integrated spectral/visual maps is performed using QGIS (geographic information system software). Georeferencing techniques transform individual instrument data maps into a layered co-registered data cube for analysis across spectral and spatial scales. The body of data in MIND is intended to serve as a permanent, reliable, and expanding database of deep-UV spectroscopy datasets generated by this unique suite of JPL-based instruments on samples of broad planetary science interest.

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

PubMed

Liu, Cong; Liu, Yen-Liang; Perillo, Evan P; Dunn, Andrew K; Yeh, Hsin-Chih

2016-01-01

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mangum, John S.; Chan, Lisa H.; Schmidt, Ute

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In thismore » work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice.« less

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection

PubMed Central

Liu, Cong; Liu, Yen-Liang; Perillo, Evan P.; Dunn, Andrew K.; Yeh, Hsin-Chih

2016-01-01

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges. PMID:27660404

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs.

PubMed

Mangum, John S; Chan, Lisa H; Schmidt, Ute; Garten, Lauren M; Ginley, David S; Gorman, Brian P

2018-05-01

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In this work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice. Copyright © 2018 Elsevier B.V. All rights reserved.

Correlative Raman spectroscopy and focused ion beam for targeted phase boundary analysis of titania polymorphs

DOE PAGES

Mangum, John S.; Chan, Lisa H.; Schmidt, Ute; ...

2018-02-23

Site-specific preparation of specimens using focused ion beam instruments for transmission electron microscopy is at the forefront of targeting regions of interest for nanoscale characterization. Typical methods of pinpointing desired features include electron backscatter diffraction for differentiating crystal structures and energy-dispersive X-Ray spectroscopy for probing compositional variations. Yet there are situations, notably in the titanium dioxide system, where these techniques can fail. Differentiating between the brookite and anatase polymorphs of titania is either excessively laborious or impossible with the aforementioned techniques. However, due to differences in bonding structure, Raman spectroscopy serves as an ideal candidate for polymorph differentiation. In thismore » work, a correlative approach utilizing Raman spectroscopy for targeted focused ion beam specimen preparation was employed. Dark field imaging and diffraction in the transmission electron microscope confirmed the region of interest located via Raman spectroscopy and demonstrated the validity of this new method. Correlative Raman spectroscopy, scanning electron microscopy, and focused ion beam is shown to be a promising new technique for identifying site-specific preparation of nanoscale specimens in cases where conventional approaches do not suffice.« less

A Simplified, Low-Cost Method for Polarized Light Microscopy

PubMed Central

Maude, Richard J.; Buapetch, Wanchana; Silamut, Kamolrat

2009-01-01

Malaria pigment is an intracellular inclusion body that appears in blood and tissue specimens on microscopic examination and can help in establishing the diagnosis of malaria. In simple light microscopy, it can be difficult to discern from cellular background and artifacts. It has long been known that if polarized light microscopy is used, malaria pigment can be much easier to distinguish. However, this technique is rarely used because of the need for a relatively costly polarization microscope. We describe a simple and economical technique to convert any standard light microscope suitable for examination of malaria films into a polarization microscope. PMID:19861611

Localization microscopy of DNA in situ using Vybrant{sup ®} DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant{sup ®} DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei ofmore » fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10{sup 6} signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100 nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy. - Highlights: • Super-resolution imaging of nuclear DNA with Vybrant Violet and blue excitation. • 90nm resolution images of DNA structures in optically thick eukaryotic nuclei. • Enhanced resolution confirms the existence of DNA-free regions inside the nucleus. • Optimized imaging conditions enable multicolor super-resolution imaging.« less

Direct Microscopy: A Useful Tool to Diagnose Oral Candidiasis in Children and Adolescents.

PubMed

Marty, Mathieu; Bourrat, Emmanuelle; Vaysse, Frédéric; Bonner, Mark; Bailleul-Forestier, Isabelle

2015-12-01

Oral candidiasis is one of the most common opportunistic fungal infections of the oral cavity in human. Among children, this condition represents one of the most frequent affecting the mucosa. Although most diagnoses are made based on clinical signs and features, a microbiological analysis is sometimes necessary. We performed a literature review on the diagnosis of oral candidiasis to identify the techniques most commonly employed in routine clinical practice. A Medline-PubMed search covering the last 10 years was performed. Microbiological techniques were used in cases requiring confirmation of the clinical diagnosis. In such cases, direct microscopy was the method most commonly used for diagnosing candidiasis. Direct microscopy appears as the method of choice for confirming clinical diagnosis and could become a routine chair-side technique.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies.

PubMed

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia ( pml ) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology.

Field-Flow Fractionation of Carbon Nanotubes and Related Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

John P. Selegue

During the grant period, we carried out FFF studies of carbonaceous soot, single-walled and multi-walled carbon nanotubes, carbon nano-onions and polyoxometallates. FFF alone does not provide enough information to fully characterize samples, so our suite of characterization techniques grew to include light scattering (especially Photon Correlation Spectroscopy), scanning and transmission electron microscopy, thermogravimetric analysis and spectroscopic methods. We developed convenient techniques to deposit and examine minute FFF fractions by electron microscopy. In collaboration with Arthur Cammers (University of Kentucky), we used Flow Field-Flow Fractionation (Fl-FFF) to monitor the solution-phase growth of keplerates, a class of polyoxometallate (POM) nanoparticles. We monitoredmore » the evolution of Mo-POM nanostructures over the course of weeks by by using flow field-flow fractionation and corroborated the nanoparticle structures by using transmission electron microscopy (TEM). Total molybdenum in the solution and precipitate phases was monitored by using inductively coupled plasma analyses, and total Mo-POM concentration by following the UV-visible spectra of the solution phase. We observe crystallization-driven formation of (Mo132) keplerate and solution phase-driven evolution of structurally related nanoscopic species (3-60 nm). FFF analyses of other classes of materials were less successful. Attempts to analyze platelets of layered materials, including exfoliated graphite (graphene) and TaS2 and MoS2, were disappointing. We were not able to optimize flow conditions for the layered materials. The metal sulfides react with the aqueous carrier liquid and settle out of suspension quickly because of their high density.« less

Test chamber and forensic microscopy investigation of the transfer of brominated flame retardants into indoor dust via abrasion of source materials.

PubMed

Rauert, C; Harrad, S; Suzuki, G; Takigami, H; Uchida, N; Takata, K

2014-09-15

Brominated flame retardants (BFRs) have been detected in indoor dust in many studies, at concentrations spanning several orders of magnitude. Limited information is available on the pathways via which BFRs migrate from treated products into dust, yet the different mechanisms hypothesized to date may provide an explanation for the range of reported concentrations. In particular, transfer of BFRs to dust via abrasion of particles or fibers from treated products may explain elevated concentrations (up to 210 mg g(-1)) of low volatility BFRs like decabromodiphenyl ether (BDE-209). In this study, an indoor dust sample containing a low concentration of hexabromocyclododecane, or HBCD, (110 ng g(-1) ΣHBCDs) was placed on the floor of an in-house test chamber. A fabric curtain treated with HBCDs was placed on a mesh shelf 3 cm above the chamber floor and abrasion induced using a stirrer bar. This induced abrasion generated fibers of the curtain, which contaminated the dust, and ΣHBCD concentrations in the dust increased to between 4020 and 52 500 ng g(-1) for four different abrasion experiment times. The highly contaminated dust (ΣHBCD at 52 500 ng g(-1)) together with three archived dust samples from various UK microenvironments, were investigated with forensic microscopy techniques. These techniques included Micro X-ray fluorescent spectroscopy, scanning emission microscopy coupled with an energy dispersive X-ray spectrometer, Fourier transform infrared spectroscopy with further BFR analysis on LC-MS/MS. Using these techniques, fibers or particles abraded from a product treated with BFRs were identified in all dust samples, thereby accounting for the elevated concentrations detected in the original dust (3500 to 88 800 ng g(-1) ΣHBCD and 24 000 to 1,438 000 ng g(-1) for BDE-209). This study shows how test chamber experiments alongside forensic microscopy techniques, can provide valuable insights into the pathways via which BFRs contaminate indoor dust. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the Chondromalacia Patella Using a Microscopy Coil: Comparison of the Two-Dimensional Fast Spin Echo Techniques and the Three-Dimensional Fast Field Echo Techniques

PubMed Central

Kim, Hyun-joo; Kang, Chang Ho; Ryu, Jeong Ah; Shin, Myung Jin; Cho, Kyung-Ja; Cho, Woo Shin

2011-01-01

Objective We wanted to compare the two-dimensional (2D) fast spin echo (FSE) techniques and the three-dimensional (3D) fast field echo techniques for the evaluation of the chondromalacia patella using a microscopy coil. Materials and Methods Twenty five patients who underwent total knee arthroplasty were included in this study. Preoperative MRI evaluation of the patella was performed using a microscopy coil (47 mm). The proton density-weighted fast spin echo images (PD), the fat-suppressed PD images (FS-PD), the intermediate weighted-fat suppressed fast spin echo images (iw-FS-FSE), the 3D balanced-fast field echo images (B-FFE), the 3D water selective cartilage scan (WATS-c) and the 3D water selective fluid scan (WATS-f) were obtained on a 1.5T MRI scanner. The patellar cartilage was evaluated in nine areas: the superior, middle and the inferior portions that were subdivided into the medial, central and lateral facets in a total of 215 areas. Employing the Noyes grading system, the MRI grade 0-I, II and III lesions were compared using the gross and microscopic findings. The sensitivity, specificity and accuracy were evaluated for each sequence. The significance of the differences for the individual sequences was calculated using the McNemar test. Results The gross and microscopic findings demonstrated 167 grade 0-I lesions, 40 grade II lesions and eight grade III lesions. Iw-FS-FSE had the highest accuracy (sensitivity/specificity/accuracy = 88%/98%/96%), followed by FS-PD (78%/98%/93%, respectively), PD (76%/98%/93%, respectively), B-FFE (71%/100%/93%, respectively), WATS-c (67%/100%/92%, respectively) and WATS-f (58%/99%/89%, respectively). There were statistically significant differences for the iw-FS-FSE and WATS-f and for the PD-FS and WATS-f (p < 0.01). Conclusion The iw-FS-FSE images obtained with a microscopy coil show best diagnostic performance among the 2D and 3D GRE images for evaluating the chondromalacia patella. PMID:21228943

Evaluation of the chondromalacia patella using a microscopy coil: comparison of the two-dimensional fast spin echo techniques and the three-dimensional fast field echo techniques.

PubMed

Kim, Hyun-joo; Lee, Sang Hoon; Kang, Chang Ho; Ryu, Jeong Ah; Shin, Myung Jin; Cho, Kyung-Ja; Cho, Woo Shin

2011-01-01

We wanted to compare the two-dimensional (2D) fast spin echo (FSE) techniques and the three-dimensional (3D) fast field echo techniques for the evaluation of the chondromalacia patella using a microscopy coil. Twenty five patients who underwent total knee arthroplasty were included in this study. Preoperative MRI evaluation of the patella was performed using a microscopy coil (47 mm). The proton density-weighted fast spin echo images (PD), the fat-suppressed PD images (FS-PD), the intermediate weighted-fat suppressed fast spin echo images (iw-FS-FSE), the 3D balanced-fast field echo images (B-FFE), the 3D water selective cartilage scan (WATS-c) and the 3D water selective fluid scan (WATS-f) were obtained on a 1.5T MRI scanner. The patellar cartilage was evaluated in nine areas: the superior, middle and the inferior portions that were subdivided into the medial, central and lateral facets in a total of 215 areas. Employing the Noyes grading system, the MRI grade 0-I, II and III lesions were compared using the gross and microscopic findings. The sensitivity, specificity and accuracy were evaluated for each sequence. The significance of the differences for the individual sequences was calculated using the McNemar test. The gross and microscopic findings demonstrated 167 grade 0-I lesions, 40 grade II lesions and eight grade III lesions. Iw-FS-FSE had the highest accuracy (sensitivity/specificity/accuracy = 88%/98%/96%), followed by FS-PD (78%/98%/93%, respectively), PD (76%/98%/93%, respectively), B-FFE (71%/100%/93%, respectively), WATS-c (67%/100%/92%, respectively) and WATS-f (58%/99%/89%, respectively). There were statistically significant differences for the iw-FS-FSE and WATS-f and for the PD-FS and WATS-f (p < 0.01). The iw-FS-FSE images obtained with a microscopy coil show best diagnostic performance among the 2D and 3D GRE images for evaluating the chondromalacia patella.

Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

NASA Astrophysics Data System (ADS)

Chia, Thomas H.

Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning multiphoton laser excitation to sample a ˜4 mm2 region from 54 genuine Reserve Notes. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

Practical applications of nondestructive materials characterization

NASA Astrophysics Data System (ADS)

Green, Robert E., Jr.

1992-10-01

Nondestructive evaluation (NDE) techniques are reviewed for applications to the industrial production of materials including microstructural, physical, and chemical analyses. NDE techniques addressed include: (1) double-pulse holographic interferometry for sealed-package leak testing; (2) process controls for noncontact metals fabrication; (3) ultrasonic detections of oxygen contamination in titanium welds; and (4) scanning acoustic microscopy for the evaluation of solder bonds. The use of embedded sensors and emerging NDE concepts provides the means for controlling the manufacturing and quality of quartz crystal resonators, nickel single-crystal turbine blades, and integrated circuits. Advances in sensor technology and artificial intelligence algorithms and the use of embedded sensors combine to make NDE technology highly effective in controlling industrial materials manufacturing and the quality of the products.

Micromechanical Characterization and Texture Analysis of Direct Cast Titanium Alloys Strips

NASA Technical Reports Server (NTRS)

2000-01-01

This research was conducted to determine a post-processing technique to optimize mechanical and material properties of a number of Titanium based alloys and aluminides processed via Melt Overflow Solidification Technique (MORST). This technique was developed by NASA for the development of thin sheet titanium and titanium aluminides used in high temperature applications. The materials investigated in this study included conventional titanium alloy strips and foils, Ti-1100, Ti-24Al-11Nb (Alpha-2), and Ti-48Al-2Ta (Gamma). The methodology used included micro-characterization, heat-treatment, mechanical processing and mechanical testing. Characterization techniques included optical, electron microscopy, and x-ray texture analysis. The processing included heat-treatment and mechanical deformation through cold rolling. The initial as-cast materials were evaluated for their microstructure and mechanical properties. Different heat-treatment and rolling steps were chosen to process these materials. The properties were evaluated further and a processing relationship was established in order to obtain an optimum processing condition. The results showed that the as-cast material exhibited a Widmanstatten (fine grain) microstructure that developed into a microstructure with larger grains through processing steps. The texture intensity showed little change for all processing performed in this investigation.

Synthesis, characterization, and antimicrobial properties of copper nanoparticles

PubMed Central

Usman, Muhammad Sani; Zowalaty, Mohamed Ezzat El; Shameli, Kamyar; Zainuddin, Norhazlin; Salama, Mohamed; Ibrahim, Nor Azowa

2013-01-01

Copper nanoparticle synthesis has been gaining attention due to its availability. However, factors such as agglomeration and rapid oxidation have made it a difficult research area. In the present work, pure copper nanoparticles were prepared in the presence of a chitosan stabilizer through chemical means. The purity of the nanoparticles was authenticated using different characterization techniques, including ultraviolet visible spectroscopy, transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and field emission scanning electron microscopy. The antibacterial as well as antifungal activity of the nanoparticles were investigated using several microorganisms of interest, including methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella choleraesuis, and Candida albicans. The effect of a chitosan medium on growth of the microorganism was studied, and this was found to influence growth rate. The size of the copper nanoparticles obtained was in the range of 2–350 nm, depending on the concentration of the chitosan stabilizer. PMID:24293998

Key Role of M.G. Nakhodkin’s Insight and Inspiration in Development of UHV STM-Related Techniques and Methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyubinetsky, Igor

2015-02-15

In this contribution I briefly describe my joint efforts and experiences with M.G. Nakhodkin in the field of scanning tunneling microscopy (STM) including a construction of the home-built microscopes, application of this technique in various scientific endevours, as well as fruitfull and enlightening discusions. Our co-operation was focused on the novel aspects of STM probes preparation and conditioning, coupling the STM junction with laser irradiation, STM-based nanolithography, and also on collaboration at the international scale with M.G. Nakhodkin and members of his scientific group.

Fluorescence lifetime in cardiovascular diagnostics

PubMed Central

Marcu, Laura

2010-01-01

We review fluorescence lifetime techniques including time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and fluorescence lifetime imaging microscopy (FLIM) instrumentation and associated methodologies that allow for characterization and diagnosis of atherosclerotic plaques. Emphasis is placed on the translational research potential of TR-LIFS and FLIM and on determining whether intrinsic fluorescence signals can be used to provide useful contrast for the diagnosis of high-risk atherosclerotic plaque. Our results demonstrate that these techniques allow for the discrimination of important biochemical features involved in atherosclerotic plaque instability and rupture and show their potential for future intravascular applications. PMID:20210432

A comparative review of optical surface contamination assessment techniques

NASA Technical Reports Server (NTRS)

Heaney, James B.

1987-01-01

This paper will review the relative sensitivities and practicalities of the common surface analytical methods that are used to detect and identify unwelcome adsorbants on optical surfaces. The compared methods include visual inspection, simple reflectometry and transmissiometry, ellipsometry, infrared absorption and attenuated total reflectance spectroscopy (ATR), Auger electron spectroscopy (AES), scanning electron microscopy (SEM), secondary ion mass spectrometry (SIMS), and mass accretion determined by quartz crystal microbalance (QCM). The discussion is biased toward those methods that apply optical thin film analytical techniques to spacecraft optical contamination problems. Examples are cited from both ground based and in-orbit experiments.

Biophotonic techniques for manipulation and characterization of drug delivery nanosystems in cancer therapy.

PubMed

Spyratou, E; Makropoulou, M; Mourelatou, E A; Demetzos, C

2012-12-31

Reactive oxygen species (ROS) are usually involved in two opposite procedures related to cancer: initiation, progression and metastasis of cancer, as well as in all non-surgical therapeutic approaches for cancer, including chemotherapy, radiotherapy and photodynamic therapy. This review is concentrated in new therapeutic strategies that take advantage of increased ROS in cancer cells to enhance therapeutic activity and selectivity. Novel biophotonic techniques for manipulation and characterization of drug delivery nanosystems in cancer therapy are discussed, including optical tweezers and atomic force microscopy. This review highlights how these techniques are playing a critical role in recent and future cancer fighting applications. We can conclude that Biophotonics and nanomedicine are the future for cancer biology and disease management, possessing unique potential for early detection, accurate diagnosis, dosimetry and personalized treatment of biomedical applications targeting cancer. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Biological applications of confocal fluorescence polarization microscopy

NASA Astrophysics Data System (ADS)

Bigelow, Chad E.

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed.

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection

PubMed Central

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E.

2018-01-01

Titanium dioxide (TiO2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here. PMID:29541425

Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.

PubMed

Brown, Koshonna; Thurn, Ted; Xin, Lun; Liu, William; Bazak, Remon; Chen, Si; Lai, Barry; Vogt, Stefan; Jacobsen, Chris; Paunesku, Tatjana; Woloschak, Gayle E

2018-01-01

Titanium dioxide (TiO 2 ) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. Herein, we describe two in situ post-treatment labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyne-conjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.

Total internal reflection and dynamic light scattering microscopy of gels

NASA Astrophysics Data System (ADS)

Gregor, Brian F.

Two different techniques which apply optical microscopy in novel ways to the study of biological systems and materials were built and applied to several samples. The first is a system for adapting the well-known technique of dynamic light scattering (DLS) to an optical microscope. This can detect and scatter light from very small volumes, as compared to standard DLS which studies light scattering from volumes 1000x larger. The small scattering volume also allows for the observation of nonergodic dynamics in appropriate samples. Porcine gastric mucin (PGM) forms a gel at low pH which lines the epithelial cell layer and acts as a protective barrier against the acidic stomach environment. The dynamics and microscopic viscosity of PGM at different pH levels is studied using polystyrene microspheres as tracer particles. The microscopic viscosity and microrheological properties of the commercial basement membrane Matrigel are also studied with this instrument. Matrigel is frequently used to culture cells and its properties remain poorly determined. Well-characterized and purely synthetic Matrigel substitutes will need to have the correct rheological and morphological characteristics. The second instrument designed and built is a microscope which uses an interferometry technique to achieve an improvement in resolution 2.5x better in one dimension than the Abbe diffraction limit. The technique is based upon the interference of the evanescent field generated on the surface of a prism by a laser in a total internal reflection geometry. The enhanced resolution is demonstrated with fluorescent samples. Additionally. Raman imaging microscopy is demonstrated using the evanescent field in resonant and non-resonant samples, although attempts at applying the enhanced resolution technique to the Raman images were ultimately unsuccessful. Applications of this instrument include high resolution imaging of cell membranes and macroscopic structures in gels and proteins. Finally, a third section incorporating previous research on simulations of complex fluids is included. Two dimensional simulations of oil, water, and surfactant mixtures were computed with a lattice gas method. The simulated systems were randomly mixed and then the temperature was quenched to a predetermined point. Spontaneous micellization is observed for a narrow range of temperature quenches, and the overall growth rate of macroscopic structure is found to follow a Vogel-Fulcher growth law.

Quantitative photothermal phase imaging of red blood cells using digital holographic photothermal microscope.

PubMed

Vasudevan, Srivathsan; Chen, George C K; Lin, Zhiping; Ng, Beng Koon

2015-05-10

Photothermal microscopy (PTM), a noninvasive pump-probe high-resolution microscopy, has been applied as a bioimaging tool in many biomedical studies. PTM utilizes a conventional phase contrast microscope to obtain highly resolved photothermal images. However, phase information cannot be extracted from these photothermal images, as they are not quantitative. Moreover, the problem of halos inherent in conventional phase contrast microscopy needs to be tackled. Hence, a digital holographic photothermal microscopy technique is proposed as a solution to obtain quantitative phase images. The proposed technique is demonstrated by extracting phase values of red blood cells from their photothermal images. These phase values can potentially be used to determine the temperature distribution of the photothermal images, which is an important study in live cell monitoring applications.

Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

DOE PAGES

Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; ...

2016-09-16

We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yieldmore » can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.« less

Curcumin Inhibits Tau Aggregation and Disintegrates Preformed Tau Filaments in vitro.

PubMed

Rane, Jitendra Subhash; Bhaumik, Prasenjit; Panda, Dulal

2017-01-01

The pathological aggregation of tau is a common feature of most of the neuronal disorders including frontotemporal dementia, Parkinson's disease, and Alzheimer's disease. The inhibition of tau aggregation is considered to be one of the important strategies for treating these neurodegenerative diseases. Curcumin, a natural polyphenolic molecule, has been reported to have neuroprotective ability. In this work, curcumin was found to bind to adult tau and fetal tau with a dissociation constant of 3.3±0.4 and 8±1 μM, respectively. Molecular docking studies indicated a putative binding site of curcumin in the microtubule-binding region of tau. Using several complementary techniques, including dynamic light scattering, thioflavin S fluorescence, 90° light scattering, electron microscopy, and atomic force microscopy, curcumin was found to inhibit the aggregation of tau. The dynamic light scattering analysis and atomic force microscopic images revealed that curcumin inhibits the oligomerization of tau. Curcumin also disintegrated preformed tau oligomers. Using Far-UV circular dichroism, curcumin was found to inhibit the β-sheets formation in tau indicating that curcumin inhibits an initial step of tau aggregation. In addition, curcumin inhibited tau fibril formation. Furthermore, the effect of curcumin on the preformed tau filaments was analyzed by atomic force microscopy, transmission electron microscopy, and 90° light scattering. Curcumin treatment disintegrated preformed tau filaments. The results indicated that curcumin inhibited the oligomerization of tau and could disaggregate tau filaments.

Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing.

PubMed

Oommen, Seema; Banaji, Nandita

2017-01-01

There have been rapid technological advances in the detection of Mycobacterium tuberculosis and its drug susceptibility in clinical samples. These include advances in microscopic examination, in vitro culture and application of molecular techniques. The World Health Organization (WHO) has played a large role in evaluating these technologies for their efficacy and feasibility, especially in the developing countries. Amongst these, the Revised National Tuberculosis Control Programme (RNTCP), through its national network of designated microscopy centres and intermediate reference laboratories, has adopted certain technologies that are currently implemented in India. Advances in microscopy technology include fluorescent microscopy using light-emitting diode source, sodium hypochlorite microscopy and vital fluorescent staining of sputum smears. Automation of in vitro culture has markedly reduced the turnaround time (TAT), even in smear-negative samples, and permits simultaneous detection of resistant mutants. Molecular detection of drug resistance has further reduced the TAT, and the cartridge-based nucleic acid amplification test with its performance convenience and rapid results, appears poised to become the future of tuberculosis (TB) diagnosis in all settings, provided the cost of testing is reduced especially for use in private diagnostic settings. This article reviews technologies currently available for the diagnosis of TB, keeping in mind the WHO recommendations and the RNTCP practices. This is a thematic synthesis of data available on diagnosis in literature, preserving the conclusions of the primary studies.

Two-photon speckle illumination for super-resolution microscopy.

PubMed

Negash, Awoke; Labouesse, Simon; Chaumet, Patrick C; Belkebir, Kamal; Giovannini, Hugues; Allain, Marc; Idier, Jérôme; Sentenac, Anne

2018-06-01

We present a numerical study of a microscopy setup in which the sample is illuminated with uncontrolled speckle patterns and the two-photon excitation fluorescence is collected on a camera. We show that, using a simple deconvolution algorithm for processing the speckle low-resolution images, this wide-field imaging technique exhibits resolution significantly better than that of two-photon excitation scanning microscopy or one-photon excitation bright-field microscopy.

Individual Human Cell Responses to Low Doses of Chemicals and Radiation Studied by Synchrotron Infrared Spectromicroscopy

NASA Astrophysics Data System (ADS)

Martin, Michael C.; Holman, Hoi-Ying N.; Blakely, Eleanor A.; Goth-Goldstein, Regine; McKinney, Wayne R.

2000-03-01

Vibrational spectroscopy, when combined with synchrotron radiation-based (SR) microscopy, is a powerful new analytical tool with high spatial resolution for detecting biochemical changes in individual living cells. In contrast to other microscopy methods that require fixing, drying, staining or labeling, SR FTIR microscopy probes intact living cells providing a composite view of all of the molecular responses and the ability to monitor the responses over time in the same cell. Observed spectral changes include all types of lesions induced in that cell as well as cellular responses to external and internal stresses. These spectral changes combined with other analytical tools may provide a fundamental understanding of the key molecular mechanisms induced in response to stresses created by low-doses of radiation and chemicals. In this study we used high spatial-resolution SR FTIR vibrational spectromicroscopy at ALS Beamline 1.4.3 as a sensitive analytical tool to detect chemical- and radiation-induced changes in individual human cells. Our preliminary spectral measurements indicate that this technique is sensitive enough to detect changes in nucleic acids and proteins of cells treated with environmentally relevant concentrations of oxidative stresses: bleomycin, hydrogen peroxide, and X-rays. We observe spectral changes that are unique to each exogenous stressor. This technique has the potential to distinguish changes from exogenous or endogenous oxidative processes. Future development of this technique will allow rapid monitoring of cellular processes such as drug metabolism, early detection of disease, bio-compatibility of implant materials, cellular repair mechanisms, self assembly of cellular apparatus, cell differentiation and fetal development.

A Comparison of Honey Bee-Collected Pollen From Working Agricultural Lands Using Light Microscopy and ITS Metabarcoding.

PubMed

Smart, M D; Cornman, R S; Iwanowicz, D D; McDermott-Kubeczko, M; Pettis, J S; Spivak, M S; Otto, C R V

2017-02-01

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010-2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiaoliang Sunney

Single-molecule spectroscopy has made considerable impact on many disciplines including chemistry, physics, and biology. To date, most single-molecule spectroscopy work is accomplished by detecting fluorescence. On the other hand, many naturally occurring chromophores, such as retinal, hemoglobin and cytochromes, do not have detectable fluorescence. There is an emerging need for single-molecule spectroscopy techniques that do not require fluorescence. In the last proposal period, we have successfully demonstrated stimulated emission microscopy, single molecule absorption, and stimulated Raman microscopy based on a high-frequency modulation transfer technique. These first-of-a- kind new spectroscopy/microscopy methods tremendously improved our ability to observe molecules that fluorescence weakly,more » even to the limit of single molecule detection for absorption measurement. All of these methods employ two laser beams: one (pump beam) excites a single molecule to a real or virtual excited state, and the other (probe beam) monitors the absorption/emission property of the single. We extract the intensity change of the probe beam with high sensitivity by implementing a high-frequency phase-sensitive detection scheme, which offers orders of magnitude improvement in detection sensitivity over direct absorption/emission measurement. However, single molecule detection based on fluorescence or absorption is fundamentally limited due to their broad spectral response. It is important to explore other avenues in single molecule detection and imaging which provides higher molecular specificity for studying a wide variety of heterogeneous chemical and biological systems. This proposal aimed to achieve single-molecule detection sensitivity with near resonance stimulated Raman scattering (SRS) microscopy. SRS microscopy was developed in our lab as a powerful technique for imaging heterogeneous samples based on their intrinsic vibrational contrasts, which provides much higher molecular specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.« less

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy

PubMed Central

Cisek, Richard; Wein, Marc N.; Turcotte, Raphaël; Haase, Christa; Yeh, Shu-Chi A.; Bharadwaj, Srinidhi; Raphael, Anthony P.; Paudel, Hari; Alt, Clemens; Liu, Tzu-Ming; Kronenberg, Henry M.; Lin, Charles P.

2017-01-01

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning. PMID:29065178

Developing best practice for fungal specimen management: audit of UK microbiology laboratories.

PubMed

Lasseter, G; Palmer, M; Morgan, J; Watts, J; Yoxall, H; Kibbler, C; McNulty, C

2011-01-01

This study represents an audit of microbiology laboratories in the UK to ascertain whether they are aware of, or follow, the Health Protection Agency (HPA) National Standard Methods Standard Operating Procedure (NSM SOP) for the investigation of dermatological specimens for superficial mycoses, or use a locally adapted version. A questionnaire audit was distributed to 179 NHS microbiology laboratories throughout England, Wales, Scotland and Northern Ireland. The NSM SOP was followed by 92% of laboratories for the microscopy of dermatological samples; light microscopy/ KOH digestion was used by 63% and fluorescence microscopy/KOH digestion by 29% of laboratories. Preliminary reports post-microscopy were issued by 98% of laboratories, with 93% issuing reports within 48 hours. Adherence to the NSM SOP guidelines for culture was low; only 34% of laboratories incubated microscopy-negative specimens for the recommended 14 days, while approximately 60% incubated microscopy-positive specimens for 21 days. The culture medium recommended by the NSM SOP was used in 82% of laboratories. Comments were added to culture reports by 51% of laboratories; most were added manually and comments varied between laboratories. Nail samples were the most common sample received from primary care, followed by skin and hair. These results show no significant difference in the rate of microscopy positives versus culture positives. Microscopy and culture are the easiest and cheapest methods available to UK laboratories for the investigation of suspected superficial fungal infections. Although most laboratories included in this audit claimed to follow the NSM SOP for microscopy and culture, these results show that the techniques used vary throughout the UK. To maximise the service provided to primary care, UK laboratories should use standardise methods based on the NSM SOP.

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

PubMed

Havrdova, M; Polakova, K; Skopalik, J; Vujtek, M; Mokdad, A; Homolkova, M; Tucek, J; Nebesarova, J; Zboril, R

2014-12-01

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Two-photon optical microscopy imaging of endothelial keratoplasty grafts.

PubMed

Lombardo, Marco; Parekh, Mohit; Serrao, Sebastiano; Ruzza, Alessandro; Ferrari, Stefano; Lombardo, Giuseppe

2017-03-01

To investigate the microstructure of endothelial keratoplasty grafts using two-photon optical microscopy. Six endothelial keratoplasty grafts obtained from human donor corneoscleral tissues and prepared by submerged hydrodissection technique were imaged by two-photon optical microscopy. In each graft, two liquid bubbles were created in order to investigate the presence of a conserved cleavage plane regardless of the volume of posterior stroma that remained attached to Descemet's membrane (DM); the first bubble (bubble A) was generated under DM and the second bubble (bubble B) injection was done in order to obtain a layer of deep stroma that kept the two bubbles separated. Six human donor corneoscleral tissues were used as controls. Second harmonic generation and two-photon emitted fluorescence signals were collected from each specimen. Dissection of stroma occurred along the posterior collagen lamellae at variable distance from DM, which ranged between 3 and 16 μm in bubble A and between 23 and 41 μm in bubble B. The residual stroma included, anteriorly, bands of collagen lamellae, and thin bundles of stromal collagen fibrils, posteriorly, which were tightly intertwining with the underlying DM. There was no anatomically distinct plane of separation between these pre-Descemetic stromal collagen bundles and the overlying collagen lamellae with this hydrodissection technique. Two-photon optical microscopy provided label-free high-resolution imaging of endothelial keratoplasty grafts, showing that the most posterior stroma changes organization at approximately 10 μm above the DM. The pre-Descemetic stromal collagen fibrils form an intertwined complex with DM, which cannot be separated using hydrodissection.

Comparison of microscopy, ELISA, and real-time PCR for detection of Giardia intestinalis in human stool specimens

PubMed

Beyhan, Yunus Emre; Taş Cengiz, Zeynep

2017-08-23

Background/aim: This study included patients who had digestive system complaints between August 2015 and October 2015. The research was designed to compare conventional microscopy with an antigen detection ELISA kit and the TaqMan-based real-time PCR (RT-PCR) technique for detection of Giardia intestinalis in human stool specimens. Materials and methods: Samples were concentrated by formalin-ether sedimentation technique and microscopic examinations were carried out on wet mount slides. A commercially available ELISA kit (Giardia CELISA, Cellabs, Brookvale, Australia) was used for immunoassay. DNA was extracted from fecal samples of about 200 mg using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and the LightCycler Nano system (Roche Diagnostics, Mannheim, Germany) was used for the TaqMan-based RT-PCR assay. Results: A total of 94 stool samples, 38 of them diagnosed positive (40.4%) and 56 of them diagnosed negative by microscopy, were selected for evaluation by antigen detection and molecular assays. The prevalence of G. intestinalis infection was found as 46.8% (n: 44) and 79.8% (n: 75) by ELISA and RT-PCR, respectively. RT-PCR revealed by far the highest positivity rate compared to the other two methods. The difference between these methods was found to be statistically significant (P < 0.05). In comparison to PCR, the sensitivity and specificity of microscopy and ELISA were 50.7% and 100% and 53.3% and 79%, respectively. Conclusion: RT-PCR seems to be much more sensitive and beneficial for rapid and accurate diagnosis of G. intestinalis in human stools.

Determination of the electrical resistivity of vertically aligned carbon nanotubes by scanning probe microscopy

NASA Astrophysics Data System (ADS)

Ageev, O. A.; Il'in, O. I.; Rubashkina, M. V.; Smirnov, V. A.; Fedotov, A. A.; Tsukanova, O. G.

2015-07-01

Techniques are developed to determine the resistance per unit length and the electrical resistivity of vertically aligned carbon nanotubes (VA CNTs) using atomic force microscopy (AFM) and scanning tunneling microscopy (STM). These techniques are used to study the resistance of VA CNTs. The resistance of an individual VA CNT calculated with the AFM-based technique is shown to be higher than the resistance of VA CNTs determined by the STM-based technique by a factor of 200, which is related to the influence of the resistance of the contact of an AFM probe to VA CNTs. The resistance per unit length and the electrical resistivity of an individual VA CNT 118 ± 39 nm in diameter and 2.23 ± 0.37 μm in height that are determined by the STM-based technique are 19.28 ± 3.08 kΩ/μm and 8.32 ± 3.18 × 10-4 Ω m, respectively. The STM-based technique developed to determine the resistance per unit length and the electrical resistivity of VA CNTs can be used to diagnose the electrical parameters of VA CNTs and to create VA CNT-based nanoelectronic elements.

Nondestructive evaluation of structural ceramics by photoacoustic microscopy

NASA Technical Reports Server (NTRS)

Khandelwal, Pramod K.

1987-01-01

A photoacoustic microscopy (PAM) digital imaging system was developed and utilized to characterize silicon nitride material at the various stages of the ceramic fabrication process. Correlation studies revealed that photoacoustic microscopy detected failure initiating defects in substantially more specimens than microradiography and ultrasonic techniques. Photoacoustic microscopy detected 10 to 100 micron size surface and subsurface pores and inclusions, respectively, up to 80 microns below the interrogating surface in machined sintered silicon nitride. Microradiography detected 50 micron diameter fracture controlling pores and inclusions. Subsurface holes were detected up to a depth of 570 microns and 1.00 mm in sintered silicon nitride and silicon carbide, respectively. Seeded voids of 20 to 30 micron diameters at the surface and 50 microns below the interrogating surface were detected by photoacoustic microscopy and microradiography with 1 percent X-ray thickness sensitivity. Tight surface cracks of 96 micron length x 48 micron depth were detected by photoacoustic microscopy. PAM volatilized and removed material in the green state which resulted in linear shallow microcracks after sintering. This significantly limits the use of PAM as an in-process NDE technique.

In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy.

PubMed

Littlejohn, George R; Mansfield, Jessica C; Parker, David; Lind, Rob; Perfect, Sarah; Seymour, Mark; Smirnoff, Nicholas; Love, John; Moger, Julian

2015-05-01

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles. © 2015 American Society of Plant Biologists. All Rights Reserved.

New advances in scanning microscopy and its application to study parasitic protozoa.

PubMed

de Souza, Wanderley; Attias, Marcia

2018-07-01

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities. Copyright © 2018. Published by Elsevier Inc.

A Novel Microcharacterization Technique in the Measurement of Strain and Orientation Gradient in Advanced Materials

NASA Technical Reports Server (NTRS)

Garmestai, H.; Harris, K.; Lourenco, L.

1997-01-01

Representation of morphology and evolution of the microstructure during processing and their relation to properties requires proper experimental techniques. Residual strains, lattice distortion, and texture (micro-texture) at the interface and the matrix of a layered structure or a functionally gradient material and their variation are among parameters important in materials characterization but hard to measure with present experimental techniques. Current techniques available to measure changes in interred material parameters (residual stress, micro-texture, microplasticity) produce results which are either qualitative or unreliable. This problem becomes even more complicated in the case of a temperature variation. These parameters affect many of the mechanical properties of advanced materials including stress-strain relation, ductility, creep, and fatigue. A review of some novel experimental techniques using recent advances in electron microscopy is presented here to measure internal stress, (micro)texture, interracial strength and (sub)grain formation and realignment. Two of these techniques are combined in the chamber of an Environmental Scanning Electron Microscope to measure strain and orientation gradients in advanced materials. These techniques which include Backscattered Kikuchi Diffractometry (BKD) and Microscopic Strain Field Analysis are used to characterize metallic and intermetallic matrix composites and superplastic materials. These techniques are compared with the more conventional x-ray diffraction and indentation techniques.

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

Scanning probe recognition microscopy investigation of tissue scaffold properties

PubMed Central

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis. PMID:18203431

Scanning probe recognition microscopy investigation of tissue scaffold properties.

PubMed

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.

A study of reduced chromium content in a nickel-base superalloy via element substitution and rapid solidification processing. Ph.D. ThesisFinal Report

NASA Technical Reports Server (NTRS)

Powers, William O.

1987-01-01

A study of reduced chromium content in a nickel base superalloy via element substitution and rapid solidification processing was performed. The two elements used as partial substitutes for chromium were Si and Zr. The microstructure of conventionally solidified materials was characterized using microscopy techniques. These alloys were rapidly solidified using the chill block melt spinning technique and the rapidly solidified microstructures were characterized using electron microscopy. The spinning technique and the rapidly solidified microstructures was assessed following heat treatments at 1033 and 1272 K. Rapidly solidified material of three alloys was reduced to particulate form and consolidated using hot isostatic pressing (HIP). The consolidated materials were also characterized using microscopy techniques. In order to evaluate the relative strengths of the consolidated alloys, compression tests were performed at room temperature and 1033 K on samples of as-HIPed and HIPed plus solution treated material. Yield strength, porosity, and oxidation resistance characteristics are given and compared.

Infrared and Raman Microscopy in Cell Biology

PubMed Central

Matthäus, Christian; Bird, Benjamin; Miljković, Miloš; Chernenko, Tatyana; Romeo, Melissa; Diem, Max

2009-01-01

This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cell’s biochemical composition is collected at a spatial resolution of typically 25 mm. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes. PMID:19118679

A simple approach to characterizing block copolymer assemblies: graphene oxide supports for high contrast multi-technique imaging†

PubMed Central

Patterson, Joseph P.; Sanchez, Ana M.; Petzetakis, Nikos; Smart, Thomas P.; Epps, Thomas H.; Portman, Ian

2013-01-01

Block copolymers are well-known to self-assemble into a range of 3-dimensional morphologies. However, due to their nanoscale dimensions, resolving their exact structure can be a challenge. Transmission electron microscopy (TEM) is a powerful technique for achieving this, but for polymeric assemblies chemical fixing/staining techniques are usually required to increase image contrast and protect specimens from electron beam damage. Graphene oxide (GO) is a robust, water-dispersable, and nearly electron transparent membrane: an ideal support for TEM. We show that when using GO supports no stains are required to acquire high contrast TEM images and that the specimens remain stable under the electron beam for long periods, allowing sample analysis by a range of electron microscopy techniques. GO supports are also used for further characterization of assemblies by atomic force microscopy. The simplicity of sample preparation and analysis, as well as the potential for significantly increased contrast background, make GO supports an attractive alternative for the analysis of block copolymer assemblies. PMID:24049544

Extending the knowledge in histochemistry and cell biology.

PubMed

Heupel, Wolfgang-Moritz; Drenckhahn, Detlev

2010-01-01

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird's- up to worm's-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in "histo-biochemical" techniques and their manifold applications.

Correlative fractography: combining scanning electron microscopy and light microscopes for qualitative and quantitative analysis of fracture surfaces.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-04-01

Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

Morphology of one-time coated palladium-alumina composite membrane prepared by sol-gel process and electroless plating technique

NASA Astrophysics Data System (ADS)

Sari, R.; Dewi, R.; Pardi; Hakim, L.; Diana, S.

2018-03-01

Palladium coated porous alumina ceramic membrane tube was obtained using a combination of sol-gel process and electroless plating technique. The thickness, structure and composition of palladium-alumina composite membrane were analyzed by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and atomic force microscopy (AFM). Palladium particle size was 6.18 to 7.64 nm. Palladium membrane with thickness of approximately 301.5 to 815.1 nm was formed at the outer surface of the alumina layer. EDX data confirmed the formation of palladium-alumina membrane containing 45% of palladium. From this research it shows the combination of sol-gel process and electroless plating technique with one-time coating can produce a homogeneous and smoother palladium nano layer film on alumina substrate.

A combined confocal and magnetic resonance microscope for biological studies

NASA Astrophysics Data System (ADS)

Majors, Paul D.; Minard, Kevin R.; Ackerman, Eric J.; Holtom, Gary R.; Hopkins, Derek F.; Parkinson, Christopher I.; Weber, Thomas J.; Wind, Robert A.

2002-12-01

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of cell function in health and disease, particularly when results acquired with different methodologies can be correlated in time and space. In this article, a novel microscope is described for studying live cells simultaneously with both confocal scanning laser fluorescence optical microscopy and magnetic resonance microscopy. The various design considerations necessary for integrating these two complementary techniques are discussed, the layout and specifications of the instrument are given, and examples of confocal and magnetic resonance images of large frog cells and model tumor spheroids obtained with the compound microscope are presented.

Single-shot optical sectioning using two-color probes in HiLo fluorescence microscopy.

PubMed

Muro, Eleonora; Vermeulen, Pierre; Ioannou, Andriani; Skourides, Paris; Dubertret, Benoit; Fragola, Alexandra; Loriette, Vincent

2011-06-08

We describe a wide-field fluorescence microscope setup which combines HiLo microscopy technique with the use of a two-color fluorescent probe. It allows one-shot fluorescence optical sectioning of thick biological moving sample which is illuminated simultaneously with a flat and a structured pattern at two different wavelengths. Both homogenous and structured fluorescence images are spectrally separated at detection and combined similarly with the HiLo microscopy technique. We present optically sectioned full-field images of Xenopus laevis embryos acquired at 25 images/s frame rate. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A facile synthesis of metal nanoparticle - graphene composites for better absorption of solar radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Bindu; Mulla, Rafiq; Rabinal, M. K., E-mail: mkrabinal@yahoo.com

2015-06-24

Herein, a facile chemical approach has been adopted to prepare silver nanoparticles (AgNPs)- graphene (G) composite to study photothermal effect. Sodium borohydride (SBH), a strong reducing agent has been selected for this work. Effect of SBH concentrations on optical behavior of AgNPs-G composite was also investigated. Resultant materials were characterized by various techniques including X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), optical absorption, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM micrographs confirm wrapping of AgNPs into graphene whereas XRD analysis reveals their particle size variation between 47 nm to 69 nm. Optical studies throw a light on theirmore » strong absorption behavior towards solar radiation.« less

Spinel lithium manganese oxide nanoparticles: unique molten salt synthesis strategy and excellent electrochemical performances.

PubMed

Wang, Xiong; Zhu, Juanjuan; Liu, Yingjie

2009-11-01

As a promising candidate cathode material, spinel lithium manganese oxide nanoparticles were successfully synthesized through a novel molten salt synthesis route at relatively low temperature, using manganese dioxide nanowires as precursor. A variety of techniques were applied to characterize the spinel nanomaterial, including X-ray diffraction, transmission electron microscopy, field-emission scanning electron microscopy, and X-ray photoelectron spectroscopy. The average particle size of the resulting spinel nanoparticles was about 80 nm with narrow distribution. As cathode material for rechargeable lithium ion battery, the electrochemical properties were investigated. All the results show that the electrochemical performances of the homogeneous spinel nanoparticles were improved, which might be ascribed to large specific surface area, fairly narrow size distribution, and the unique synthesis strategy.

TLM-Tracker: software for cell segmentation, tracking and lineage analysis in time-lapse microscopy movies.

PubMed

Klein, Johannes; Leupold, Stefan; Biegler, Ilona; Biedendieck, Rebekka; Münch, Richard; Jahn, Dieter

2012-09-01

Time-lapse imaging in combination with fluorescence microscopy techniques enable the investigation of gene regulatory circuits and uncovered phenomena like culture heterogeneity. In this context, computational image processing for the analysis of single cell behaviour plays an increasing role in systems biology and mathematical modelling approaches. Consequently, we developed a software package with graphical user interface for the analysis of single bacterial cell behaviour. A new software called TLM-Tracker allows for the flexible and user-friendly interpretation for the segmentation, tracking and lineage analysis of microbial cells in time-lapse movies. The software package, including manual, tutorial video and examples, is available as Matlab code or executable binaries at http://www.tlmtracker.tu-bs.de.

Synthesis of gold nanoflowers using deep eutectic solvent with high surface enhanced Raman scattering properties

NASA Astrophysics Data System (ADS)

Aghakhani Mahyari, Farzaneh; Tohidi, Maryam; Safavi, Afsaneh

2016-09-01

A facile, seed-less and one-pot method was developed for synthesis of gold nanoflowers with multiple tips through reduction of HAuCl4 with deep eutectic solvent at room temperature. This solvent is eco-friendly, low-cost, non-toxic and biodegradable and can act as both reducing and shape-controlling agent. In this protocol, highly branched and stable gold nanoflowers were obtained without using any capping agent. The obtained products were characterized by different techniques including, field emission scanning electron microscopy, transmission electron microscopy, x-ray diffraction and UV-vis spectroscopy. The as-prepared gold nanoflowers exhibit efficient surface-enhanced Raman scattering (SERS) properties which can be used as excellent substrates for SERS.

On-Chip Biomedical Imaging

PubMed Central

Göröcs, Zoltán; Ozcan, Aydogan

2012-01-01

Lab-on-a-chip systems have been rapidly emerging to pave the way toward ultra-compact, efficient, mass producible and cost-effective biomedical research and diagnostic tools. Although such microfluidic and micro electromechanical systems achieved high levels of integration, and are capable of performing various important tasks on the same chip, such as cell culturing, sorting and staining, they still rely on conventional microscopes for their imaging needs. Recently several alternative on-chip optical imaging techniques have been introduced, which have the potential to substitute conventional microscopes for various lab-on-a-chip applications. Here we present a critical review of these recently emerging on-chip biomedical imaging modalities, including contact shadow imaging, lensfree holographic microscopy, fluorescent on-chip microscopy and lensfree optical tomography. PMID:23558399

Microbiologically influenced corrosion: looking to the future.

PubMed

Videla, Héctor A; Herrera, Liz K

2005-09-01

This review discusses the state-of-the-art of research into biocorrosion and the biofouling of metals and alloys of industrial usage. The key concepts needed to understand the main effects of microorganisms on metal decay, and current trends in monitoring and control strategies to mitigate the deleterious effects of biocorrosion and biofouling are also described. Several relevant cases of biocorrosion studied by our research group are provided as examples: (i) biocorrosion of aluminum and its alloys by fungal contaminants of jet fuels; (ii) sulfate-reducing bacteria (SRB)-induced corrosion of steel; (iii) biocorrosion and biofouling interactions in the marine environment; (iv) monitoring strategies for assessing biocorrosion in industrial water systems; (v) microbial inhibition of corrosion; (vi) use and limitations of electrochemical techniques for evaluating biocorrosion effects. Future prospects in the field are described with respect to the potential of innovative techniques in microscopy (environmental scanning electron microscopy, confocal scanning laser microscopy, atomic force microscopy), new spectroscopic techniques for the study of corrosion products and biofilms (energy dispersion X-ray analysis, X-ray photoelectron spectroscopy, electron microprobe analysis) and electrochemistry (electrochemical impedance spectroscopy, electrochemical noise analysis).

Review of combined isotopic and optical nanoscopy

PubMed Central

Richter, Katharina N.; Rizzoli, Silvio O.; Jähne, Sebastian; Vogts, Angela; Lovric, Jelena

2017-01-01

Abstract. Investigating the detailed substructure of the cell is beyond the ability of conventional optical microscopy. Electron microscopy, therefore, has been the only option for such studies for several decades. The recent implementation of several super-resolution optical microscopy techniques has rendered the investigation of cellular substructure easier and more efficient. Nevertheless, optical microscopy only provides an image of the present structure of the cell, without any information on its long-temporal changes. These can be investigated by combining super-resolution optics with a nonoptical imaging technique, nanoscale secondary ion mass spectrometry, which investigates the isotopic composition of the samples. The resulting technique, combined isotopic and optical nanoscopy, enables the investigation of both the structure and the “history” of the cellular elements. The age and the turnover of cellular organelles can be read by isotopic imaging, while the structure can be analyzed by optical (fluorescence) approaches. We present these technologies, and we discuss their implementation for the study of biological samples. We conclude that, albeit complex, this type of technology is reliable enough for mass application to cell biology. PMID:28466025

Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples

PubMed Central

Yan, Wei; Yang, Yanlong; Tan, Yu; Chen, Xun; Li, Yang; Qu, Junle; Ye, Tong

2018-01-01

Stimulated emission depletion microscopy (STED) is one of far-field optical microscopy techniques that can provide sub-diffraction spatial resolution. The spatial resolution of the STED microscopy is determined by the specially engineered beam profile of the depletion beam and its power. However, the beam profile of the depletion beam may be distorted due to aberrations of optical systems and inhomogeneity of specimens’ optical properties, resulting in a compromised spatial resolution. The situation gets deteriorated when thick samples are imaged. In the worst case, the sever distortion of the depletion beam profile may cause complete loss of the super resolution effect no matter how much depletion power is applied to specimens. Previously several adaptive optics approaches have been explored to compensate aberrations of systems and specimens. However, it is hard to correct the complicated high-order optical aberrations of specimens. In this report, we demonstrate that the complicated distorted wavefront from a thick phantom sample can be measured by using the coherent optical adaptive technique (COAT). The full correction can effectively maintain and improve the spatial resolution in imaging thick samples. PMID:29400356

Orbital angular momentum light in microscopy

PubMed Central

2017-01-01

Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue ‘Optical orbital angular momentum’. PMID:28069768

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

PubMed

Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

2012-09-01

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Application of microscopy technique and high performance liquid chromatography for quality assessment of Polygonum multiflorum Thunb. (Heshouwu)

PubMed Central

Liang, Li; Zhao, Zhongzhen; Kang, Tingguo

2014-01-01

Background: The technique of microscopy has been applied for identification of Chinese materia medica (CMM) since decades. However, very few scientific publications report the combination of conventional microscopy and high performance liquid chromatography (HPLC) techniques for further application to quality assessment of CMM. Objective: The objective of this study is to analyze the quality of the dried root tuber of Polygonum multiflorum Thunb. (Heshouwu) and to establish the relationships between 2,3,5,4’-tetrahydroxystilbene-2-O-β-glucoside, combined anthraquinone (CAQ) and quantity of clusters of calcium oxalate. Materials and Methods: In this study, microscopy and HPLC techniques were applied to assess the quality of P. multiflorum Thunb., and SPSS software was used to establish the relationship between microscopic characteristics and chemical components. Results: The results showed close and direct correlations between the quantity of clusters of calcium oxalate in P. multiflorum Thunb. and the contents of 2,3,5,4’-tetrahydroxystilbene-2-O-β-glucoside and CAQ. From these results, it can be deduced that Polygoni Multiflori Radix with a higher quantity of clusters of calcium oxalate should be of better quality. Conclusion: The established method can be helpful for evaluating the quality of CMM based upon the identification and quantitation of chemical and ergastic substance of cells. PMID:25422540

A classification system of intraocular lens dislocation sites under operating microscopy, and the surgical techniques and outcomes of exchange surgery.

PubMed

Hayashi, Ken; Ogawa, Soichiro; Manabe, Shin-Ichi; Hirata, Akira; Yoshimura, Koichi

2016-03-01

The aim of this study was to examine the recent status of intraocular lens (IOL) dislocation according to a classification system based on vertical dislocation position, as well as the surgical techniques and outcomes of IOL exchange surgery. The medical records of 230 eyes from 214 consecutive patients who experienced IOL dislocation and underwent exchange surgery between 2006 and 2014 were reviewed. Vertical dislocation sites observed preoperatively under operating microscopy were examined, along with the surgical techniques and outcomes of IOL exchange. Dislocation sites included (1) the anterior chamber (12.2 %), (2) pseudophakodonesis (19.1 %), (3) the anterior vitreous cavity (47.4 %), (4) trap door-like dislocation (dangling in the peripheral vitreous cavity; 16.1 %), and (5) the retinal surface (5.2 %). The IOL retained in the anterior segment was moved onto the iris by pulling it up through the limbal side ports with an anterior vitrectomy (67.8 %), or by pushing it up from the pars plana with an anterior vitrectomy (26.5 %), while the IOL dropped on the retina was lifting it up from the retina after pars plana vitrectomy (5.7 %). Mean uncorrected and distance-corrected visual acuity significantly improved postoperatively (p < 0.0001). Major complications included a marked elevation in intraocular pressure (7.8 %), pupillary capture (6.5 %), and vitreous hemorrhage (2.6 %). Based on the classification system, approximately 95 % of dislocated IOLs were retained in the anterior segment, and these IOLs were exchanged using an anterior approach through limbal incisions with an anterior vitrectomy. Visual acuity improved significantly, and serious complications were uncommon, probably because the IOL exchange techniques were standardized and simplified without pars plana vitrectomy.

Coherent Anti-Stokes Raman Scattering Spectroscopy of Single Molecules in Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunney Xie, Wei Min, Chris Freudiger, Sijia Lu

2012-01-18

During this funding period, we have developed two breakthrough techniques. The first is stimulated Raman scattering microscopy, providing label-free chemical contrast for chemical and biomedical imaging based on vibrational spectroscopy. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. We developed a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-freemore » and readily interpretable chemical contrast. We demonstrated a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis. This technology offers exciting prospect for medical imaging. The second technology we developed is stimulated emission microscopy. Many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. We use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, as a new contrast mechanism for optical microscopy. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distribu- tions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging. Although we did not accomplish the original goal of detecting single-molecule by CARS, our quest for high sensitivity of nonlinear optical microscopy paid off in providing the two brand new enabling technologies. Both techniques were greatly benefited from the use of high frequency modulation for microscopy, which led to orders of magnitude increase in sensitivity. Extensive efforts have been made on optics and electronics to accomplish these breakthroughs.« less

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically-important Chrysomya rufifacies (Diptera: Calliphoridae)*

PubMed Central

Flores, Danielle; Miller, Amy L.; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M.N.; Sung, Carl; Stokes, Alexander J.; Tomberlin, Jeffrey K.; Carter, David O.; Turner, Helen

2016-01-01

Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. PMID:27706817

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Recent advances in magnetic resonance microscopy to the physical structure characterization of carbonaceous and inorganic materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregory, D.M.; Gerald, R.E.; Cody, G.D.

1997-04-01

Magnetic resonance microscopy (MRM) techniques have been employed to study the molecular architectures and properties of structural polymers, fossil fuels, microporous carbons and inorganic catalysts.

Lensless microscopy technique for static and dynamic colloidal systems.

PubMed

Alvarez-Palacio, D C; Garcia-Sucerquia, J

2010-09-15

We present the application of a lensless microscopy technique known as digital in-line holographic microscopy (DIHM) to image dynamic and static colloidal systems of microspheres. DIHM has been perfected up to the point that submicrometer lateral resolution with several hundreds of micrometers depth of field is achieved with visible light; it is shown that the lateral resolution of DIHM is enough to resolve self-assembled colloidal monolayers built up from polystyrene spheres with submicrometer diameters. The time resolution of DIHM is of the order of 4 frames/s at 2048 x 2048 pixels, which represents an overall improvement of 16 times the time resolution of confocal scanning microscopy. This feature is applied to the visualization of the migration of dewetting fronts in dynamic colloidal systems and the formation of front-like arrangements of particles. Copyright 2010 Elsevier Inc. All rights reserved.

Setting up and running an advanced light microscopy and imaging facility.

PubMed

Sánchez, Carlos; Muñoz, Ma Ángeles; Villalba, Maite; Labrador, Verónica; Díez-Guerra, F Javier

2011-07-01

During the last twenty years, interest in light microscopy and imaging techniques has grown in various fields, such as molecular and cellular biology, developmental biology, and neurobiology. In addition, the number of scientific articles and journals using these techniques is rapidly increasing. Nowadays, most research institutions require sophisticated microscopy systems to cover their investigation demands. In general, such instruments are too expensive and complex to be purchased and managed by a single laboratory or research group, so they have to be shared with other groups and supervised by specialized personnel. This is the reason why microscopy and imaging facilities are becoming so important at research institutions nowadays. In this unit, we have gathered and presented a number of issues and considerations from our own experience that we hope will be helpful when planning or setting up a new facility.

Not all that glitters is gold-Electron microscopy study on uptake of gold nanoparticles in Daphnia magna and related artifacts.

PubMed

Jensen, Louise Helene Søgaard; Skjolding, Lars Michael; Thit, Amalie; Sørensen, Sara Nørgaard; Købler, Carsten; Mølhave, Kristian; Baun, Anders

2017-06-01

Increasing use of engineered nanoparticles has led to extensive research into their potential hazards to the environment and human health. Cellular uptake from the gut is sparsely investigated, and microscopy techniques applied for uptake studies can result in misinterpretations. Various microscopy techniques were used to investigate internalization of 10-nm gold nanoparticles in Daphnia magna gut lumen and gut epithelial cells following 24-h exposure and outline potential artifacts (i.e., high-contrast precipitates from sample preparation related to these techniques). Light sheet microscopy confirmed accumulation of gold nanoparticles in the gut lumen. Scanning transmission electron microscopy and elemental analysis revealed gold nanoparticles attached to the microvilli of gut cells. Interestingly, the peritrophic membrane appeared to act as a semipermeable barrier between the lumen and the gut epithelium, permitting only single particles through. Structures resembling nanoparticles were also observed inside gut cells. Elemental analysis could not verify these to be gold, and they were likely artifacts from the preparation, such as osmium and iron. Importantly, gold nanoparticles were found inside holocrine cells with disrupted membranes. Thus, false-positive observations of nanoparticle internalization may result from either preparation artifacts or mistaking disrupted cells for intact cells. These findings emphasize the importance of cell integrity and combining elemental analysis with the localization of internalized nanoparticles using transmission electron microscopy. Environ Toxicol Chem 2017;36:1503-1509. © 2016 SETAC. © 2016 SETAC.

Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine

PubMed Central

Vielreicher, M.; Schürmann, S.; Detsch, R.; Schmidt, M. A.; Buttgereit, A.; Boccaccini, A.; Friedrich, O.

2013-01-01

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. PMID:23864499

The application of laser scanning confocal microscopy to the examination of hairs and textile fibers: an initial investigation.

PubMed

Kirkbride, K Paul; Tridico, Silvana R

2010-02-25

An initial investigation of the application of laser scanning confocal microscopy to the examination of hairs and fibers has been conducted. This technique allows the production of virtual transverse and longitudinal cross-sectional images of a wide range of hairs and fibers. Special mounting techniques are not required; specimens that have been mounted for conventional microscopy require no further treatment. Unlike physical cross-sectioning, in which it is difficult to produce multiple cross-sections from a single hair or fiber and the process is destructive, confocal microscopy allows the examiner to image the cross-section at any point in the field of view along the hair or fiber and it is non-destructive. Confocal microscopy is a fluorescence-based technique. The images described in this article were collected using only the autofluorescence exhibited by the specimen (i.e. fluorescence staining was not necessary). Colorless fibers generally and hairs required excitation at 405 nm in order to stimulate useful autofluorescence; longer wavelength excitation was suitable for dyed fibers. Although confocal microscopy was found to be generally applicable to the generation virtual transverse cross-sections from a wide range of hairs and fibers, on some occasions the autofluorescence signal was attenuated by heavy pigmentation or the presence of an opaque medulla in hairs, and by heavy delustering or the presence of air-filled voids in the case of fibers. In these situations only partial cross-sections were obtained. 2009 Elsevier Ireland Ltd. All rights reserved.

Crystal structure of stacking faults in InGaAs/InAlAs/InAs heterostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trunkin, I. N.; Presniakov, M. Yu.; Vasiliev, A. L., E-mail: a.vasiliev56@gmail.com

Stacking faults and dislocations in InGaAs/InAlAs/InAs heterostructures have been studied by electron microscopy. The use of different techniques of transmission electron microscopy (primarily, highresolution dark-field scanning transmission electron microscopy) has made it possible to determine the defect structure at the atomic level.

Detailed Investigation of Core-Shell Precipitates in a Cu-Containing High Entropy Alloy

NASA Astrophysics Data System (ADS)

Alam, T.; Gwalani, B.; Viswanathan, G.; Fraser, H.; Banerjee, R.

2018-05-01

Due to the competing influences of configurational entropy and enthalpy of mixing, in recent years, secondary (including intermetallic) phases have been reported in many high entropy alloy (HEA) systems. These secondary phases offer great potential in terms of strengthening the HEA beyond the solid solution strengthening effects, and as such are of great interest in regards to alloy design for engineering applications. The present research investigates novel nano-scale core-shell precipitates forming within the disordered bcc matrix phase of an Al2CrCuFeNi2 HEA, utilizing complementary high-resolution microscopy techniques of atom probe tomography (APT) and transmission electron microscopy (TEM). The size, morphology, and local chemistry of these core-shell precipitates was measured by APT, and the composition was further corroborated by high-resolution scanning transmission electron microscopy-energy dispersive spectroscopy in an aberration-corrected TEM. Furthermore, high-resolution TEM imaging of the core-shell structure indicates that the Cu-rich core exhibits a bcc crystal structure.

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

PubMed

Blancett, Candace D; Fetterer, David P; Koistinen, Keith A; Morazzani, Elaine M; Monninger, Mitchell K; Piper, Ashley E; Kuehl, Kathleen A; Kearney, Brian J; Norris, Sarah L; Rossi, Cynthia A; Glass, Pamela J; Sun, Mei G

2017-10-01

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

TEMPy: a Python library for assessment of three-dimensional electron microscopy density fits.

PubMed

Farabella, Irene; Vasishtan, Daven; Joseph, Agnel Praveen; Pandurangan, Arun Prasad; Sahota, Harpal; Topf, Maya

2015-08-01

Three-dimensional electron microscopy is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy (template and electron microscopy comparison using Python) is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate-to-low resolution maps, both globally and locally. It also provides procedures for single-fit assessment, ensemble generation of fits, clustering, and multiple and consensus scoring, as well as plots and output files for visualization purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool suitable for both novice and expert structural biologists.

The analysis of colored acrylic, cotton, and wool textile fibers using micro-Raman spectroscopy. Part 2: comparison with the traditional methods of fiber examination.

PubMed

Buzzini, Patrick; Massonnet, Genevieve

2015-05-01

In the second part of this survey, the ability of micro-Raman spectroscopy to discriminate 180 fiber samples of blue, black, and red cottons, wools, and acrylics was compared to that gathered with the traditional methods for the examination of textile fibers in a forensic context (including light microscopy methods, UV-vis microspectrophotometry and thin-layer chromatography). This study shows that the Raman technique plays a complementary and useful role to obtain further discriminations after the application of light microscopy methods and UV-vis microspectrophotometry and assure the nondestructive nature of the analytical sequence. These additional discriminations were observed despite the lower discriminating powers of Raman data considered individually, compared to those of light microscopy and UV-vis MSP. This study also confirms that an instrument equipped with several laser lines is necessary for an efficient use as applied to the examination of textile fibers in a forensic setting. © 2015 American Academy of Forensic Sciences.

Ecology of micro-organisms in a small closed system - Potential benefits and problems for Space Station

NASA Technical Reports Server (NTRS)

Rodgers, E. B.; Seale, D. B.; Boraas, M. E.; Sommer, C. V.

1989-01-01

The probable sources and implications of microbial contamination on the proposed Space Station are discussed. Because of the limited availability of material, facilities and time on the Space Station, we are exploring the feasibility of replacing traditional incubation methods for assessing microbial contamination with rapid, automated methods. Some possibilities include: ATP measurement, microscopy and telecommunications, and molecular techniques such as DNA probes or monoclonal antibodies. Some of the important ecological factors that could alter microbes in space include microgravity, exposure to radiation, and antibiotic resistance.

Plutonium weathering on Johnston Atoll

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, S.E.; Bates, J.K.; Buck, E.C.

1995-12-31

Johnston Atoll was contaminated with transuranic elements, particularly plutonium, by atmospheric nuclear weapons tests and aborted nuclear devices. Initial cleanup operations and and an extensive soil remediation program were performed. However, many areas contained a low-level continuum of activity, and subsurface contamination has been detected. Discrete hot particles and contaminated soil were characterized to determine whether the spread of activity was caused by weathering. Analytical techniques included gamma spectrometry, alpha spectrometry, and inductively coupled plasma-mass spectrometry to determine transuranic elemental and isotopic composition. Ultrafiltration and small-particle handling techniques were employed to isolate individual particles. Optical microscopy, scanning electron microscopy, analyticalmore » transmission electron microscopy, energy dispersive X-ray spectroscopy, and electron energy loss spectroscopy were used to characterize individual particles. Analyses of the hot particles showed that they are aborted nuclear warhead fragments that been melted and weathered in the presence of water and CaCO{sub 3}. It was concluded that the formation of aqueous ionic (Pu/Am)-CO{sub 3} coordinated complexes, during environmental exposure to large volumes of rainwater and carbonate-satured seawater, enhanced the solubility of transuranic elements. The (Pu/Am)-CO{sub 3} complexes sorbed onto colloidal CaCO{sub 3} and coral soil surfaces as they were exposed to rain and seawater. This mechanism led to greater dispersal of plutonium and americium than would be expected by physical transport of discrete hot particles alone.« less

Atomic Force Microscopy: A Powerful Tool to Address Scaffold Design in Tissue Engineering.

PubMed

Marrese, Marica; Guarino, Vincenzo; Ambrosio, Luigi

2017-02-13

Functional polymers currently represent a basic component of a large range of biological and biomedical applications including molecular release, tissue engineering, bio-sensing and medical imaging. Advancements in these fields are driven by the use of a wide set of biodegradable polymers with controlled physical and bio-interactive properties. In this context, microscopy techniques such as Atomic Force Microscopy (AFM) are emerging as fundamental tools to deeply investigate morphology and structural properties at micro and sub-micrometric scale, in order to evaluate the in time relationship between physicochemical properties of biomaterials and biological response. In particular, AFM is not only a mere tool for screening surface topography, but may offer a significant contribution to understand surface and interface properties, thus concurring to the optimization of biomaterials performance, processes, physical and chemical properties at the micro and nanoscale. This is possible by capitalizing the recent discoveries in nanotechnologies applied to soft matter such as atomic force spectroscopy to measure surface forces through force curves. By tip-sample local interactions, several information can be collected such as elasticity, viscoelasticity, surface charge densities and wettability. This paper overviews recent developments in AFM technology and imaging techniques by remarking differences in operational modes, the implementation of advanced tools and their current application in biomaterials science, in terms of characterization of polymeric devices in different forms (i.e., fibres, films or particles).

High-pressure freezing and freeze substitution of Arabidopsis for electron microscopy.

PubMed

Austin, Jotham R

2014-01-01

The objectives of electron microscopy ultrastructural studies are to examine cellular architecture and relate the cell's structural machinery to dynamic functional roles. This aspiration is difficult to achieve if specimens have not been adequately preserved in a "living state"; hence specimen preparation is of the utmost importance for the success of any electron micrographic study. High-pressure freezing (HPF)/freeze substitution (FS) has long been recognized as the primer technique for the preservation of ultrastructure in biological samples. In most cases a basic HPF/freeze substitution protocol is sufficient to obtain superior ultrastructural preservation and structural contrast, which allows one to use more advanced microscopy techniques such as 3D electron tomography. However, for plant tissues, which have a thick cell wall, large water-filled vacuoles, and air spaces (all of which are detrimental to cryopreservation), these basic HPF/FS protocols often yield undesirable results. In particular, ice crystal artifacts and the staining of membrane systems are often poorly or negatively stained, which make 3D segmentation of a tomogram difficult. To overcome these problems, various aspects of the HPF/FS protocol can be altered, including the cryo-filler(s) used, freeze substitution cocktail, and the resin infiltration process. This chapter will describe these modifications for the preparation of plant tissues for routine electron microscopic studies, immunocytochemistry, and 3D tomographic electron imaging.

Developing single-laser sources for multimodal coherent anti-Stokes Raman scattering microscopy

NASA Astrophysics Data System (ADS)

Pegoraro, Adrian Frank

Coherent anti-Stokes Raman scattering (CARS) microscopy has developed rapidly and is opening the door to new types of experiments. This work describes the development of new laser sources for CARS microscopy and their use for different applications. It is specifically focused on multimodal nonlinear optical microscopy—the simultaneous combination of different imaging techniques. This allows us to address a diverse range of applications, such as the study of biomaterials, fluid inclusions, atherosclerosis, hepatitis C infection in cells, and ice formation in cells. For these applications new laser sources are developed that allow for practical multimodal imaging. For example, it is shown that using a single Ti:sapphire oscillator with a photonic crystal fiber, it is possible to develop a versatile multimodal imaging system using optimally chirped laser pulses. This system can perform simultaneous two photon excited fluorescence, second harmonic generation, and CARS microscopy. The versatility of the system is further demonstrated by showing that it is possible to probe different Raman modes using CARS microscopy simply by changing a time delay between the excitation beams. Using optimally chirped pulses also enables further simplification of the laser system required by using a single fiber laser combined with nonlinear optical fibers to perform effective multimodal imaging. While these sources are useful for practical multimodal imaging, it is believed that for further improvements in CARS microscopy sensitivity, new excitation schemes are necessary. This has led to the design of a new, high power, extended cavity oscillator that should be capable of implementing new excitation schemes for CARS microscopy as well as other techniques. Our interest in multimodal imaging has led us to other areas of research as well. For example, a fiber-coupling scheme for signal collection in the forward direction is demonstrated that allows for fluorescence lifetime imaging without significant temporal distortion. Also highlighted is an imaging artifact that is unique to CARS microscopy that can alter image interpretation, especially when using multimodal imaging. By combining expertise in nonlinear optics, laser development, fiber optics, and microscopy, we have developed systems and techniques that will be of benefit for multimodal CARS microscopy.

Contributions of in situ microscopy to the current understanding of stone biodeterioration.

PubMed

de Los Ríos, Asunción; Ascaso, Carmen

2005-09-01

In situ microscopy consists of simultaneously applying several microscopy techniques without separating the biological component from its habitat. Over the past few years, this strategy has allowed characterization of the biofilms involved in biodeterioration processes affecting stone monuments and has revealed the biogeophysical and biogeochemical impact of the microbiota present. In addition, through in situ microscopy diagnosis, appropriate treatments can be designed to resolve the problems related to microbial colonization of stone monuments.

Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination.

PubMed

Chu, Kengyeh K; Lim, Daryl; Mertz, Jerome

2007-10-01

We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

PubMed Central

Novak, Pavel; Li, Chao; Shevchuk, Andrew I.; Stepanyan, Ruben; Caldwell, Matthew; Hughes, Simon; Smart, Trevor G.; Gorelik, Julia; Ostanin, Victor P.; Lab, Max J.; Moss, Guy W. J.; Frolenkov, Gregory I.; Klenerman, David; Korchev, Yuri E.

2009-01-01

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions. PMID:19252505

Deposition And Characterization of (Ti,Zr)N Thin Films Grown Through PAPVD By The Pulsed Arc Technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marulanda, D. M.; Trujillo, O.; Devia, A.

The Plasma Assisted Physic Vapor Deposition (PAPVD) by the pulsed arc technique has been used for deposition of Titanium Zirconium Nitride (Ti,Zr)N coatings, using a segmented target of TiZr. The deposition was performed in a vacuum chamber with two faced electrodes (target and substrate) using nitrogen as working gas, and a power-controlled source used to produce the arc discharges. Films were deposited on stainless steel 304, and they were characterized using the X-Ray Photoelectron Spectroscopy (XPS), X-Ray Diffraction (XRD), Energy Dispersion Spectroscopy (EDS) and Scanning Probe Microscopy (SPM) techniques. The XRD patterns show different planes in which the film grows.more » Through SPM, using Atomic Force Microscopy (AFM) and Lateral Force Microscopy (LFM) modes, a nanotribologic study of the thin film was made, determining hardness and friction coefficient.« less

Two-dimensional dopant profiling of gallium nitride p-n junctions by scanning capacitance microscopy

NASA Astrophysics Data System (ADS)

Lamhamdi, M.; Cayrel, F.; Frayssinet, E.; Bazin, A. E.; Yvon, A.; Collard, E.; Cordier, Y.; Alquier, D.

2016-04-01

Two-dimensional imaging of dopant profiles for n and p-type regions are relevant for the development of new power semiconductors, especially for gallium nitride (GaN) for which classical profiling techniques are not adapted. This is a challenging task since it needs a technique with simultaneously good sensitivity, high spatial resolution and high dopant gradient resolution. To face these challenges, scanning capacitance microscopy combined with Atomic Force Microscopy is a good candidate, presenting reproducible results, as demonstrated in literature. In this work, we attempt to distinguish reliably and qualitatively the various doping concentrations and type at p-n and unipolar junctions. For both p-n and unipolar junctions three kinds of samples were prepared and measured separately. The space-charge region of the p-n metallurgical junction, giving rise to different contrasts under SCM imaging, is clearly observed, enlightening the interest of the SCM technique.

Single-molecule imaging of cytoplasmic dynein in vivo.

PubMed

Ananthanarayanan, Vaishnavi; Tolić, Iva M

2015-01-01

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior. Copyright © 2015 Elsevier Inc. All rights reserved.

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

PubMed

Horne, R W; Wildy, P

1979-09-01

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.

Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

DOE PAGES

Wang, Xueju; Pan, Zhipeng; Fan, Feifei; ...

2015-09-10

We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for themore » analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.« less

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

A review of demodulation techniques for amplitude-modulation atomic force microscopy

PubMed Central

Harcombe, David M; Ragazzon, Michael R P; Moheimani, S O Reza; Fleming, Andrew J

2017-01-01

In this review paper, traditional and novel demodulation methods applicable to amplitude-modulation atomic force microscopy are implemented on a widely used digital processing system. As a crucial bandwidth-limiting component in the z-axis feedback loop of an atomic force microscope, the purpose of the demodulator is to obtain estimates of amplitude and phase of the cantilever deflection signal in the presence of sensor noise or additional distinct frequency components. Specifically for modern multifrequency techniques, where higher harmonic and/or higher eigenmode contributions are present in the oscillation signal, the fidelity of the estimates obtained from some demodulation techniques is not guaranteed. To enable a rigorous comparison, the performance metrics tracking bandwidth, implementation complexity and sensitivity to other frequency components are experimentally evaluated for each method. Finally, the significance of an adequate demodulator bandwidth is highlighted during high-speed tapping-mode atomic force microscopy experiments in constant-height mode. PMID:28900596

Optical Spectroscopy for Noninvasive Monitoring of Stem Cell Differentiation

PubMed Central

Downes, Andrew; Mouras, Rabah; Elfick, Alistair

2010-01-01

There is a requirement for a noninvasive technique to monitor stem cell differentiation. Several candidates based on optical spectroscopy are discussed in this review: Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and coherent anti-Stokes Raman scattering (CARS) microscopy. These techniques are briefly described, and the ability of each to distinguish undifferentiated from differentiated cells is discussed. FTIR spectroscopy has demonstrated its ability to distinguish between stem cells and their derivatives. Raman spectroscopy shows a clear reduction in DNA and RNA concentrations during embryonic stem cell differentiation (agreeing with the well-known reduction in the nucleus to cytoplasm ratio) and also shows clear increases in mineral content during differentiation of mesenchymal stem cells. CARS microscopy can map these DNA, RNA, and mineral concentrations at high speed, and Mutliplex CARS spectroscopy/microscopy is highlighted as the technique with most promise for future applications. PMID:20182537

Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

NASA Astrophysics Data System (ADS)

Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C.

2011-03-01

Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

Surface science analysis of GaAs photocathodes following sustained electron beam delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlos Hernandez-Garcia, Fay Hannon, Marcy Stutzman, V. Shutthanandan, Z. Zhu, M. Nandasri, S. V. Kuchibhatla, S. Thevuthasan, W. P. Hess

2012-06-01

Degradation of the photocathode materials employed in photoinjectors represents a challenge for sustained operation of nuclear physics accelerators and high power Free Electron Lasers (FEL). Photocathode quantum efficiency (QE) degradation is due to residual gasses in the electron source vacuum system being ionized and accelerated back to the photocathode. These investigations are a first attempt to characterize the nature of the photocathode degradation, and employ multiple surface and bulk analysis techniques to investigate damage mechanisms including sputtering of the Cs-oxidant surface monolayer, other surface chemistry effects, and ion implantation. Surface and bulk analysis studies were conducted on two GaAs photocathodes,more » which were removed from the JLab FEL DC photoemission gun after delivering electron beam, and two control samples. The analysis techniques include Helium Ion Microscopy (HIM), Rutherford Backscattering Spectrometry (RBS), Atomic Force Microscopy (AFM) and Secondary Ion Mass Spectrometry (SIMS). In addition, two high-polarization strained superlattice GaAs photocathode samples, one removed from the Continuous Electron Beam Accelerator Facility (CEBAF) photoinjector and one unused, were also analyzed using Transmission Electron Microscopy (TEM) and SIMS. It was found that heat cleaning the FEL GaAs wafer introduces surface roughness, which seems to be reduced by prolonged use. The bulk GaAs samples retained a fairly well organized crystalline structure after delivering beam but shows evidence of Cs depletion on the surface. Within the precision of the SIMS and RBS measurements the data showed no indication of hydrogen implantation or lattice damage from ion back bombardment in the bulk GaAs wafers. In contrast, SIMS and TEM measurements of the strained superlattice photocathode show clear crystal damage in the wafer from ion back bombardment.« less

Electron Microscope Studies of Cadmium Mercury Telluride

NASA Astrophysics Data System (ADS)

Lyster, Martin

Available from UMI in association with The British Library. Requires signed TDF. Epitaxial layers of Cd_{x }Hg_{(1-x)}Te grown on various substrates by liquid phase epitaxy and metallo-organic vapour phase epitaxy have been studied using transmission and scanning electron microscopy, in a variety of contrast modes. Wavelength-dispersive X-ray microanalysis has been used to study interfaces in epitaxial specimens, and the results are used to derive diffusion coefficients for a range of values of x in Cd_ {x}Hg_{(1-x)} Te. Extensive use has been made of back-scattered electron contrast in the SEM as a means of compositional mapping, and defect structures are imaged by this technique. The back-scattered electron contrast at interfaces has been studied in detail and is modelled using the Monte Carlo approach. The modelling is combined with calculations and practical measurements of the probe size in the SEM instrument used in the work, to arrive at a quantitative explanation of this contrast. The SEM and scintillator detector used allow a spatial resolution of better than 1000A, but it is shown that improvements in this are possible with present technology. Scanning infra-red microscopy (SIRM) and high -resolution transmission electron microscopy (HREM) have been applied to the study of CdTe. SIRM images reveal information about Te precipitation, including particle size and density. HREM images provide results concerning dislocation structures in CdTe. Selected-area diffraction contrast TEM results are presented which illustrate the microstructure of LPE and MOVPE material; and TEM foil preparation techniques are discussed, including the choice of ion species for milling cross-sectional specimens. In view of the results obtained, suggestions are made for future work in this field.

Multimodal Light Microscopy Approaches to Reveal Structural and Functional Properties of Promyelocytic Leukemia Nuclear Bodies

PubMed Central

Hoischen, Christian; Monajembashi, Shamci; Weisshart, Klaus; Hemmerich, Peter

2018-01-01

The promyelocytic leukemia (pml) gene product PML is a tumor suppressor localized mainly in the nucleus of mammalian cells. In the cell nucleus, PML seeds the formation of macromolecular multiprotein complexes, known as PML nuclear bodies (PML NBs). While PML NBs have been implicated in many cellular functions including cell cycle regulation, survival and apoptosis their role as signaling hubs along major genome maintenance pathways emerged more clearly. However, despite extensive research over the past decades, the precise biochemical function of PML in these pathways is still elusive. It remains a big challenge to unify all the different previously suggested cellular functions of PML NBs into one mechanistic model. With the advent of genetically encoded fluorescent proteins it became possible to trace protein function in living specimens. In parallel, a variety of fluorescence fluctuation microscopy (FFM) approaches have been developed which allow precise determination of the biophysical and interaction properties of cellular factors at the single molecule level in living cells. In this report, we summarize the current knowledge on PML nuclear bodies and describe several fluorescence imaging, manipulation, FFM, and super-resolution techniques suitable to analyze PML body assembly and function. These include fluorescence redistribution after photobleaching, fluorescence resonance energy transfer, fluorescence correlation spectroscopy, raster image correlation spectroscopy, ultraviolet laser microbeam-induced DNA damage, erythrocyte-mediated force application, and super-resolution microscopy approaches. Since most if not all of the microscopic equipment to perform these techniques may be available in an institutional or nearby facility, we hope to encourage more researches to exploit sophisticated imaging tools for their research in cancer biology. PMID:29888200

Hyperspectral imaging with laser-scanning sum-frequency generation microscopy

PubMed Central

Hanninen, Adam; Shu, Ming Wai; Potma, Eric O.

2017-01-01

Vibrationally sensitive sum-frequency generation (SFG) microscopy is a chemically selective imaging technique sensitive to non-centrosymmetric molecular arrangements in biological samples. The routine use of SFG microscopy has been hampered by the difficulty of integrating the required mid-infrared excitation light into a conventional, laser-scanning nonlinear optical (NLO) microscope. In this work, we describe minor modifications to a regular laser-scanning microscope to accommodate SFG microscopy as an imaging modality. We achieve vibrationally sensitive SFG imaging of biological samples with sub-μm resolution at image acquisition rates of 1 frame/s, almost two orders of magnitude faster than attained with previous point-scanning SFG microscopes. Using the fast scanning capability, we demonstrate hyperspectral SFG imaging in the CH-stretching vibrational range and point out its use in the study of molecular orientation and arrangement in biologically relevant samples. We also show multimodal imaging by combining SFG microscopy with second-harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) on the same imaging platfrom. This development underlines that SFG microscopy is a unique modality with a spatial resolution and image acquisition time comparable to that of other NLO imaging techniques, making point-scanning SFG microscopy a valuable member of the NLO imaging family. PMID:28966861

Two-photon excitation fluorescence bioassays.

PubMed

Hänninen, Pekka; Soukka, Jori; Soini, Juhani T

2008-01-01

Application of two-photon excitation of fluorescence in microscopy is one of the major discoveries of the "renaissance" of light microscopy that started in the 1980s. The technique derives its advantages from the biologically "smooth" wavelength of the excitation light and the confinement of the excitation. Difficult, and seemingly nontransparent, samples may be imaged with the technique with good resolution. Although the bioresearch has been concentrating mostly on the positive properties of the technique for imaging, the same properties may be applied successfully to nonimaging bioassays. This article focuses on the development path of two-photon excitation-based assay system.

Hybrid-coded 3D structured illumination imaging with Bayesian estimation (Conference Presentation)

NASA Astrophysics Data System (ADS)

Chen, Hsi-Hsun; Luo, Yuan; Singh, Vijay R.

2016-03-01

Light induced fluorescent microscopy has long been developed to observe and understand the object at microscale, such as cellular sample. However, the transfer function of lense-based imaging system limits the resolution so that the fine and detailed structure of sample cannot be identified clearly. The techniques of resolution enhancement are fascinated to break the limit of resolution for objective given. In the past decades, the resolution enhancement imaging has been investigated through variety of strategies, including photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), stimulated emission depletion (STED), and structure illuminated microscopy (SIM). In those methods, only SIM can intrinsically improve the resolution limit for a system without taking the structure properties of object into account. In this paper, we develop a SIM associated with Bayesian estimation, furthermore, with optical sectioning capability rendered from HiLo processing, resulting the high resolution through 3D volume. This 3D SIM can provide the optical sectioning and resolution enhancement performance, and be robust to noise owing to the Data driven Bayesian estimation reconstruction proposed. For validating the 3D SIM, we show our simulation result of algorithm, and the experimental result demonstrating the 3D resolution enhancement.

Tumor tissue characterization using polarization-sensitive second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Tokarz, Danielle; Cisek, Richard; Golaraei, Ahmad; Krouglov, Serguei; Navab, Roya; Niu, Carolyn; Sakashita, Shingo; Yasufuku, Kazuhiro; Tsao, Ming-Sound; Asa, Sylvia L.; Barzda, Virginijus; Wilson, Brian C.

2015-06-01

Changes in the ultrastructure of collagen in various tumor and non-tumor human tissues including lung, pancreas and thyroid were investigated ex vivo by a polarization-sensitive second harmonic generation (SHG) microscopy technique referred to as polarization-in, polarization-out (PIPO) SHG. This involves measuring the orientation of the linear polarization of outgoing SHG as a function of the linear polarization orientation of incident laser radiation. From the PIPO SHG data, the second-order nonlinear optical susceptibility tensor component ratio, χ(2) ZZZ'/χ(2) ZXX', for each pixel of the SHG image was obtained and presented as color-coded maps. Further, the orientation of collagen fibers in the tissue was deduced. Since the χ(2) ZZZ'/χ(2) ZXX' values represent the organization of collagen in the tissue, theses maps revealed areas of altered collagen structure (not simply concentration) within tissue sections. Statistically-significant differences in χ(2) ZZZ'/χ(2) ZXX' were found between tumor and non-tumor tissues, which varied from organ to organ. Hence, PIPO SHG microscopy could potentially be used to aid pathologists in diagnosing cancer. Additionally, PIPO SHG microscopy could aid in characterizing the structure of collagen in other collagen-related biological processes such as wound repair.

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Koshonna; Thurn, Ted; Xin, Lun

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Intracellular in situ labeling of TiO 2 nanoparticles for fluorescence microscopy detection

DOE PAGES

Brown, Koshonna; Thurn, Ted; Xin, Lun; ...

2017-07-19

Titanium dioxide (TiO 2) nanoparticles are produced for many different purposes, including development of therapeutic and diagnostic nanoparticles for cancer detection and treatment, drug delivery, induction of DNA double-strand breaks, and imaging of specific cells and subcellular structures. Currently, the use of optical microscopy, an imaging technique most accessible to biology and medical pathology, to detect TiO 2 nanoparticles in cells and tissues ex vivo is limited with low detection limits, while more sensitive imaging methods (transmission electron microscopy, X-ray fluorescence microscopy, etc.) have low throughput and technical and operational complications. In this paper, we describe two in situ posttreatmentmore » labeling approaches to stain TiO 2 nanoparticles taken up by the cells. The first approach utilizes fluorescent biotin and fluorescent streptavidin to label the nanoparticles before and after cellular uptake; the second approach is based on the copper-catalyzed azide-alkyne cycloaddition, the so-called Click chemistry, for labeling and detection of azide-conjugated TiO 2 nanoparticles with alkyneconjugated fluorescent dyes such as Alexa Fluor 488. To confirm that optical fluorescence signals of these nanoparticles match the distribution of the Ti element, we used synchrotron X-ray fluorescence microscopy (XFM) at the Advanced Photon Source at Argonne National Laboratory. Titanium-specific XFM showed excellent overlap with the location of optical fluorescence detected by confocal microscopy. Finally and therefore, future experiments with TiO 2 nanoparticles may safely rely on confocal microscopy after in situ nanoparticle labeling using approaches described here.« less

Measuring surface-area-to-volume ratios in soft porous materials using laser-polarized xenon interphase exchange nuclear magnetic resonance

NASA Technical Reports Server (NTRS)

Butler, J. P.; Mair, R. W.; Hoffmann, D.; Hrovat, M. I.; Rogers, R. A.; Topulos, G. P.; Walsworth, R. L.; Patz, S.

2002-01-01

We demonstrate a minimally invasive nuclear magnetic resonance (NMR) technique that enables determination of the surface-area-to-volume ratio (S/V) of soft porous materials from measurements of the diffusive exchange of laser-polarized 129Xe between gas in the pore space and 129Xe dissolved in the solid phase. We apply this NMR technique to porous polymer samples and find approximate agreement with destructive stereological measurements of S/V obtained with optical confocal microscopy. Potential applications of laser-polarized xenon interphase exchange NMR include measurements of in vivo lung function in humans and characterization of gas chromatography columns.

Visualizing and quantifying the in vivo structure and dynamics of the Arabidopsis cortical cytoskeleton using CLSM and VAEM.

PubMed

Rosero, Amparo; Zárský, Viktor; Cvrčková, Fatima

2014-01-01

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

Determination of the Subcellular Distribution of Liposomes Using Confocal Microscopy.

PubMed

Solomon, Melani A

2017-01-01

It is being increasingly recognized that therapeutics need to be delivered to specific organelle targets within cells. Liposomes are versatile lipid-based drug delivery vehicles that can be surface-modified to deliver the loaded cargo to specific subcellular locations within the cell. Hence, the development of such technology requires a means of measuring the subcellular distribution possibly by utilizing imaging techniques that can visualize and quantitate the extent of this subcellular localization. The apparent increase of resolution along the Z-axis offered by confocal microscopy makes this technique suitable for such studies. In this chapter, we describe the application of confocal laser scanning microscopy (CLSM) to determine the subcellular distribution of fluorescently labeled mitochondriotropic liposomes.

Material properties of viral nanocages explored by atomic force microscopy.

PubMed

van Rosmalen, Mariska G M; Roos, Wouter H; Wuite, Gijs J L

2015-01-01

Single-particle nanoindentation by atomic force microscopy (AFM) is an emergent technique to characterize the material properties of nano-sized proteinaceous systems. AFM uses a very small tip attached to a cantilever to scan the surface of the substrate. As a result of the sensitive feedback loop of AFM, the force applied by the tip on the substrate during scanning can be controlled and monitored. By accurately controlling this scanning force, topographical maps of fragile substrates can be acquired to study the morphology of the substrate. In addition, mechanical properties of the substrate like stiffness and breaking point can be determined by using the force spectroscopy capability of AFM. Here we discuss basics of AFM operation and how this technique is used to determine the structure and mechanical properties of protein nanocages, in particular viral particles. Knowledge of morphology as well as mechanical properties is essential for understanding viral life cycles, including genome packaging, capsid maturation, and uncoating, but also contributes to the development of diagnostics, vaccines, imaging modalities, and targeted therapeutic devices based on viruslike particles.

Sublimation-assisted graphene transfer technique based on small polyaromatic hydrocarbons

NASA Astrophysics Data System (ADS)

Chen, Mingguang; Stekovic, Dejan; Li, Wangxiang; Arkook, Bassim; Haddon, Robert C.; Bekyarova, Elena

2017-06-01

Advances in the chemical vapor deposition (CVD) growth of graphene have made this material a very attractive candidate for a number of applications including transparent conductors, electronics, optoeletronics, biomedical devices and energy storage. The CVD method requires transfer of graphene on a desired substrate and this is most commonly accomplished with polymers. The removal of polymer carriers is achieved with organic solvents or thermal treatment which makes this approach inappropriate for application to plastic thin films such as polyethylene terephthalate substrates. An ultraclean graphene transfer method under mild conditions is highly desired. In this article, we report a naphthalene-assisted graphene transfer technique which provides a reliable route to residue-free transfer of graphene to both hard and flexible substrates. The quality of the transferred graphene was characterized with atomic force microscopy, scanning electron microscopy, and Raman spectroscopy. Field effect transistors, based on the naphthalene-transfered graphene, were fabricated and characterized. This work has the potential to broaden the applications of CVD graphene in fields where ultraclean graphene and mild graphene transfer conditions are required.

Minerals and aligned collagen fibrils in tilapia fish scales: structural analysis using dark-field and energy-filtered transmission electron microscopy and electron tomography.

PubMed

Okuda, Mitsuhiro; Ogawa, Nobuhiro; Takeguchi, Masaki; Hashimoto, Ayako; Tagaya, Motohiro; Chen, Song; Hanagata, Nobutaka; Ikoma, Toshiyuki

2011-10-01

The mineralized structure of aligned collagen fibrils in a tilapia fish scale was investigated using transmission electron microscopy (TEM) techniques after a thin sample was prepared using aqueous techniques. Electron diffraction and electron energy loss spectroscopy data indicated that a mineralized internal layer consisting of aligned collagen fibrils contains hydroxyapatite crystals. Bright-field imaging, dark-field imaging, and energy-filtered TEM showed that the hydroxyapatite was mainly distributed in the hole zones of the aligned collagen fibrils structure, while needle-like materials composed of calcium compounds including hydroxyapatite existed in the mineralized internal layer. Dark-field imaging and three-dimensional observation using electron tomography revealed that hydroxyapatite and needle-like materials were mainly found in the matrix between the collagen fibrils. It was observed that hydroxyapatite and needle-like materials were preferentially distributed on the surface of the hole zones in the aligned collagen fibrils structure and in the matrix between the collagen fibrils in the mineralized internal layer of the scale.

Structure and Dynamics of Domains in Ferroelectric Nanostructures. In-situ TEM Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Xiaoqing

2015-06-30

The goal of this project was to explore the structure and dynamic behaviors of ferroelectric domains in ferroelectric thin films and nanostructures by advanced transmission electron microscopy (TEM) techniques in close collaboration with phase field modeling. The experimental techniques used include aberration-corrected sub-Å resolution TEM and in-situ TEM using a novel scanning tunneling microscopy (STM) - TEM holder that allows the direct observation of nucleation and dynamic evolution of ferroelectric domains under applied electric field. Specifically, this project was aimed to (1) to study the roles of static electrical boundary conditions and electrical charge in controlling the equilibrium domain structuresmore » of BiFeO 3 thin films with controlled substrate constraints, (2) to explore the fundamental mechanisms of ferroelectric domain nucleation, growth, and switching under an applied electric field in both uniform thin films and nanostructures, and to understand the roles of crystal defects such as dislocations and interfaces in these processes, (3) to understand the physics of ferroelectric domain walls and the influence of defects on the electrical switching of ferroelectric domains.« less

Visualizing molecular polar order in tissues via electromechanical coupling

PubMed Central

Denning, Denise; Alilat, Sofiane; Habelitz, Stefan; Fertala, Andrzej; Rodriguez, Brian J.

2015-01-01

Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides. PMID:22985991

DURIP: Super-Resolution Module for Confocal Microscopy of Reconfigurable Matter

DTIC Science & Technology

2014-09-28

Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 superresolution microscopy, colloidal particles, self-assembly REPORT...previously have been resolved by optical microscopy. Results of Super Resolution Technique Evaluation Commercially available superresolution imaging...Weaknesses of the method are that is fundamentally a measurement that can only be deployed for fixed samples. Because superresolution is obtained by

Nobel Prize Recipient Eric Betzig Presents Lecture on Efforts to Improve High-Resolution Microscopy | Poster

Cancer.gov

Eric Betzig, Ph.D., a 2014 recipient of the Nobel Prize in Chemistry and a scientist at Janelia Research Campus (JRC), Howard Hughes Medical Institute, in Ashburn, Va., visited NCI at Frederick on Sept. 10 to present a Distinguished Scientist lecture and discuss the latest high-resolution microscopy techniques. Betzig co-invented photoactivation localization microscopy (PALM)

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

PubMed

Kaji, Yuichi; Akiyama, Toshihiro; Segawa, Hiroki; Oshika, Tetsuro; Kano, Hideaki

2017-11-01

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells

PubMed Central

Gonzalez Carter, Daniel A.; Motskin, Michael; Pienaar, Ilse S.; Chen, Shu; Hu, Sheng; Ruenraroengsak, Pakatip; Ryan, Mary P.; Shaffer, Milo S. P.; Dexter, David T.

2016-01-01

Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNT, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 hrs exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 hours. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic, quantitative and multi-scale techniques in understanding the different cellular processing routes of functionalised nanomaterials. This correlative approach has wide implications for assessing the biopersistence of MWNT aggregates elsewhere in the body, in particular their interaction with macrophages in the lung. PMID:26298523

Dark-field X-ray microscopy for multiscale structural characterization

NASA Astrophysics Data System (ADS)

Simons, H.; King, A.; Ludwig, W.; Detlefs, C.; Pantleon, W.; Schmidt, S.; Snigireva, I.; Snigirev, A.; Poulsen, H. F.

2015-01-01

Many physical and mechanical properties of crystalline materials depend strongly on their internal structure, which is typically organized into grains and domains on several length scales. Here we present dark-field X-ray microscopy; a non-destructive microscopy technique for the three-dimensional mapping of orientations and stresses on lengths scales from 100 nm to 1 mm within embedded sampling volumes. The technique, which allows ‘zooming’ in and out in both direct and angular space, is demonstrated by an annealing study of plastically deformed aluminium. Facilitating the direct study of the interactions between crystalline elements is a key step towards the formulation and validation of multiscale models that account for the entire heterogeneity of a material. Furthermore, dark-field X-ray microscopy is well suited to applied topics, where the structural evolution of internal nanoscale elements (for example, positioned at interfaces) is crucial to the performance and lifetime of macro-scale devices and components thereof.

Magnetic resonance microscopy: concepts, challenges, and state-of-the-art.

PubMed

Gimi, Barjor

2006-01-01

Recent strides in targeted therapy and regenerative medicine have created a need to identify molecules and metabolic pathways implicated in a disease and its treatment. These molecules and pathways must be discerned at the cellular level to meaningfully reveal the biochemical underpinnings of the disease and to identify key molecular targets for therapy. Magnetic resonance (MR) techniques are well suited for molecular and functional imaging because of their noninvasive nature and their versatility in extracting physiological, biochemical, and functional information over time. However, MR is an insensitive technique; MR microscopy seeks to increase detection sensitivity, thereby localizing biochemical and functional information at the level of single cells or small cellular clusters. Here, we discuss some of the challenges facing MR microscopy and the technical and phenomenological strategies used to overcome these challenges. Some of the applications of MR microscopy are highlighted in this chapter.

Acousto-optical tunable filter for combined wideband, spectral, and optical coherence microscopy.

PubMed

Machikhin, Alexander S; Pozhar, Vitold E; Viskovatykh, Alexander V; Burmak, Ludmila I

2015-09-01

A multimodal technique for inspection of microscopic objects by means of wideband optical microscopy, spectral microscopy, and optical coherence microscopy is described, implemented, and tested. The key feature is the spectral selection of light in the output arm of an interferometer with use of the specialized imaging acousto-optical tunable filter. In this filter, two interfering optical beams are diffracted via the same ultrasound wave without destruction of interference image structure. The basic requirements for the acousto-optical tunable filter are defined, and mathematical formulas for calculation of its parameters are derived. Theoretical estimation of the achievable accuracy of the 3D image reconstruction is presented and experimental proofs are given. It is demonstrated that spectral imaging can also be accompanied by measurement of the quantitative reflectance spectra. Examples of inspection of optically transparent and nontransparent samples demonstrate the applicability of the technique.

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Numerical tilting compensation in microscopy based on wavefront sensing using transport of intensity equation method

NASA Astrophysics Data System (ADS)

Hu, Junbao; Meng, Xin; Wei, Qi; Kong, Yan; Jiang, Zhilong; Xue, Liang; Liu, Fei; Liu, Cheng; Wang, Shouyu

2018-03-01

Wide-field microscopy is commonly used for sample observations in biological research and medical diagnosis. However, the tilting error induced by the oblique location of the image recorder or the sample, as well as the inclination of the optical path often deteriorates the imaging quality. In order to eliminate the tilting in microscopy, a numerical tilting compensation technique based on wavefront sensing using transport of intensity equation method is proposed in this paper. Both the provided numerical simulations and practical experiments prove that the proposed technique not only accurately determines the tilting angle with simple setup and procedures, but also compensates the tilting error for imaging quality improvement even in the large tilting cases. Considering its simple systems and operations, as well as image quality improvement capability, it is believed the proposed method can be applied for tilting compensation in the optical microscopy.

Wavefront correction using machine learning methods for single molecule localization microscopy

NASA Astrophysics Data System (ADS)

Tehrani, Kayvan F.; Xu, Jianquan; Kner, Peter

2015-03-01

Optical Aberrations are a major challenge in imaging biological samples. In particular, in single molecule localization (SML) microscopy techniques (STORM, PALM, etc.) a high Strehl ratio point spread function (PSF) is necessary to achieve sub-diffraction resolution. Distortions in the PSF shape directly reduce the resolution of SML microscopy. The system aberrations caused by the imperfections in the optics and instruments can be compensated using Adaptive Optics (AO) techniques prior to imaging. However, aberrations caused by the biological sample, both static and dynamic, have to be dealt with in real time. A challenge for wavefront correction in SML microscopy is a robust optimization approach in the presence of noise because of the naturally high fluctuations in photon emission from single molecules. Here we demonstrate particle swarm optimization for real time correction of the wavefront using an intensity independent metric. We show that the particle swarm algorithm converges faster than the genetic algorithm for bright fluorophores.

Fabrication and optical characterization of imaging fiber-based nanoarrays.

PubMed

Tam, Jenny M; Song, Linan; Walt, David R

2005-09-15

In this paper, we present a technique for fabricating arrays containing a density at least 90 times higher than previously published. Specifically, we discuss the fabrication of two imaging fiber-based nanoarrays, one with 700nm features, another with 300nm features. With arrays containing up to 4.5x10(6) array elements/mm(2), these nanoarrays have an ultra-high packing density. A straightforward etching protocol is used to create nanowells into which beads can be deposited. These beads comprise the sensing elements of the nanoarray. Deposition of the nanobeads into the nanowells using two techniques is described. The surface characteristics of the etched arrays are examined with atomic force microscopy and scanning electron microscopy. Fluorescence microscopy was used to observe the arrays. The 300nm array features and the 500nm center-to-center distance approach the minimum feature sizes viewable using conventional light microscopy.

Scanning electron microscopy of cells and tissues under fully hydrated conditions

PubMed Central

Thiberge, Stephan; Nechushtan, Amotz; Sprinzak, David; Gileadi, Opher; Behar, Vered; Zik, Ory; Chowers, Yehuda; Michaeli, Shulamit; Schlessinger, Joseph; Moses, Elisha

2004-01-01

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers. PMID:14988502

Comparing high-resolution microscopy techniques for potential intraoperative use in guiding low-grade glioma resections.

PubMed

Meza, Daphne; Wang, Danni; Wang, Yu; Borwege, Sabine; Sanai, Nader; Liu, Jonathan T C

2015-04-01

Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy. Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality). HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast. Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning. © 2015 Wiley Periodicals, Inc.

Microstructural Characterization of Irradiated U0.7ZrH1.6 Using Ultrasonic Techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramuhalli, Pradeep; Jacob, Richard E.; MacFarlan, Paul J.

In recent years, there has been an increased level of effort to understand the changes in microstructure that occur due to irradiation of nuclear fuel. The primary driver for this increased effort is the potential for designing new fuels that are safer and more reliable, in turn enabling new and improved reactor technologies. Much of the data on microstructural change in irradiated fuels is generated through a host of post irradiation examination techniques such as optical microscopy (OM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) to determine grain structure, porosity, crack geometry, etc. in irradiated fuels. Such “traditional”more » examination techniques were recently used to characterize a novel new fuel consisting of U0.17ZrH1.6 pellets bonded to zircaloy-2 cladded with lead-bismuth eutectic before and after irradiation. However, alternative methods such as ultrasonic inspection can provide an opportunity for nondestructively assessing microstructure in both in-pile and post-irradiation examinations. In this paper, we briefly describe initial results of ultrasonic examination of the U0.17ZrH1.6 pellets (unirradiated and irradiated), in a post-irradiation examination study. Data indicate some correlation with microstructural changes due to irradiation; however, it is not clear what the specific microstructural changes are that are influencing the ultrasonic measurements. Interestingly, specimens with nominally identical burnup show differences in ultrasonic signatures, indicating apparent microstructural differences between these specimens. A summary of the experimental study, preliminary data and findings are presented in this short paper. Additional details of the analysis will be included in the presentation.« less

Recent Advances in Fiber Lasers for Nonlinear Microscopy

PubMed Central

Xu, C.; Wise, F. W.

2013-01-01

Nonlinear microscopy techniques developed over the past two decades have provided dramatic new capabilities for biological imaging. The initial demonstrations of nonlinear microscopies coincided with the development of solid-state femtosecond lasers, which continue to dominate applications of nonlinear microscopy. Fiber lasers offer attractive features for biological and biomedical imaging, and recent advances are leading to high-performance sources with the potential for robust, inexpensive, integrated instruments. This article discusses recent advances, and identifies challenges and opportunities for fiber lasers in nonlinear bioimaging. PMID:24416074

Super-resolution optical microscopy for studying membrane structure and dynamics.

PubMed

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Introduction to Modern Methods in Light Microscopy.

PubMed

Ryan, Joel; Gerhold, Abby R; Boudreau, Vincent; Smith, Lydia; Maddox, Paul S

2017-01-01

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.

Sequential Super-Resolution Imaging of Bacterial Regulatory Proteins: The Nucleoid and the Cell Membrane in Single, Fixed E. coli Cells.

PubMed

Spahn, Christoph; Glaesmann, Mathilda; Gao, Yunfeng; Foo, Yong Hwee; Lampe, Marko; Kenney, Linda J; Heilemann, Mike

2017-01-01

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

Bessel light sheet structured illumination microscopy

NASA Astrophysics Data System (ADS)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in confocal quality images in thick tissue. The technique was applied to live transgenic zebra fish tg(kdrl:GFP), and the sub-cellular structure of fish vasculature genetically labeled with GFP was captured in 3D. The superior speed of the microscope enables us to acquire signal from 200 layers of a thick sample in 4 minutes. The compact microscope uses exclusively off-the-shelf components and offers a low-cost imaging solution for studying small animal models or tissue samples.

Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions

NASA Astrophysics Data System (ADS)

Hirsch, Michelle S.; Svoboda, Kathy K. H.

1994-04-01

Hyaline cartilage is composed of chondrocytes that reside in lacunae surrounded by extracellular matrix molecules. Microscopic and histochemical features of cartilage have been studied with many techniques. Many of these techniques can be time consuming and may alter natural cartilage characteristics. In addition, the orientation and order of sectioned tissue must be maintained to create 3D reconstructions. We show that confocal laser scanning microscopy may replace traditional methods for studying cartilage.