Analysis of off-axis incoherent digital holographic microscopy

NASA Astrophysics Data System (ADS)

Quan, Xiangyu; Matoba, Osamu; Awatsuji, Yasuhiro

2017-05-01

Off-axis incoherent digital holography that enables single-shot three-dimensional (3D) distribution is introduced in the paper. Conventional fluorescence microscopy images 3D fields by sectioning, this prevents instant imaging of fast reactions of living cells. In order to realize digital holography from incoherent light, we adapted common path configuration to achieve the best temporal coherence. And by introducing gratings, we shifted the direction of each light to achieve off-axis interference. Simulations and preliminary experiments using LED light have confirmed the results. We expect to use this method to realize 3D phase imaging and fluorescent imaging at the same time from the same biological sample.

Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

NASA Astrophysics Data System (ADS)

Candeo, Alessia; Sana, Ilenia; Ferrari, Eleonora; Maiuri, Luigi; D'Andrea, Cosimo; Valentini, Gianluca; Bassi, Andrea

2016-05-01

Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.

Light microscopy morphological characteristics of the sperm flagellum may be related to axonemal abnormalities.

PubMed

Mitchell, V; Sigala, J; Ballot, C; Jumeau, F; Barbotin, A L; Duhamel, A; Rives, N; Rigot, J M; Escalier, D; Peers, M C

2015-03-01

Although electron microscopy provides a detailed analysis of ultrastructural abnormalities, this technique is not available in all laboratories. We sought to determine whether certain characteristics of the flagellum as assessed by light microscopy were related to axonemal abnormalities. Forty-one patients with an absence of outer dynein arms (type I), a lack of a central complex (type III) and an absence of peripheral doublets (type IV) were studied. Sperm morphology was scored according to David's modified classification. Flagella with an irregular thickness were classified as being of normal length, short or broken. There were correlations between missing outer dynein arms and abnormal, short or coiled flagellum. Type III patients showed the highest flagellar defects (a short (P = 0.0027) or an absent flagellum (P = 0.011)). Just over 68% of the irregular flagella were short in Type III patients, whereas this value was only 34.5% in type I and 26.4% in type IV (P = 0.002). There was a negative correlation between misassembly and spermatozoa of irregular flagella (r = -0.79; P = 0.019). It is concluded that light microscopy analysis of flagellum abnormalities may help provide a correct diagnosis, identify sperm abnormalities with fertility potentials and outcomes in assisted reproduction technologies and assess the genetic risk. © 2014 Blackwell Verlag GmbH.

Histological Analysis of the Arabidopsis Gynoecium and Ovules Using Chloral Hydrate Clearing and Differential Interference Contrast Light Microscopy.

PubMed

Franks, Robert G

2016-01-01

The use of chloral hydrate optical clearing paired with differential interference contrast microscopy allows the analysis of internal structures of developing plant organs without the need for paraffin embedding and sectioning. This approach is appropriate for the analysis of the developing gynoecium or seedpod of the flowering plant Arabidopsis thaliana and many other types of fixed plant material. Early stages of ovule development are observable with this approach.

Detection of local chemical states of lithium and their spatial mapping by scanning transmission electron microscopy, electron energy-loss spectroscopy and hyperspectral image analysis.

PubMed

Muto, Shunsuke; Tatsumi, Kazuyoshi

2017-02-08

Advancements in the field of renewable energy resources have led to a growing demand for the analysis of light elements at the nanometer scale. Detection of lithium is one of the key issues to be resolved for providing guiding principles for the synthesis of cathode active materials, and degradation analysis after repeated use of those materials. We have reviewed the different techniques currently used for the characterization of light elements such as high-resolution transmission electron microscopy, scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS). In the present study, we have introduced a methodology to detect lithium in solid materials, particularly for cathode active materials used in lithium-ion battery. The chemical states of lithium were isolated and analyzed from the overlapping multiple spectral profiles, using a suite of STEM, EELS and hyperspectral image analysis. The method was successfully applied in the chemical state analyses of hetero-phases near the surface and grain boundary regions of the active material particles formed by chemical reactions between the electrolyte and the active materials. © The Author 2016. 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.

The Pathologist 2.0: An Update on Digital Pathology in Veterinary Medicine.

PubMed

Bertram, Christof A; Klopfleisch, Robert

2017-09-01

Using light microscopy to describe the microarchitecture of normal and diseased tissues has changed very little since the middle of the 19th century. While the premise of histologic analysis remains intact, our relationship with the microscope is changing dramatically. Digital pathology offers new forms of visualization, and delivery of images is facilitated in unprecedented ways. This new technology can untether us entirely from our light microscopes, with many pathologists already performing their jobs using virtual microscopy. Several veterinary colleges have integrated virtual microscopy in their curriculum, and some diagnostic histopathology labs are switching to virtual microscopy as their main tool for the assessment of histologic specimens. Considering recent technical advancements of slide scanner and viewing software, digital pathology should now be considered a serious alternative to traditional light microscopy. This review therefore intends to give an overview of the current digital pathology technologies and their potential in all fields of veterinary pathology (ie, research, diagnostic service, and education). A future integration of digital pathology in the veterinary pathologist's workflow seems to be inevitable, and therefore it is proposed that trainees should be taught in digital pathology to keep up with the unavoidable digitization of the profession.

Analysis of Cutmarks on Bone: Can Light Microscopy Be of Any Help?

PubMed

Cerutti, Elisa; Spagnoli, Laura; Araujo, Nadezhda; Gibelli, Daniele; Mazzarelli, Debora; Cattaneo, Cristina

2016-12-01

One of the main issues in forensic anthropology consists of the identification of signs of trauma in skeletal remains, including sharp-force injuries. So far, several studies have been performed to assess differences between injuries caused by different instruments, not, however, through light microscopy.In this study, 152 sharp-force injuries were performed by 5 different tools through 2 different orientations on 2 humeral diaphyses and were analyzed by stereo and light microscopy to assess possible morphological differences.This study showed that although W-shaped injuries are frequently reported in cases of wood-cutting saws, other shapes are often observed; lesions due to metal-cutting saws are almost always U shaped, whereas injuries caused by knives are V shaped. Although cut marks may represent a variable range of features, the present study was able to highlight typical profiles that may be of some help for the diagnosis of weapon and the intentionality of the action.

Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes.

PubMed

Shen, Ying; Betzendahl, Ilse; Tinneberg, Hans-Rudolf; Eichenlaub-Ritter, Ursula

2008-03-12

Chromosomal non-disjunction in female meiosis gives rise to reduced fertility and trisomy in humans. Human oocytes, especially from aged women, appear especially susceptible to non-disjunction. The oocyte spindle is crucial for high fidelity of chromosome segregation at meiotic divisions, and alterations in spindle morphology are therefore indicators of adverse conditions during oocyte development that may result in meiotic aneuploidy. In the past, oocytes had to be fixed for spindle analysis, precluding direct non-invasive identification of aneugens and adverse maturation conditions that affect spindle integrity and chromosome behaviour. Aneuploidy research for detection of spindle aberrations was therefore mainly focused on in vivo or in vitro exposed, fixed animal oocytes or cytogenetic analysis of spread oocytes. Orientation independent enhanced polarizing microscopy with nearly circularly polarized light and electronically controlled liquid crystal compensator optics is a new tool to study spindle morphology non-invasively in vivo for qualitative as well as quantitative analysis. Image generation by polarization microscopy depends on the intrinsic optical properties of the spindle with its paracrystalline microtubule lattice. When polarized light passes through such a lattice it induces a splitting of the beam and shift in the plane of vibration and retardation of light (termed birefringence and retardance). Studies of animal oocytes and follicle-cell denuded human oocytes fertilized by intracytoplasmic sperm injection for assisted conception have demonstrated the safety and efficacy of enhanced polarization microscopy. The method can be employed in aneuploidy research for non-invasive dose-response studies to detect spindle aberrations, for instance, in combination with cytogenetic analysis. Due to the non-invasive nature of the technique it may be employed in routine analysis of human oocytes to assess risks by lifestyle factors, and occupational and adverse environmental exposures.

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

Photoassisted Kelvin probe force microscopy at GaN surfaces: The role of polarity

NASA Astrophysics Data System (ADS)

Wei, J. D.; Li, S. F.; Atamuratov, A.; Wehmann, H.-H.; Waag, A.

2010-10-01

The behavior of GaN surfaces during photoassisted Kelvin probe force microscopy is demonstrated to be strongly dependant on surface polarity. The surface photovoltage of GaN surfaces illuminated with above-band gap light is analyzed as a function of time and light intensity. Distinct differences between Ga-polar and N-polar surfaces could be identified, attributed to photoinduced chemisorption of oxygen during illumination. These differences can be used for a contactless, nondestructive, and easy-performable analysis of the polarity of GaN surfaces.

Comparative study of three Plumbago L. species (Plumbaginaceae) by microscopy, UPLC–UV and HPTLC analyses

USDA-ARS?s Scientific Manuscript database

This paper presents a comparative study of anatomy of leaves, stems and roots of three species of Plumbago, namely P. auriculata Lam., P. indica L. and P. zeylanica L. by light microscopy. The paper also provides qualitative and quantitative analysis of the naphthoquinone, plumbagin, a major constit...

Preparation, characterization and photocatalytic behavior of WO3-fullerene/TiO2 catalysts under visible light

PubMed Central

2011-01-01

WO3-treated fullerene/TiO2 composites (WO3-fullerene/TiO2) were prepared using a sol-gel method. The composite obtained was characterized by BET surface area measurements, X-ray diffraction, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, and UV-vis analysis. A methyl orange (MO) solution under visible light irradiation was used to determine the photocatalytic activity. Excellent photocatalytic degradation of a MO solution was observed using the WO3-fullerene, fullerene-TiO2, and WO3-fullerene/TiO2 composites under visible light. An increase in photocatalytic activity was observed, and WO3-fullerene/TiO2 has the best photocatalytic activity; it may attribute to the increase of the photo-absorption effect by the fullerene and the cooperative effect of the WO3. PMID:21774800

Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

PubMed

Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

2012-11-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

PubMed Central

Lai, Ken; Wu, Xiaojuan; Yong, Jim L. C.; Lee, C. Soon

2012-01-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images

PubMed Central

Salas, Desirée; Le Gall, Antoine; Fiche, Jean-Bernard; Valeri, Alessandro; Ke, Yonggang; Bron, Patrick; Bellot, Gaetan

2017-01-01

Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions. PMID:28811371

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.

Structured light optical microscopy for three-dimensional reconstruction of technical surfaces

NASA Astrophysics Data System (ADS)

Kettel, Johannes; Reinecke, Holger; Müller, Claas

2016-04-01

In microsystems technology quality control of micro structured surfaces with different surface properties is playing an ever more important role. The process of quality control incorporates three-dimensional (3D) reconstruction of specularand diffusive reflecting technical surfaces. Due to the demand on high measurement accuracy and data acquisition rates, structured light optical microscopy has become a valuable solution to solve this problem providing high vertical and lateral resolution. However, 3D reconstruction of specular reflecting technical surfaces still remains a challenge to optical measurement principles. In this paper we present a measurement principle based on structured light optical microscopy which enables 3D reconstruction of specular- and diffusive reflecting technical surfaces. It is realized using two light paths of a stereo microscope equipped with different magnification levels. The right optical path of the stereo microscope is used to project structured light onto the object surface. The left optical path is used to capture the structured illuminated object surface with a camera. Structured light patterns are generated by a Digital Light Processing (DLP) device in combination with a high power Light Emitting Diode (LED). Structured light patterns are realized as a matrix of discrete light spots to illuminate defined areas on the object surface. The introduced measurement principle is based on multiple and parallel processed point measurements. Analysis of the measured Point Spread Function (PSF) by pattern recognition and model fitting algorithms enables the precise calculation of 3D coordinates. Using exemplary technical surfaces we demonstrate the successful application of our measurement principle.

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

Identification of a Multicomponent Traditional Herbal Medicine by HPLC-MS and Electron and Light Microscopy.

PubMed

Liu, Ju-Han; Cheng, Yung-Yi; Hsieh, Chen-Hsi; Tsai, Tung-Hu

2017-12-15

Commercial pharmaceutical herbal products have enabled people to take traditional Chinese medicine (TCM) in a convenient and accessible form. However, the quantity and quality should be additionally inspected. To address the issue, a combination of chemical and physical inspection methods were developed to evaluate the amount of an herbal formula, Xiang-Sha-Liu-Jun-Zi-Tang (XSLJZT), in clinical TCM practice. A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method with electrospray ionization was developed to measure the herbal biomarkers of guanosine, atractylenolide III, glycyrrhizic acid, dehydrocostus lactone, hesperidin, and oleanolic acid from XSLJZT. Scanning electron microscopy (SEM) photographs and light microscopy photographs with Congo red and iodine-KI staining were used to identify the cellulose fibers and starch content. Furthermore, solubility analysis, swelling power test, and crude fiber analysis were contributed to measure the starch additive in pharmaceutical products. The results demonstrated large variations in the chemical components of different pharmaceutical brands. The SEM photographs revealed that the starch was oval, smooth, and granular, and that the raw herbal powder appears stripy, stretched, and filiform. The stained light microscopy photographs of all of the pharmaceutical products showed added starch and raw herbal powder as extenders. The developed chemical and physical methods provide a standard operating procedure for the quantity control of the herbal pharmaceutical products of XSLJZT.

A facile hydrothermal approach to synthesize rGO/BiVO4 photocatalysts for visible light induced degradation of RhB dye

NASA Astrophysics Data System (ADS)

Pal, Shreyasi; Dutta, Shibsankar; De, Sukanta

2018-05-01

RGO/BiVO4 composites were synthesized by a simple hydrothermal method. The samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) and surface analysis (BET). The photocatalytic activity of the as-prepared samples was evaluated by studying the degradation of model dyes rhodamine B (RhB) under visible light. The prepared rGO/BiVO4 composites exhibited higher photocatalytic activity for the degradation of RhB with a maximum removal rate of 86% under visible light irradiation under visible-light irradiation than pure BiVO4 nanoparticles (63%). This behavior could be associated to their higher specific surface area (BET), increased light absorption intensity and the degradation of electron-hole pair recombination in BiVO4 with the introduction of the rGO.

5D imaging via light sheet microscopy reveals cell dynamics during the eye-antenna disc primordium formation in Drosophila

NASA Astrophysics Data System (ADS)

Huang, Yu Shan; Ku, Hui Yu; Tsai, Yun Chi; Chang, Chin Hao; Pao, Sih Hua; Sun, Y. Henry; Chiou, Arthur

2017-03-01

5D images of engrailed (en) and eye gone (eyg) gene expressions during the course of the eye-antenna disc primordium (EADP) formation of Drosophila embryos from embryonic stages 13 through 16 were recorded via light sheet microscopy and analyzed to reveal the cell dynamics involved in the development of the EADP. Detailed analysis of the time-lapsed images revealed the process of EADP formation and its invagination trajectory, which involved an inversion of the EADP anterior-posterior axis relative to the body. Furthermore, analysis of the en-expression pattern in the EADP provided strong evidence that the EADP is derived from one of the en-expressing head segments.

Restoration of uneven illumination in light sheet microscopy images.

PubMed

Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

2011-08-01

Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

Concepts in Light Microscopy of Viruses

PubMed Central

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Concepts in Light Microscopy of Viruses.

PubMed

Witte, Robert; Andriasyan, Vardan; Georgi, Fanny; Yakimovich, Artur; Greber, Urs F

2018-04-18

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Automated Diatom Analysis Applied to Traditional Light Microscopy: A Proof-of-Concept Study

NASA Astrophysics Data System (ADS)

Little, Z. H. L.; Bishop, I.; Spaulding, S. A.; Nelson, H.; Mahoney, C.

2017-12-01

Diatom identification and enumeration by high resolution light microscopy is required for many areas of research and water quality assessment. Such analyses, however, are both expertise and labor-intensive. These challenges motivate the need for an automated process to efficiently and accurately identify and enumerate diatoms. Improvements in particle analysis software have increased the likelihood that diatom enumeration can be automated. VisualSpreadsheet software provides a possible solution for automated particle analysis of high-resolution light microscope diatom images. We applied the software, independent of its complementary FlowCam hardware, to automated analysis of light microscope images containing diatoms. Through numerous trials, we arrived at threshold settings to correctly segment 67% of the total possible diatom valves and fragments from broad fields of view. (183 light microscope images were examined containing 255 diatom particles. Of the 255 diatom particles present, 216 diatoms valves and fragments of valves were processed, with 170 properly analyzed and focused upon by the software). Manual analysis of the images yielded 255 particles in 400 seconds, whereas the software yielded a total of 216 particles in 68 seconds, thus highlighting that the software has an approximate five-fold efficiency advantage in particle analysis time. As in past efforts, incomplete or incorrect recognition was found for images with multiple valves in contact or valves with little contrast. The software has potential to be an effective tool in assisting taxonomists with diatom enumeration by completing a large portion of analyses. Benefits and limitations of the approach are presented to allow for development of future work in image analysis and automated enumeration of traditional light microscope images containing diatoms.

Comparison of LED and Conventional Fluorescence Microscopy for Detection of Acid Fast Bacilli in a Low-Incidence Setting

PubMed Central

Minion, Jessica; Pai, Madhukar; Ramsay, Andrew; Menzies, Dick; Greenaway, Christina

2011-01-01

Introduction Light emitting diode fluorescence microscopes have many practical advantages over conventional mercury vapour fluorescence microscopes, which would make them the preferred choice for laboratories in both low- and high-resource settings, provided performance is equivalent. Methods In a nested case-control study, we compared diagnostic accuracy and time required to read slides with the Zeiss PrimoStar iLED, LW Scientific Lumin, and a conventional fluorescence microscope (Leica DMLS). Mycobacterial culture was used as the reference standard, and subgroup analysis by specimen source and organism isolated were performed. Results There was no difference in sensitivity or specificity between the three microscopes, and agreement was high for all comparisons and subgroups. The Lumin and the conventional fluorescence microscope were equivalent with respect to time required to read smears, but the Zeiss iLED was significantly time saving compared to both. Conclusions Light emitting diode microscopy should be considered by all tuberculosis diagnostic laboratories, including those in high income countries, as a replacement for conventional fluorescence microscopes. Our findings provide support to the recent World Health Organization policy recommending that conventional fluorescence microscopy be replaced by light emitting diode microscopy using auramine staining in all settings where fluorescence microscopy is currently used. PMID:21811622

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

Quantification of Bacterial Twitching Motility in Dense Colonies Using Transmitted Light Microscopy and Computational Image Analysis.

PubMed

Smith, Benjamin; Li, Jianfang; Metruccio, Matteo; Wan, Stephanie; Evans, David; Fleiszig, Suzanne

2018-04-20

A method was developed to allow the quantification and mapping of relative bacterial twitching motility in dense samples, where tracking of individual bacteria was not feasible. In this approach, movies of bacterial films were acquired using differential interference contrast microscopy (DIC), and bacterial motility was then indirectly quantified by the degree to which the bacteria modulated the intensity of light in the field-of-view over time. This allowed the mapping of areas of relatively high and low motility within a single field-of-view, and comparison of the total distribution of motility between samples.

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.

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Onset of molar incisor hypomineralization (MIH).

PubMed

Fagrell, Tobias G; Salmon, Phil; Melin, Lisa; Norén, Jörgen G

2013-01-01

The etiological factors and timing of the onset of molar incisor hypomineralization (MIH) are still not clear. The aim of this study was to examine ground radial and sagittal sections from teeth diagnosed with MIH using light microscopy, polarized light microscopy and X-ray micro-computed tomography (XMCT) and to estimate the onset and timing of the MIH and to relate the hypomineralized enamel to the incremental lines. Thirteen extracted permanent first molars diagnosed MIH, were analyzed with light microscopy and XMCT. The hypomineralized areas were mainly located in the mesio-buccal cusps, starting at the enamel-dentin-junction and continuing towards the enamel surface. In a relative gray scale analysis the values decreased from the EDJ towards the enamel surface. The findings indicate that the ameloblasts in the hypomineralized enamel are capable of forming an enamel of normal thickness, but with a substantial reduction of their capacity for maturation of enamel. Chronologically, it is estimated that the timing of the disturbance is at a period during the first 6-7 months of age.

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.

Evanescent Waves in High Numerical Aperture Aplanatic Solid Immersion Microscopy: Effects of Forbidden Light on Subsurface Imaging (Open Access, Publisher’s Version)

DTIC Science & Technology

2014-03-24

of the aSIL microscopy for semiconductor failure analysis and is applicable to imaging in quantum optics [18], biophotonics [19] and metrology [20...is usually of interest, the model can be adapted to applications in fields such as quantum optics and biophotonics for which the non-resonant

Diagnostic accuracy of same-day microscopy versus standard microscopy for pulmonary tuberculosis: a systematic review and meta-analysis.

PubMed

Davis, J Lucian; Cattamanchi, Adithya; Cuevas, Luis E; Hopewell, Philip C; Steingart, Karen R

2013-02-01

Sputum smear microscopy is the most widely available diagnostic test for pulmonary tuberculosis in countries with a high burden of the disease. Improving its accuracy is crucial to achievement of case-detection targets established by the Millennium Development Goals. Unfortunately, many patients are unable to submit all of the specimens needed for examination or to return for treatment because standard sputum collection and reporting requires several clinic visits. To inform policy recommendations by a WHO-convened Expert Group, we aimed to assess the accuracy of sputum smear examination with strategies for obtaining sputum on 1 day compared with strategies for obtaining sputum over 2 days. We did a systematic review and meta-analysis of research articles comparing the accuracy of front-loaded or same-day microscopy and standard sputum smear microscopy for diagnosis of culture-confirmed pulmonary tuberculosis. We searched Medline, Embase, Biosis, and Web of Science for articles published between Jan 1, 2005, and Feb 14, 2012. Two investigators identified eligible articles and extracted data for individual study sites. We generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when four or more studies were available. We identified eight relevant studies from five articles enrolling 7771 patients with suspected tuberculosis in low-income countries. Compared with the standard approach of examination of two smears with Ziehl-Neelsen light microscopy over 2 days, examination of two smears taken on the same day had much the same sensitivity (64% [95% CI 60 to 69] for standard microscopy vs 63% [58 to 68] for same-day microscopy) and specificity (98% [97 to 99] vs 98% [97 to 99]). We noted similar results for studies employing light-emitting diode fluorescence microscopy and for studies examining three smears, whether they were compared with two-smear strategies or with one another. Same-day sputum smear microscopy is as accurate as standard smear microscopy. Data from tuberculosis programmes are needed to document the changes required in the health system to successfully implement the strategy and understand its effects. WHO and US National Institutes of Health. Copyright © 2013 Elsevier Ltd. All rights reserved.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Tissue and cellular localization of tannins in Tunisian dates (Phoenix dactylifera L.) by light and transmission electron microscopy.

PubMed

Hammouda, Hédi; Alvarado, Camille; Bouchet, Brigitte; Kalthoum-Chérif, Jamila; Trabelsi-Ayadi, Malika; Guyot, Sylvain

2014-07-16

A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa.

SEM-EDX analysis of an unknown "known" white powder found in a shipping container from Peru

NASA Astrophysics Data System (ADS)

Albright, Douglas C.

2009-05-01

In 2008, an unknown white powder was discovered spilled inside of a shipping container of whole kernel corn during an inspection by federal inspectors in the port of Baltimore, Maryland. The container was detained and quarantined while a sample of the powder was collected and sent to a federal laboratory where it was screened using chromatography for the presence of specific poisons and pesticides with negative results. Samples of the corn kernels and the white powder were forwarded to the Food and Drug Administration, Forensic Chemistry Center for further analysis. Stereoscopic Light Microscopy (SLM), Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDX), and Polarized Light Microscopy/Infrared Spectroscopy (PLM-IR) were used in the analysis of the kernels and the unknown powder. Based on the unique particle analysis by SLM and SEM as well as the detection of the presence of aluminum and phosphorous by EDX, the unknown was determined to be consistent with reacted aluminum phosphide (AlP). While commonly known in the agricultural industry, aluminum phosphide is relatively unknown in the forensic community. A history of the use and acute toxicity of this compound along with some very unique SEM/EDX analysis characteristics of aluminum phosphide will be discussed.

Evaluation of mobile digital light-emitting diode fluorescence microscopy in Hanoi, Viet Nam.

PubMed

Chaisson, L H; Reber, C; Phan, H; Switz, N; Nilsson, L M; Myers, F; Nhung, N V; Luu, L; Pham, T; Vu, C; Nguyen, H; Nguyen, A; Dinh, T; Nahid, P; Fletcher, D A; Cattamanchi, A

2015-09-01

Hanoi Lung Hospital, Hanoi, Viet Nam. To compare the accuracy of CellScopeTB, a manually operated mobile digital fluorescence microscope, with conventional microscopy techniques. Patients referred for sputum smear microscopy to the Hanoi Lung Hospital from May to September 2013 were included. Ziehl-Neelsen (ZN) smear microscopy, conventional light-emitting diode (LED) fluorescence microscopy (FM), CellScopeTB-based LED FM and Xpert(®) MTB/RIF were performed on sputum samples. The sensitivity and specificity of microscopy techniques were determined in reference to Xpert results, and differences were compared using McNemar's paired test of proportions. Of 326 patients enrolled, 93 (28.5%) were Xpert-positive for TB. The sensitivity of ZN microscopy, conventional LED FM, and CellScopeTB-based LED FM was respectively 37.6% (95%CI 27.8-48.3), 41.9% (95%CI 31.8-52.6), and 35.5% (95%CI 25.8-46.1). The sensitivity of CellScopeTB was similar to that of conventional LED FM (difference -6.5%, 95%CI -18.2 to 5.3, P = 0.33) and ZN microscopy (difference -2.2%, 95%CI -9.2 to 4.9, P = 0.73). The specificity was >99% for all three techniques. CellScopeTB performed similarly to conventional microscopy techniques in the hands of experienced TB microscopists. However, the sensitivity of all sputum microscopy techniques was low. Options enabled by digital microscopy, such as automated imaging with real-time computerized analysis, should be explored to increase sensitivity.

Effects of Er:YAG laser irradiation on human dentin: polarizing microscopic, light microscopic and microradiographic observations, and FT-IR analysis.

PubMed

Ishizaka, Yaeko; Eguro, Toru; Maeda, Toru; Tanaka, Hisayoshi

2002-01-01

The effects of Er:YAG laser irradiation on dentin have not been sufficiently investigated. The purpose of this study was to investigate the effects of Er:YAG laser irradiation on dentin. After cavities were prepared using Er:YAG laser irradiation or rotary cutting instruments, histological observations of cavity-floor dentin utilizing polarizing microscopy, microradiography and light microscopy, and analysis of composition of cavity-floor dentin using Fourier-transformed (FT-IR) spectrometry were conducted. In the laser-treated side, a deeply stained basophilic layer was observed. The number of odontoblastic processes present was obviously less in the laser-treated side than in the bur-treated side. FT-IR analysis revealed that compared to the bur-treated side, a broad background peak at around 1,600 cm(-1) was present. Er:YAG laser irradiation might have denatured the organic materials of dentin. Copyright 2002 Wiley-Liss, Inc.

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.

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

Type 1 diabetes mellitus effects on dental enamel formation revealed by microscopy and microanalysis.

PubMed

Silva, Bruna Larissa Lago; Medeiros, Danila Lima; Soares, Ana Prates; Line, Sérgio Roberto Peres; Pinto, Maria das Graças Farias; Soares, Telma de Jesus; do Espírito Santo, Alexandre Ribeiro

2018-03-01

Type 1 diabetes mellitus (T1DM) largely affects children, occurring therefore at the same period of deciduous and permanent teeth development. The aim of this work was to investigate birefringence and morphology of the secretory stage enamel organic extracellular matrix (EOECM), and structural and mechanical features of mature enamel from T1DM rats. Adult Wistar rats were maintained alive for a period of 56 days after the induction of experimental T1DM with a single dose of streptozotocin (60 mg/kg). After proper euthanasia of the animals, fixed upper incisors were accurately processed, and secretory stage EOECM and mature enamel were analyzed by transmitted polarizing and bright field light microscopies (TPLM and BFLM), energy-dispersive x-ray (EDX) analysis, scanning electron microscopy (SEM), and microhardness testing. Bright field light microscopies and transmitted polarizing light microscopies showed slight morphological changes in the secretory stage EOECM from diabetic rats, which also did not exhibit statistically significant alterations in birefringence brightness when compared to control animals (P > .05). EDX analysis showed that T1DM induced statistically significant little increases in the amount of calcium and phosphorus in outer mature enamel (P < .01) with preservation of calcium/phosphorus ratio in that structure (P > .05). T1DM also caused important ultrastructural alterations in mature enamel as revealed by SEM and induced a statistically significant reduction of about 13.67% in its microhardness at 80 μm from dentin-enamel junction (P < .01). This study shows that T1DM may disturb enamel development, leading to alterations in mature enamel ultrastructure and in its mechanical features. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

PubMed

Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

2016-07-01

High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

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

Two-photon confocal microscopy in wound healing

NASA Astrophysics Data System (ADS)

Navarro, Fernando A.; So, Peter T. C.; Driessen, Antoine; Kropf, Nina; Park, Christine S.; Huertas, Juan C.; Lee, Hoon B.; Orgill, Dennis P.

2001-04-01

Advances in histopathology and immunohistochemistry have allowed for precise microanatomic detail of tissues. Two Photon Confocal Microscopy (TPCM) is a new technology useful in non-destructive analysis of tissue. Laser light excites the natural florophores, NAD(P)H and NADP+ and the scattering patterns of the emitted light are analyzed to reconstruct microanatomic features. Guinea pig skin was studied using TPCM and skin preparation methods including chemical depilation and tape striping. Results of TPCM were compared with conventional hematoxylin and eosin microscopy. Two-dimensional images were rendered from the three dimensional reconstructions. Images of deeper layers including basal cells and the dermo-epidermal junction improved after removing the stratum corneum with chemical depilation or tape stripping. TCPM allows good resolution of corneocytes, basal cells and collagen fibers and shows promise as a non-destructive method to study wound healing.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Research and application on imaging technology of line structure light based on confocal microscopy

NASA Astrophysics Data System (ADS)

Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

2009-11-01

In 2005, the theory of line structure light confocal microscopy was put forward firstly in China by Xingyu Gao and Zexin Xiao in the Institute of Opt-mechatronics of Guilin University of Electronic Technology. Though the lateral resolution of line confocal microscopy can only reach or approach the level of the traditional dot confocal microscopy. But compared with traditional dot confocal microscopy, it has two advantages: first, by substituting line scanning for dot scanning, plane imaging only performs one-dimensional scanning, with imaging velocity greatly improved and scanning mechanism simplified, second, transfer quantity of light is greatly improved by substituting detection hairline for detection pinhole, and low illumination CCD is used directly to collect images instead of photoelectric intensifier. In order to apply the line confocal microscopy to practical system, based on the further research on the theory of the line confocal microscopy, imaging technology of line structure light is put forward on condition of implementation of confocal microscopy. Its validity and reliability are also verified by experiments.

The e-evolution of microscopy in dental education.

PubMed

Farah, Camile S; Maybury, Terrence S

2009-08-01

Recent technological innovation has now made it possible to turn the computer into a microscope. This has entailed a shift from light microscopy to virtual microscopy. This development then foregrounds the issue of the pedagogy involved in this move from the analogue technology of the light microscope to the digital, computerized instance of virtual microscopy. In order to address this issue, undergraduate students enrolled in the Bachelor of Dental Science program at the University of Queensland School of Dentistry were surveyed to ascertain their preference for light or virtual microscopy. The value of this study is that it was conducted on the same cohort of students in two separate courses in 2006 and 2008, giving it longitudinal validity. The responses were overwhelmingly in favor of virtual microscopy. When it came to completely replacing the light microscope with virtual microscopy, however, students were much more ambivalent about such a wholesale change although this was less of an issue in the senior year. This shift from light to virtual microscopy signals larger changes in the tertiary sector from print-literate to electronic forms of knowledge and from teacher-centered to student-focused frames of learning. In short, we are in the midst of the e-evolution of microscopy in dental education.

Development of image analysis software for quantification of viable cells in microchips.

PubMed

Georg, Maximilian; Fernández-Cabada, Tamara; Bourguignon, Natalia; Karp, Paola; Peñaherrera, Ana B; Helguera, Gustavo; Lerner, Betiana; Pérez, Maximiliano S; Mertelsmann, Roland

2018-01-01

Over the past few years, image analysis has emerged as a powerful tool for analyzing various cell biology parameters in an unprecedented and highly specific manner. The amount of data that is generated requires automated methods for the processing and analysis of all the resulting information. The software available so far are suitable for the processing of fluorescence and phase contrast images, but often do not provide good results from transmission light microscopy images, due to the intrinsic variation of the acquisition of images technique itself (adjustment of brightness / contrast, for instance) and the variability between image acquisition introduced by operators / equipment. In this contribution, it has been presented an image processing software, Python based image analysis for cell growth (PIACG), that is able to calculate the total area of the well occupied by cells with fusiform and rounded morphology in response to different concentrations of fetal bovine serum in microfluidic chips, from microscopy images in transmission light, in a highly efficient way.

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.

Thermal analysis and microstructural characterization of Mg-Al-Zn system alloys

NASA Astrophysics Data System (ADS)

Król, M.; Tański, T.; Sitek, W.

2015-11-01

The influence of Zn amount and solidification rate on the characteristic temperature of the evaluation of magnesium dendrites during solidification at different cooling rates (0.6-2.5°C) were examined by thermal derivative analysis (TDA). The dendrite coherency point (DCP) is presented with a novel approach based on second derivative cooling curve. Solidification behavior was examined via one thermocouple thermal analysis method. Microstructural assessments were described by optical light microscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. These studies showed that utilization of d2T/dt2 vs. the time curve methodology provides for analysis of the dendrite coherency point

The role of light microscopy in aerospace analytical laboratories

NASA Technical Reports Server (NTRS)

Crutcher, E. R.

1977-01-01

Light microscopy has greatly reduced analytical flow time and added new dimensions to laboratory capability. Aerospace analytical laboratories are often confronted with problems involving contamination, wear, or material inhomogeneity. The detection of potential problems and the solution of those that develop necessitate the most sensitive and selective applications of sophisticated analytical techniques and instrumentation. This inevitably involves light microscopy. The microscope can characterize and often identify the cause of a problem in 5-15 minutes with confirmatory tests generally less than one hour. Light microscopy has and will make a very significant contribution to the analytical capabilities of aerospace laboratories.

Transmission Electron Microscopy Analysis of Skin Lesions from Sporotrichosis Epidemic in Rio de Janeiro, Brazil

PubMed Central

Porto Ferreira, Cassio; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-01-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. PMID:25653392

Super-resolution binding activated localization microscopy through reversible change of DNA conformation.

PubMed

Szczurek, Aleksander; Birk, Udo; Knecht, Hans; Dobrucki, Jurek; Mai, Sabine; Cremer, Christoph

2018-01-01

Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine.

Super-resolution binding activated localization microscopy through reversible change of DNA conformation

PubMed Central

Knecht, Hans; Dobrucki, Jurek; Mai, Sabine

2018-01-01

ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future applications in biology and medicine. PMID:29297245

Optical properties of metamorphic GaAs/InAlGaAs/InGaAs heterostructures with InAs/InGaAs quantum wells, emitting light in the 1250–1400-nm spectral range

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

Egorov, A. Yu., E-mail: anton@beam.ioffe.ru; Karachinsky, L. Ya.; Novikov, I. I.

It is demonstrated that metamorphic GaAs/InAlGaAs/InGaAs heterostructures with InAs/InGaAs quantum wells, which emit light in the 1250–1400 nm spectral range, can be fabricated by molecular-beam epitaxy. The structural and optical properties of the heterostructures are studied by X-ray diffraction analysis, transmission electron microscopy, and the photoluminescence method. Comparative analysis of the integrated photoluminescence intensity of the heterostructures and a reference sample confirm the high efficiency of radiative recombination in the heterostructures. It is confirmed by transmission electron microscopy that dislocations do not penetrate into the active region of the metamorphic heterostructures, where the radiative recombination of carriers occurs.

Refractive index measurements of single, spherical cells using digital holographic microscopy.

PubMed

Schürmann, Mirjam; Scholze, Jana; Müller, Paul; Chan, Chii J; Ekpenyong, Andrew E; Chalut, Kevin J; Guck, Jochen

2015-01-01

In this chapter, we introduce digital holographic microscopy (DHM) as a marker-free method to determine the refractive index of single, spherical cells in suspension. The refractive index is a conclusive measure in a biological context. Cell conditions, such as differentiation or infection, are known to yield significant changes in the refractive index. Furthermore, the refractive index of biological tissue determines the way it interacts with light. Besides the biological relevance of this interaction in the retina, a lot of methods used in biology, including microscopy, rely on light-tissue or light-cell interactions. Hence, determining the refractive index of cells using DHM is valuable in many biological applications. This chapter covers the main topics that are important for the implementation of DHM: setup, sample preparation, and analysis. First, the optical setup is described in detail including notes and suggestions for the implementation. Following that, a protocol for the sample and measurement preparation is explained. In the analysis section, an algorithm for the determination of quantitative phase maps is described. Subsequently, all intermediate steps for the calculation of the refractive index of suspended cells are presented, exploiting their spherical shape. In the last section, a discussion of possible extensions to the setup, further measurement configurations, and additional analysis methods are given. Throughout this chapter, we describe a simple, robust, and thus easily reproducible implementation of DHM. The different possibilities for extensions show the diverse fields of application for this technique. Copyright © 2015 Elsevier Inc. All rights reserved.

Light sheet microscopy.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail. © 2014 Elsevier Inc. All rights reserved.

How Hedstrom files fail during clinical use? A retrieval study based on SEM, optical microscopy and micro-XCT analysis.

PubMed

Zinelis, Spiros; Al Jabbari, Youssef S

2018-05-01

This study was conducted to evaluate the failure mechanism of clinically failed Hedstrom (H)-files. Discarded H-files (n=160) from #8 to #40 ISO sizes were collected from different dental clinics. Retrieved files were classified according to their macroscopic appearance and they were investigated under scanning electron microscopy (SEM) and X-ray micro-computed tomography (mXCT). Then the files were embedded in resin along their longitudinal axis and after metallographic grinding and polishing, studied under an incident light microscope. The macroscopic evaluation showed that small ISO sizes (#08-#15) failed by extensive plastic deformation, while larger sizes (≥#20) tended to fracture. Light microscopy and mXCT results coincided showing that unused and plastically deformed files were free of internal defects, while fractured files demonstrate the presence of intense cracking in the flute region. SEM analysis revealed the presence of striations attributed to the fatigue mechanism. Secondary cracks were also identified by optical microscopy and their distribution was correlated to fatigue under bending loading. Experimental results demonstrated that while overloading of cutting instruments is the predominating failure mechanism of small file sizes (#08-#15), fatigue should be considered the fracture mechanism for larger sizes (≥#20).

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.

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.

Integration of a high-NA light microscope in a scanning electron microscope.

PubMed

Zonnevylle, A C; Van Tol, R F C; Liv, N; Narvaez, A C; Effting, A P J; Kruit, P; Hoogenboom, J P

2013-10-01

We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

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.

Light microscopy applications in systems biology: opportunities and challenges

PubMed Central

2013-01-01

Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy.

PubMed

Liau, Ee Shan; Yen, Ya-Ping; Chen, Jun-An

2018-05-11

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Characterization of magnetic nanoparticle by dynamic light scattering

PubMed Central

2013-01-01

Here we provide a complete review on the use of dynamic light scattering (DLS) to study the size distribution and colloidal stability of magnetic nanoparticles (MNPs). The mathematical analysis involved in obtaining size information from the correlation function and the calculation of Z-average are introduced. Contributions from various variables, such as surface coating, size differences, and concentration of particles, are elaborated within the context of measurement data. Comparison with other sizing techniques, such as transmission electron microscopy and dark-field microscopy, revealed both the advantages and disadvantages of DLS in measuring the size of magnetic nanoparticles. The self-assembly process of MNP with anisotropic structure can also be monitored effectively by DLS. PMID:24011350

Biobeam—Multiplexed wave-optical simulations of light-sheet microscopy

PubMed Central

Weigert, Martin; Bundschuh, Sebastian T.

2018-01-01

Sample-induced image-degradation remains an intricate wave-optical problem in light-sheet microscopy. Here we present biobeam, an open-source software package that enables simulation of operational light-sheet microscopes by combining data from 105–106 multiplexed and GPU-accelerated point-spread-function calculations. The wave-optical nature of these simulations leads to the faithful reproduction of spatially varying aberrations, diffraction artifacts, geometric image distortions, adaptive optics, and emergent wave-optical phenomena, and renders image-formation in light-sheet microscopy computationally tractable. PMID:29652879

Comparative Analysis Reveals Potential Utility of Digital Microscopy in the Evaluation of Peripheral Blood Smears With Some Barriers to Implementation.

PubMed

Gomez-Gelvez, Juan C; Kryvenko, Oleksandr N; Chabot-Richards, Devon S; Foucar, Kathryn; Inamdar, Kedar V; Karner, Kristin H

2015-07-01

Evaluation of the peripheral blood smear (PBS) is an essential diagnostic test in current medical practice. We aimed to evaluate the use of digital microscopy for the examination of PBS as an option to provide expert interpretation to remote sites and in "on-call" situations. We collected 100 Wright-Giemsa-stained PBS slides representing normal and abnormal findings seen at a community-based hospital. Four hematopathologists independently evaluated the cases using conventional light and digital microscopy. When comparing digital vs light microscopy, most of the cellular features evaluated showed at least a moderate degree of agreement in at least three of the reviewers. Discrepancies in final diagnosis were identified in a minority of the cases, most of which were attributed to the poorer resolution of digital microscopy at high magnification (×400). These results support the limited use of digital microscopy for evaluation and triage of peripheral blood smears as a practical option to obtain expert opinion in locations where experienced staff is not available on site. Our results indicate that while digital microscopy is well suited for basic triage of these blood smears, limitations in quality of imaging at higher magnification as well as large file size may limit its utility in certain settings and situations. Copyright© by the American Society for Clinical Pathology.

Structural Analysis of Enamel in Teeth from Head-and-Neck Cancer Patients Who Underwent Radiotherapy.

PubMed

Madrid, Cristhian C; de Pauli Paglioni, Mariana; Line, Sergio R; Vasconcelos, Karina G; Brandão, Thaís Bianca; Lopes, Marcio A; Santos-Silva, Alan Roger; De Goes, Mario Fernando

2017-01-01

To analyze macroscopic, microscopic, and ultrastructural aspects of enamel from head-and-neck cancer patients submitted to radiotherapy. Twenty sound extracted permanent molars were used and divided into 2 groups. The experimental group consisted of 10 molars from head-and-neck cancer patients submitted to radiotherapy with total doses that ranged from 50 to 70 Gy. Ten molars from patients who did not receive radiotherapy were matched with experimental-group samples by anatomic tooth group and comprised the control group. To perform a macroscopic analysis, standardized photos of different enamel faces were taken with a camera. Teeth were subjected to longitudinal cuts and hand polished to a final thickness of 0.1 mm. Enamel was analyzed under polarized light microscopy, and optical retardation values of birefringence were calculated in cervical, cusp, and occlusal pit areas. Subsequently, the same enamel areas were analyzed by scanning electron microscopy. Data from optical retardation values were statistically analyzed by 2-way ANOVA and Fisher's test (α < 0.05). No macroscopic differences were observed between the irradiated and control groups. Polarized light microscopy analysis revealed that cervical enamel exhibited darker areas characterized by discrete birefringence patterns compared to the control enamel. Optical retardation values were only significantly different in the cervical enamel of the irradiated and control groups (p < 0.0001). Scanning electron microscopy analysis revealed more evident interprismatic spaces in the cervical and outer cusp enamel of irradiated samples. Head-and-neck radiotherapy reduced optical retardation values of birefringence in cervical enamel, and the interprismatic spaces became more evident. © 2017 S. Karger AG, Basel.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Elemental distribution analysis of urinary crystals.

PubMed

Fazil Marickar, Y M; Lekshmi, P R; Varma, Luxmi; Koshy, Peter

2009-10-01

Various crystals are seen in human urine. Some of them, particularly calcium oxalate dihydrate, are seen normally. Pathological crystals indicate crystal formation initiating urinary stones. Unfortunately, many of the relevant crystals are not recognized in light microscopic analysis of the urinary deposit performed in most of the clinical laboratories. Many crystals are not clearly identifiable under the ordinary light microscopy. The objective of the present study was to perform scanning electron microscopic (SEM) assessment of various urinary deposits and confirm the identity by elemental distribution analysis (EDAX). 50 samples of urinary deposits were collected from urinary stone clinic. Deposits containing significant crystalluria (more than 10 per HPF) were collected under liquid paraffin in special containers and taken up for SEM studies. The deposited crystals were retrieved with appropriate Pasteur pipettes, and placed on micropore filter paper discs. The fluid was absorbed by thicker layers of filter paper underneath and discs were fixed to brass studs. They were then gold sputtered to 100 A and examined under SEM (Jeol JSM 35C microscope). When crystals were seen, their morphology was recorded by taking photographs at different angles. At appropriate magnification, EDAX probe was pointed to the crystals under study and the wave patterns analyzed. Components of the crystals were recognized by utilizing the data. All the samples analyzed contained significant number of crystals. All samples contained more than one type of crystal. The commonest crystals encountered included calcium oxalate monohydrate (whewellite 22%), calcium oxalate dihydrate (weddellite 32%), uric acid (10%), calcium phosphates, namely, apatite (4%), brushite (6%), struvite (6%) and octocalcium phosphate (2%). The morphological appearances of urinary crystals described were correlated with the wavelengths obtained through elemental distribution analysis. Various urinary crystals that are not reported under light microscopy could be recognized by SEM-EDAX combination. EDAX is a significant tool for recognizing unknown crystals not identified by ordinary light microscopy or SEM alone.

Quantitative analysis with advanced compensated polarized light microscopy on wavelength dependence of linear birefringence of single crystals causing arthritis

NASA Astrophysics Data System (ADS)

Takanabe, Akifumi; Tanaka, Masahito; Taniguchi, Atsuo; Yamanaka, Hisashi; Asahi, Toru

2014-07-01

To improve our ability to identify single crystals causing arthritis, we have developed a practical measurement system of polarized light microscopy called advanced compensated polarized light microscopy (A-CPLM). The A-CPLM system is constructed by employing a conventional phase retardation plate, an optical fibre and a charge-coupled device spectrometer in a polarized light microscope. We applied the A-CPLM system to measure linear birefringence (LB) in the visible region, which is an optical anisotropic property, for tiny single crystals causing arthritis, i.e. monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD). The A-CPLM system performance was evaluated by comparing the obtained experimental data using the A-CPLM system with (i) literature data for a standard sample, MgF2, and (ii) experimental data obtained using an established optical method, high-accuracy universal polarimeter, for the MSUM. The A-CPLM system was found to be applicable for measuring the LB spectra of the single crystals of MSUM and CPPD, which cause arthritis, in the visible regions. We quantitatively reveal the large difference in LB between MSUM and CPPD crystals. These results demonstrate the usefulness of the A-CPLM system for distinguishing the crystals causing arthritis.

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

Dual-dimensional microscopy: real-time in vivo three-dimensional observation method using high-resolution light-field microscopy and light-field display.

PubMed

Kim, Jonghyun; Moon, Seokil; Jeong, Youngmo; Jang, Changwon; Kim, Youngmin; Lee, Byoungho

2018-06-01

Here, we present dual-dimensional microscopy that captures both two-dimensional (2-D) and light-field images of an in-vivo sample simultaneously, synthesizes an upsampled light-field image in real time, and visualizes it with a computational light-field display system in real time. Compared with conventional light-field microscopy, the additional 2-D image greatly enhances the lateral resolution at the native object plane up to the diffraction limit and compensates for the image degradation at the native object plane. The whole process from capturing to displaying is done in real time with the parallel computation algorithm, which enables the observation of the sample's three-dimensional (3-D) movement and direct interaction with the in-vivo sample. We demonstrate a real-time 3-D interactive experiment with Caenorhabditis elegans. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

PubMed

Engelmann, Péter; Hayashi, Yuya; Bodó, Kornélia; Ernszt, Dávid; Somogyi, Ildikó; Steib, Anita; Orbán, József; Pollák, Edit; Nyitrai, Miklós; Németh, Péter; Molnár, László

2016-12-01

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single shot white light interference microscopy with colour fringe analysis for quantitative phase imaging of biological cells

NASA Astrophysics Data System (ADS)

Srivastava, Vishal; Mehta, D. S.

2013-02-01

To quantitatively obtain the phase map of Onion and human red blood cell (RBC) from white light interferogram we used Hilbert transform color fringe analysis technique. The three Red, Blue and Green color components are decomposed from single white light interferogram and Refractive index profile for Red, Blue and Green colour were computed in a completely non-invasive manner for Onion and human RBC. The present technique might be useful for non-invasive determination of the refractive index variation within cells and tissues and morphological features of sample with ease of operation and low cost.

Repeatability and reproducibility of intracellular molar concentration assessed by synchrotron-based x-ray fluorescence microscopy

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

Merolle, L., E-mail: lucia.merolle@elettra.eu; Gianoncelli, A.; Malucelli, E., E-mail: emil.malucelli@unibo.it

2016-01-28

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the majormore » obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.« less

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.

[Clinical pathology on the verge of virtual microscopy].

PubMed

Tolonen, Teemu; Näpänkangas, Juha; Isola, Jorma

2015-01-01

For more than 100 years, examinations of pathology specimens have relied on the use of the light microscope. The technological progress of the last few years is enabling the digitizing of histologic specimen slides and application of the virtual microscope in diagnostics. Virtual microscopy will facilitate consultation possibilities, and digital image analysis serves to enhance the level of diagnostics. Organizing and monitoring clinicopathological meetings will become easier. Digital archive of histologic specimens and the virtual microscopy network are expected to benefit training and research as well, particularly what applies to the Finnish biobank network which is currently being established.

Transmission electron microscopy analysis of skin lesions from sporotrichosis epidemic in Rio de Janeiro, Brazil.

PubMed

Ferreira, Cassio Porto; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-02-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. © The American Society of Tropical Medicine and Hygiene.

Dissociation of β-Sheet Stacking of Amyloid β Fibrils by Irradiation of Intense, Short-Pulsed Mid-infrared Laser.

PubMed

Kawasaki, Takayasu; Yaji, Toyonari; Ohta, Toshiaki; Tsukiyama, Koichi; Nakamura, Kazuhiro

2018-02-05

Structure of amyloid β (Aβ) fibrils is rigidly stacked by β-sheet conformation, and the fibril state of Aβ is profoundly related to pathogenesis of Alzheimer's disease (AD). Although mid-infrared light has been used for various biological researches, it has not yet been known whether the infrared light changes the fibril structure of Aβ. In this study, we tested the effect of irradiation of intense mid-infrared light from a free-electron laser (FEL) targeting the amide bond on the reduction of β-sheet content in Aβ fibrils. The FEL reduced entire contents of proteins exhibiting β-sheet structure in brain sections from AD model mice, as shown by synchrotron-radiation infrared microscopy analysis. Since Aβ 1-42 fibril absorbed a considerable FEL energy at amide I band (6.17 μm), we irradiated the FEL at 6.17 μm and found that β-sheet content of naked Aβ 1-42 fibril was decreased using infrared microscopic analysis. Consistent with the decrease in the β-sheet content, Congo-red signal is decreased after the irradiation to Aβ 1-42 fibril. Furthermore, electron microscopy analysis revealed that morphologies of the fibril and proto-fibril were largely changed after the irradiation. Thus, mid-infrared light dissociates β-sheet structure of Aβ fibrils, which justifies exploration of possible laser-based therapy for AD.

Sample holder for axial rotation of specimens in 3D microscopy.

PubMed

Bruns, T; Schickinger, S; Schneckenburger, H

2015-10-01

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Performance of rapid diagnostic test, blood-film microscopy and PCR for the diagnosis of malaria infection among febrile children from Korogwe District, Tanzania.

PubMed

Mahende, Coline; Ngasala, Billy; Lusingu, John; Yong, Tai-Soon; Lushino, Paminus; Lemnge, Martha; Mmbando, Bruno; Premji, Zul

2016-07-26

Rapid diagnostic tests (RDT) and light microscopy are still recommended for diagnosis to guide the clinical management of malaria despite difficult challenges in rural settings. The performance of these tests may be affected by several factors, including malaria prevalence and intensity of transmission. The study evaluated the diagnostic performance of malaria RDT, light microscopy and polymerase chain reaction (PCR) in detecting malaria infections among febrile children at outpatient clinic in Korogwe District, northeastern Tanzania. The study enrolled children aged 2-59 months with fever and/or history of fever in the previous 48 h attending outpatient clinics. Blood samples were collected for identification of Plasmodium falciparum infection using histidine-rich-protein-2 (HRP-2)-based malaria RDT, light microscopy and conventional PCR. A total of 867 febrile patients were enrolled into the study. Malaria-positive samples were 85/867 (9.8 %, 95 % CI, 7.9-12.0 %) by RDT, 72/867 (8.3 %, 95 % CI, 6.5-10.1 %) by microscopy and 79/677 (11.7 %, 95 % CI, 9.3-14.3 %) by PCR. The performance of malaria RDT compared with microscopy results had sensitivity and positive predictive value (PPV) of 88.9 % (95 % CI, 79.3-95.1 %) and 75.3 % (95 % CI, 64.8-84.0 %), respectively. Confirmation of P. falciparum infection with PCR analysis provided lower sensitivity and PPV of 88.6 % (95 % CI, 79.5-94.7 %) and 84.3 % (95 % CI, 74.7-91.4 %) for RDT compared to microscopy. Diagnosis of malaria infection is still a challenge due to variation in results among diagnostic methods. HRP-2 malaria RDT and microscopy were less sensitive than PCR. Diagnostic tools with high sensitivity are required in areas of low malaria transmission.

Analysis of multi-channel microscopy: Spectral self-interference, multi-detector confocal and 4Pi systems

NASA Astrophysics Data System (ADS)

Davis, Brynmor J.

Fluorescence microscopy is an important and ubiquitous tool in biological imaging due to the high specificity with which fluorescent molecules can be attached to an organism and the subsequent nondestructive in-vivo imaging allowed. Focused-light microscopies allow three-dimensional fluorescence imaging but their resolution is restricted by diffraction. This effect is particularly limiting in the axial dimension as the diffraction-limited focal volume produced by a lens is more extensive along the optical axis than perpendicular to it. Approaches such as confocal microscopy and 4Pi microscopy have been developed to improve the axial resolution. Spectral Self-Interference Fluorescence Microscopy (SSFM) is another high-axial-resolution technique and is the principal subject of this dissertation. Nanometer-precision localization of a single fluorescent layer has been demonstrated using SSFM. This accuracy compares favorably with the axial resolutions given by confocal and 4Pi systems at similar operating parameters (these resolutions are approximately 350nm and 80nm respectively). This theoretical work analyzes the expected performance of the SSFM system when imaging a general object, i.e. an arbitrary fluorophore density function rather than a single layer. An existing model of SSFM is used in simulations to characterize the system's resolution. Several statistically-based reconstruction methods are applied to show that the expected resolution for SSFM is similar to 4Pi microscopy for a general object but does give very high localization accuracy when the object is known to consist of a limited number of layers. SSFM is then analyzed in a linear systems framework and shown to have strong connections, both physically and mathematically, to a multi-channel 4Pi microscope. Fourier-domain analysis confirms that SSFM cannot be expected to outperform this multi-channel 4Pi instrument. Differences between the channels in spatial-scanning, multi-channel microscopies are then exploited to show that such instruments can operate at a sub-Nyquist scanning rate but still produce images largely free of aliasing effects. Multi-channel analysis is also used to show how light typically discarded in confocal and 4Pi systems can be collected and usefully incorporated into the measured image.

Plane wave analysis of coherent holographic image reconstruction by phase transfer (CHIRPT).

PubMed

Field, Jeffrey J; Winters, David G; Bartels, Randy A

2015-11-01

Fluorescent imaging plays a critical role in a myriad of scientific endeavors, particularly in the biological sciences. Three-dimensional imaging of fluorescent intensity often requires serial data acquisition, that is, voxel-by-voxel collection of fluorescent light emitted throughout the specimen with a nonimaging single-element detector. While nonimaging fluorescence detection offers some measure of scattering robustness, the rate at which dynamic specimens can be imaged is severely limited. Other fluorescent imaging techniques utilize imaging detection to enhance collection rates. A notable example is light-sheet fluorescence microscopy, also known as selective-plane illumination microscopy, which illuminates a large region within the specimen and collects emitted fluorescent light at an angle either perpendicular or oblique to the illumination light sheet. Unfortunately, scattering of the emitted fluorescent light can cause blurring of the collected images in highly turbid biological media. We recently introduced an imaging technique called coherent holographic image reconstruction by phase transfer (CHIRPT) that combines light-sheet-like illumination with nonimaging fluorescent light detection. By combining the speed of light-sheet illumination with the scattering robustness of nonimaging detection, CHIRPT is poised to have a dramatic impact on biological imaging, particularly for in vivo preparations. Here we present the mathematical formalism for CHIRPT imaging under spatially coherent illumination and present experimental data that verifies the theoretical model.

SPED light sheet microscopy: fast mapping of biological system structure and function

PubMed Central

Tomer, Raju; Lovett-Barron, Matthew; Kauvar, Isaac; Andalman, Aaron; Burns, Vanessa M.; Sankaran, Sethuraman; Grosenick, Logan; Broxton, Michael; Yang, Samuel; Deisseroth, Karl

2016-01-01

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light-sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca2+ imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function. PMID:26687363

Light Emitting Diode Flashlights as Effective and Inexpensive Light Sources for Fluorescence Microscopy

PubMed Central

Robertson, J. Brian; Zhang, Yunfei; Johnson, Carl Hirschie

2009-01-01

Summary Light-emitting diodes (LEDs) are becoming more commonly used as light sources for fluorescence microscopy. We describe the adaptation of a commercially available LED flashlight for use as a source for fluorescence excitation. This light source is long-lived, inexpensive, and is effective for excitation in the range of 440–600 nm. PMID:19772530

Cost-utility analysis of LED fluorescence microscopy in the diagnosis of pulmonary tuberculosis in Indian settings.

PubMed

Kelly, V; Sagili, K D; Satyanarayana, S; Reza, L W; Chadha, S S; Wilson, N C

2015-06-01

With support from the Stop TB Partnership's TB REACH Wave 2 Grant, diagnostic microscopy services for tuberculosis (TB) were upgraded from conventional Ziehl-Neelsen (ZN) based sputum microscopy to light emitting diode technology-based fluorescence microscopy (LED FM) in 200 high-workload microscopy centres in India as a pilot intervention. To evaluate the cost-effectiveness of LED-FM over conventional ZN microscopy to inform further scale-up. A decision-tree model was constructed to assess the cost utility of LED FM over ZN microscopy. The results were summarised using incremental cost-effectiveness ratio (ICER); one-way and probabilistic sensitivity analyses were also conducted to address uncertainty within the model. Data were analysed from 200 medical colleges in 2011 and 2012, before and after the introduction of LED microscopes. A full costing analysis was carried out from the perspective of a national TB programme. The ICER was calculated at US$14.64 per disability-adjusted life-year, with an 82% probability of being cost-effective at a willingness-to-pay threshold equivalent to Indian gross domestic product per capita. LED FM is a cost-effective intervention for detecting TB cases in India at high-workload medical college settings.

Biomolecular Analysis Capability for Cellular and Omics Research on the International Space Station

NASA Technical Reports Server (NTRS)

Guinart-Ramirez, Y.; Cooley, V. M.; Love, J. E.

2016-01-01

International Space Station (ISS) assembly complete ushered a new era focused on utilization of this state-of-the-art orbiting laboratory to advance science and technology research in a wide array of disciplines, with benefits to Earth and space exploration. ISS enabling capability for research in cellular and molecular biology includes equipment for in situ, on-orbit analysis of biomolecules. Applications of this growing capability range from biomedicine and biotechnology to the emerging field of Omics. For example, Biomolecule Sequencer is a space-based miniature DNA sequencer that provides nucleotide sequence data for entire samples, which may be used for purposes such as microorganism identification and astrobiology. It complements the use of WetLab-2 SmartCycler"TradeMark", which extracts RNA and provides real-time quantitative gene expression data analysis from biospecimens sampled or cultured onboard the ISS, for downlink to ground investigators, with applications ranging from clinical tissue evaluation to multigenerational assessment of organismal alterations. And the Genes in Space-1 investigation, aimed at examining epigenetic changes, employs polymerase chain reaction to detect immune system alterations. In addition, an increasing assortment of tools to visualize the subcellular distribution of tagged macromolecules is becoming available onboard the ISS. For instance, the NASA LMM (Light Microscopy Module) is a flexible light microscopy imaging facility that enables imaging of physical and biological microscopic phenomena in microgravity. Another light microscopy system modified for use in space to image life sciences payloads is initially used by the Heart Cells investigation ("Effects of Microgravity on Stem Cell-Derived Cardiomyocytes for Human Cardiovascular Disease Modeling and Drug Discovery"). Also, the JAXA Microscope system can perform remotely controllable light, phase-contrast, and fluorescent observations. And upcoming confocal microscopy capability will allow for optical sectioning of biological tissues to determine microanatomical localization of biomarkers. Furthermore, NASA's geneLAB effort addresses integration of genomic, epigenomic, transcriptomic, proteomic and metabolomic datasets, by applying an innovative open source science platform for multi-investigator high throughput utilization of the ISS. In sum, the expanding ISS capability for analysis of biomolecules is enabling innovative research in a broad spectrum of areas such as cellular and molecular biology, biotechnology, tissue engineering, biomedicine, and Omics, providing manifold benefits for humanity.

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Age-related adaptive responses of mitochondria of the retinal pigment epithelium to the everyday blue LED lighting.

PubMed

Serezhnikova, N B; Pogodina, L S; Lipina, T V; Trofimova, N N; Gurieva, T S; Zak, P P

2017-07-01

The effect of everyday blue light (λ = 440-460 nm) on mitochondria of the retinal pigment epithelium of different age groups of Japanese quail was studied using electron microscopy, morphometric methods, and biochemical analysis. We have found a significant increase in the number of mitochondria, including those modified, mainly in young birds. In addition, cell metabolic activity increased in response to blue lighting. These changes are assumed to reflect an adaptive response of mitochondria aimed at neutralizing the phototoxic effect of blue light caused by accumulation of lipofuscin granules.

Hue-saturation-density (HSD) model for stain recognition in digital images from transmitted light microscopy.

PubMed

van Der Laak, J A; Pahlplatz, M M; Hanselaar, A G; de Wilde, P C

2000-04-01

Transmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density. The usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels. In the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space. In the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose. Copyright 2000 Wiley-Liss, Inc.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging.

PubMed

Schieber, Nicole L; Machado, Pedro; Markert, Sebastian M; Stigloher, Christian; Schwab, Yannick; Steyer, Anna M

2017-01-01

Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy.

PubMed

Zhao, Ziqing W; Roy, Rahul; Gebhardt, J Christof M; Suter, David M; Chapman, Alec R; Xie, X Sunney

2014-01-14

Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of "transcription factories." Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells.

Evaluation of erythrocyte dysmorphism by light microscopy with lowering of the condenser lens: A simple and efficient method.

PubMed

Barros Silva, Gyl Eanes; Costa, Roberto Silva; Ravinal, Roberto Cuan; Saraiva e Silva, Jucélia; Dantas, Marcio; Coimbra, Terezila Machado

2010-03-01

To demonstrate that the evaluation of erythrocyte dysmorphism by light microscopy with lowering of the condenser lens (LMLC) is useful to identify patients with a haematuria of glomerular or non-glomerular origin. A comparative double-blind study between phase contrast microscopy (PCM) and LMLC is reported to evaluate the efficacy of these techniques. Urine samples of 39 patients followed up for 9 months were analyzed, and classified as glomerular and non-glomerular haematuria. The different microscopic techniques were compared using receiver-operator curve (ROC) analysis and area under curve (AUC). Reproducibility was assessed by coefficient of variation (CV). Specific cut-offs were set for each method according to their best rate of specificity and sensitivity as follows: 30% for phase contrast microscopy and 40% for standard LMLC, reaching in the first method the rate of 95% and 100% of sensitivity and specificity, respectively, and in the second method the rate of 90% and 100% of sensitivity and specificity, respectively. In ROC analysis, AUC for PCM was 0.99 and AUC for LMLC was 0.96. The CV was very similar in glomerular haematuria group for PCM (35%) and LMLC (35.3%). LMLC proved to be effective in contributing to the direction of investigation of haematuria, toward the nephrological or urological side. This method can substitute PCM when this equipment is not available.

Preparation of electrospun pyrochlore-structure KGdTa2O7:Eu3+ phosphor: the optical and structural properties for white light emitting diode applications.

PubMed

Yim, Chul Jin; Unithrattil, Sanjith; Chung, Woon Jin; Im, Won Bin

2013-12-01

Red emitting nanofibers, KGdTa2O7:Eu3+ were synthesized by electrospinning technique followed by heat treatment. As-prepared uniform fiber precursor with diameter ranging from about 700 nm to about 900 nm were calcined after removing organic species by calcination. The fiber surface become rough and diameter decreased to about 250-340 nm range due to decomposition of organic species and formation of inorganic phase. Morphology, structural and photoluminescent properties of fibers were analyzed using thermogravimetric and differential thermal analysis (TG-DTA), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), and photoluminescence (PL). TG-DTA analysis indicates that KGdTa2O7:Eu3+ began to crystalize at 520 degrees C. Fibers annealed at 900 degrees C formed well crystallized uniform fibers. Under ultraviolet excitation KGdTa2O7:Eu3+ exhibits red emission due to transitions in 4f states of Eu3+. The excitation band is dominated by the Eu(3+)--O2-charge transfer band peaked at 289 nm. The emission peak is in the region that is ideal for red light emission.

Blue light emitting diesel soot for photonic applications

NASA Astrophysics Data System (ADS)

Swapna, M. S.; Sankararaman, S.

2018-01-01

The present work is the first report of producing blue light emission from phosphor free and low-cost material—the diesel soot from the internal combustion engines (ICEs). The structural morphology is analyzed by field emission scanning electron microscopy and high-resolution transmission electron microscopy. The optical characterization is done by recording UV-visible spectrum and photoluminescent Spectrum. The CIE plot and the power spectrum for the sample show blue emission. This is further verified by collecting diesel soot from the ICE of different year of make. A visual confirmation of blue emission is obtained by exciting the sample with UV laser. The presence of various allotropic forms of carbon in the sample is identified by x-ray diffraction, Fourier transform infrared and Raman spectroscopic analysis.

Value of Reflected Light Microscopy in Teaching.

ERIC Educational Resources Information Center

Pasteris, Jill Dill

1983-01-01

Briefly reviews some optical and other physical properties of minerals that can be determined in reflected/incident light. Topics include optical properties of minerals, reflectance, internal reflections, color, bireflectance and reflection pleochroism, anisotropism, zonation, and reflected light microscopy as a teaching tool in undergraduate…

Ceramic femoral component fracture in total knee arthroplasty: an analysis using fractography, fourier-transform infrared microscopy, contact radiography and histology.

PubMed

Krueger, Alexander P; Singh, Gurpal; Beil, Frank Timo; Feuerstein, Bernd; Ruether, Wolfgang; Lohmann, Christoph H

2014-05-01

Ceramic components in total knee arthroplasty (TKA) are evolving. We analyze the first case of BIOLOX delta ceramic femoral component fracture. A longitudinal midline fracture in the patellar groove was present, with an intact cement mantle and no bony defects. Fractographic analysis with laser scanning microscopy and white light interferometry showed no evidence of arrest lines, hackles, wake hackles, material flaws, fatigue or crack propagation. Analysis of periprosthetic tissues with Fourier-transform infrared (FT-IR) microscopy, contact radiography, histology, and subsequent digestion and high-speed centrifugation did not show ceramic debris. A macrophage-dominated response was present around polyethylene debris. We conclude that ceramic femoral component failure in this case was related to a traumatic event. Further research is needed to determine the suitability of ceramic components in TKA. Copyright © 2014 Elsevier Inc. All rights reserved.

Biophysical mechanism of transient retinal phototropism in rod photoreceptors.

PubMed

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Gai, Shaoyan; Yao, Xincheng

2016-02-13

Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.

Biophysical mechanism of transient retinal phototropism in rod photoreceptors

NASA Astrophysics Data System (ADS)

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Gai, Shaoyan; Yao, Xincheng

2016-03-01

Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.

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.

Effect of the insertion and polymerization technique in composite resin restorations: analysis of marginal gap by atomic force microscopy.

PubMed

da Silva, Marcos Aurélio Bomfim; de Oliveira, Guilherme José Pimentel Lopes; Tonholo, Josealdo; Júnior, José Ginaldo da Silva; Santos, Lucineide de Melo; Dos Reis, José Ivo Limeira

2010-12-01

This in vitro study evaluated the marginal gap at the composite tooth/resin interface in class V cavities under the influence of two insertion techniques and a curing system by means of atomic force microscopy (AFM). Forty enamel and dentin cavities were prepared on the buccal surface in bovine teeth with quadratic forms measuring 2 mm × 2 mm and depth of 1.5 mm. The teeth were then divided into four groups: group A, 10 cavities were restored in one increment, light cured by halogen light; group B, 10 cavities filled with bulk filling, light cured by the light emitting diodes (LED); group C, 10 cavities were restored by the incremental technique, light cured by halogen light; group D, 10 cavities were restored by the incremental technique, light cured by the LED. The teeth underwent the polishing procedure and were analyzed by AFM for tooth/restoration interface evaluation. The data were compared between groups using the nonparametric Kruskall-Wallis and Mann-Whitney tests (p < 0.05). The results showed a statistically significant difference between groups A and B and groups A and C. It was concluded that no insertion and polymerization technique was able to completely seal the cavity.

Characterizing string-of-pearls colloidal silica by multidetector hydrodynamic chromatography and comparison to multidetector size-exclusion chromatography, off-line multiangle static light scattering, and transmission electron microscopy.

PubMed

Brewer, Amandaa K; Striegel, André M

2011-04-15

The string-of-pearls-type morphology is ubiquitous, manifesting itself variously in proteins, vesicles, bacteria, synthetic polymers, and biopolymers. Characterizing the size and shape of analytes with such morphology, however, presents a challenge, due chiefly to the ease with which the "strings" can be broken during chromatographic analysis or to the paucity of information obtained from the benchmark microscopy and off-line light scattering methods. Here, we address this challenge with multidetector hydrodynamic chromatography (HDC), which has the ability to determine, simultaneously, the size, shape, and compactness and their distributions of string-of-pearls samples. We present the quadruple-detector HDC analysis of colloidal string-of-pearls silica, employing static multiangle and quasielastic light scattering, differential viscometry, and differential refractometry as detection methods. The multidetector approach shows a sample that is broadly polydisperse in both molar mass and size, with strings ranging from two to five particles, but which also contains a high concentration of single, unattached "pearls". Synergistic combination of the various size parameters obtained from the multiplicity of detectors employed shows that the strings with higher degrees of polymerization have a shape similar to the theory-predicted shape of a Gaussian random coil chain of nonoverlapping beads, while the strings with lower degrees of polymerization have a prolate ellipsoidal shape. The HDC technique is contrasted experimentally with multidetector size-exclusion chromatography, where, even under extremely gentle conditions, the strings still degraded during analysis. Such degradation is shown to be absent in HDC, as evidenced by the fact that the molar mass and radius of gyration obtained by HDC with multiangle static light scattering detection (HDC/MALS) compare quite favorably to those determined by off-line MALS analysis under otherwise identical conditions. The multidetector HDC results were also comparable to those obtained by transmission electron microscopy (TEM). Unlike off-line MALS or TEM, however, multidetector HDC is able to provide complete particle analysis based on the molar mass, size, shape, and compactness and their distributions for the entire sample population in less than 20 min. © 2011 American Chemical Society

Identification of Foreign Particles in Human Tissues using Raman Microscopy.

PubMed

Campion, Alan; Smith, Kenneth J; Fedulov, Alexey V; Gregory, David; Fan, Yuwei; Godleski, John J

2018-06-12

The precise identification of foreign particles in tissue for patient care and research has been studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction. The goal of this study was to unambiguously identify particles in tissues using a combina-tion of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicron spatial resolution. We designed a model system of stained and unstained cells that contained birefringent talc particles, and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra ob-tained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely as-signed to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micron in diameter. Among commonly used coverslip mounting media, Cytoseal 60 is recommended; Permount was unacceptable due to intense background interference. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

Hyperspectral microscopy to identify foodborne bacteria with optimum lighting source

USDA-ARS?s Scientific Manuscript database

Hyperspectral microscopy is an emerging technology for rapid detection of foodborne pathogenic bacteria. Since scattering spectral signatures from hyperspectral microscopic images (HMI) vary with lighting sources, it is important to select optimal lights. The objective of this study is to compare t...

Remineralization Potential of Three Tooth Pastes on Enamel Caries.

PubMed

Singhal, Rajnish K; Rai, Balwant

2017-08-15

Different formulations of dentifrices exist in the market. Usually, single toothpaste is used by all family members including children. There is a big concern of fluoride ingestion with the toothpaste containing high fluoride content in children. Recently, new toothpaste (including toothpaste) with remineralization potential without fluoride content has been formulated. There is an urgent need to compare remineralization potential of this new formulation with the exiting dentifrices. Therefore, the present study has been undertaken to assess and compare the remineralization potential of three dentifrices with different compositions on artificially induced carious lesions in vitro by using scanning electron microscopy and polarised light microscopy. The present in vitro study was conducted on 21 healthy extracted primary central incisor teeth surfaces, which were divided into three groups and were treated by three different dentifrices. Artificial demineralization was followed by remineralization using dentifrice slurry as per the group distribution. All the samples were studied for remineralization by using scanning electron microscopy and polarised light microscopy. Data were analysed using SPSS version 11 software. A significant difference was found between the remineralization potential of incudent toothpaste and other toothpaste groups based on the analysis of polarised light microscopy and stereomicroscope. The remineralizing ability of incudent toothpaste for artificial enamel lesions was found to be significantly higher than that of Colgate® and Crest toothpaste. The limitations of this study include, being a short term study, low sample size and in vitro experiment. incudent toothpaste has exhibited a higher remineralizing potential as compared to fluoride based toothpaste in our study.

Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

PubMed

Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

2013-12-01

The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

PubMed Central

Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

2014-01-01

In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM. PMID:24876996

You can't measure what you can't see - detectors for microscopies

NASA Astrophysics Data System (ADS)

Denes, Peter

For centuries, the human eye has been the imaging detector of choice thanks to its high sensitivity, wide dynamic range, and direct connection to a built-in data recording and analysis system. The eye, however, is limited to visible light, which excludes microscopies with electrons and X-rays, and the built-in recording system stores archival information at very low rates. The former limitation has been overcome by ``indirect'' detectors, which convert probe particles to visible light, and the latter by a variety of recording techniques, from photographic film to semiconductor-based imagers. Semiconductor imagers have been used for decades as ``direct'' detectors in particle physics, and almost as long for hard X-rays. For soft X-ray microscopy, the challenge has been the small signal levels - plus getting the X-rays into the detector itself, given how quickly they are absorbed in inert layers. For electron microscopy, the challenge has been reconciling detector spatial resolution and pixel count with the large multiple scattering of electrons with energies used for microscopy. Further, a high recording rate (``movies'' rather than ``snapshots'') enables time-resolved studies, time-dependent corrections, shot-by-shot experiments and scanning techniques - at the expense of creating large data volumes. This talk will discuss solutions to these challenges, as well as an outlook towards future developments.

Quantitative light and scanning electron microscopy of ferret sperm.

PubMed

Van der Horst, G; Curry, P T; Kitchin, R M; Burgess, W; Thorne, E T; Kwiatkowski, D; Parker, M; Atherton, R W

1991-11-01

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

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

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.

3D imaging of cleared human skin biopsies using light-sheet microscopy: A new way to visualize in-depth skin structure.

PubMed

Abadie, S; Jardet, C; Colombelli, J; Chaput, B; David, A; Grolleau, J-L; Bedos, P; Lobjois, V; Descargues, P; Rouquette, J

2018-05-01

Human skin is composed of the superimposition of tissue layers of various thicknesses and components. Histological staining of skin sections is the benchmark approach to analyse the organization and integrity of human skin biopsies; however, this approach does not allow 3D tissue visualization. Alternatively, confocal or two-photon microscopy is an effective approach to perform fluorescent-based 3D imaging. However, owing to light scattering, these methods display limited light penetration in depth. The objectives of this study were therefore to combine optical clearing and light-sheet fluorescence microscopy (LSFM) to perform in-depth optical sectioning of 5 mm-thick human skin biopsies and generate 3D images of entire human skin biopsies. A benzyl alcohol and benzyl benzoate solution was used to successfully optically clear entire formalin fixed human skin biopsies, making them transparent. In-depth optical sectioning was performed with LSFM on the basis of tissue-autofluorescence observations. 3D image analysis of optical sections generated with LSFM was performed by using the Amira ® software. This new approach allowed us to observe in situ the different layers and compartments of human skin, such as the stratum corneum, the dermis and epidermal appendages. With this approach, we easily performed 3D reconstruction to visualise an entire human skin biopsy. Finally, we demonstrated that this method is useful to visualise and quantify histological anomalies, such as epidermal hyperplasia. The combination of optical clearing and LSFM has new applications in dermatology and dermatological research by allowing 3D visualization and analysis of whole human skin biopsies. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Studying aerosol light scattering based on aspect ratio distribution observed by fluorescence microscope.

PubMed

Li, Li; Zheng, Xu; Li, Zhengqiang; Li, Zhanhua; Dubovik, Oleg; Chen, Xingfeng; Wendisch, Manfred

2017-08-07

Particle shape is crucial to the properties of light scattered by atmospheric aerosol particles. A method of fluorescence microscopy direct observation was introduced to determine the aspect ratio distribution of aerosol particles. The result is comparable with that of the electron microscopic analysis. The measured aspect ratio distribution has been successfully applied in modeling light scattering and further in simulation of polarization measurements of the sun/sky radiometer. These efforts are expected to improve shape retrieval from skylight polarization by using directly measured aspect ratio distribution.

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

Mitrofanov, Oleg; Han, Zhanghua; Ding, Fei

(THz) plasmonic double-metal resonators enable enhanced light-matter coupling by utilizing strong localization of the resonant field. The closed resonator design however restricts investigations of the light-matter interaction effects. We propose and demonstrate a method for spatial mapping and spectroscopic analysis of the internal resonant THz fields in plasmonic double-metal THz resonators. We use the aperture-type scanning near-field THz time-domain microscopy and the concept of image charges to probe the THz fields confined within the resonator. The experimental method opens doors to studies of light-matter coupling in deeply sub-wavelength volumes at THz frequencies.

Redescription of Oswaldocruzia chambrieri (Strongylida: Molineidae) from Rhinella margaritifera (Anura: Bufonidae) in Caxiuanã National Forest, Brazil.

PubMed

Willkens, Yuri; Maldonado, Arnaldo; Dos Santos, Jeannie Nascimento; Maschio, Gleomar Fabiano; de Vasconcelos Melo, Francisco Tiago

2016-09-01

Oswaldocruzia chambrieri Ben Slimane et Durette-Desset, 1993 is redescribed from specimens collected from the small intestine of the South American common toad, Rhinella margaritifera, from Caxiuanã National Forest in Pará, Brazil, using light and scanning microscopy and molecular analysis of Cytochrome Oxidase I (COI) - coding regions of DNA. The discovered nematodes are characterized by a type III caudal bursa with two papillae, rays 4 with a median groove, and spicules divided into a blade, a shoe and a fork. Cervical alae are absent, the cephalic vesicle is divided into two portions, and the synlophe has low ridges without chitinous supports. The present study establishes the Caxiuanã National Forest as a new location for O. chambrieri, which had previously been reported as a parasite of R. margaritifera in Ecuador, uses light microscopy and scanning electron microscopy (SEM) to identify new morphological characters of the species and represents the second molecular sequence deposited for the Oswaldocruzia genus.

Quantitative assessment of neural outgrowth using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Lee, Young Jae; Cintora, Pati; Arikkath, Jyothi; Akinsola, Olaoluwa; Kandel, Mikhail; Popescu, Gabriel; Best-Popescu, Catherine

2017-06-01

Optimal growth as well as branching of axons and dendrites is critical for the nervous system function. Neuritic length, arborization, and growth rate determine the innervation properties of neurons and define each cell's computational capability. Thus, to investigate the nervous system function, we need to develop methods and instrumentation techniques capable of quantifying various aspects of neural network formation: neuron process extension, retraction, stability, and branching. During the last three decades, fluorescence microscopy has yielded enormous advances in our understanding of neurobiology. While fluorescent markers provide valuable specificity to imaging, photobleaching, and photoxicity often limit the duration of the investigation. Here, we used spatial light interference microscopy (SLIM) to measure quantitatively neurite outgrowth as a function of cell confluence. Because it is label-free and nondestructive, SLIM allows for long-term investigation over many hours. We found that neurons exhibit a higher growth rate of neurite length in low-confluence versus medium- and high-confluence conditions. We believe this methodology will aid investigators in performing unbiased, nondestructive analysis of morphometric neuronal parameters.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

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

King, John T.; Granick, Steve

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Inter-comparison of unrelated fiber evidence.

PubMed

Houck, Max M

2003-08-12

The foreign textile fibers recovered from one item of evidence from each of 20 unrelated crimes in three categories (bank robbery, kidnapping, and homicide) were cross-compared. The items of evidence were scraped to remove the trace evidence and a sample of the collected fibers was examined using a standard scheme of analysis. The fibers were examined with light microscopy (including polarized light microscopy), fluorescence microscopy, and microspectrophotometry. The fibers were divided into natural and manufactured groups and then categorized by color and generic (polymer) class. Cross-comparing all 2083 fibers resulted in 2,168,403 comparisons, after removing duplicate (same fiber) comparisons. Colorless and denim fibers were excluded from this study. No two fibers were found to exhibit the same microscopic characteristics and analytical properties. Therefore, it is rare to find two unrelated items that have foreign fibers that are analytically indistinguishable. These results corroborate other population studies conducted in Europe and target fiber studies conducted both in the US and in Europe.

Operating organic light-emitting diodes imaged by super-resolution spectroscopy

DOE PAGES

King, John T.; Granick, Steve

2016-06-21

Super-resolution stimulated emission depletion (STED) microscopy is adapted here for materials characterization that would not otherwise be possible. With the example of organic light-emitting diodes (OLEDs), spectral imaging with pixel-by-pixel wavelength discrimination allows us to resolve local-chain environment encoded in the spectral response of the semi-conducting polymer, and correlate chain packing with local electroluminescence by using externally applied current as the excitation source. We observe nanoscopic defects that would be unresolvable by traditional microscopy. They are revealed in electroluminescence maps in operating OLEDs with 50 nm spatial resolution. We find that brightest emission comes from regions with more densely packedmore » chains. Conventional microscopy of an operating OLED would lack the resolution needed to discriminate these features, while traditional methods to resolve nanoscale features generally cannot be performed when the device is operating. As a result, this points the way towards real-time analysis of materials design principles in devices as they actually operate.« less

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

PubMed

Malucelli, Emil; Procopio, Alessandra; Fratini, Michela; Gianoncelli, Alessandra; Notargiacomo, Andrea; Merolle, Lucia; Sargenti, Azzurra; Castiglioni, Sara; Cappadone, Concettina; Farruggia, Giovanna; Lombardo, Marco; Lagomarsino, Stefano; Maier, Jeanette A; Iotti, Stefano

2018-01-01

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

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

Organic petrology of selected oil shale samples from lower Carboniferous Albert Formation, New Brunswick, Canada

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

Kalkreuth, W.; Macauley, G.

1984-04-01

Incident light microscopy was used to describe maturation and composition of organic material in oil shale samples from the Lower Carboniferous Albert Formation of New Brunswick. The maturation level was determined in normal (white) light by measuring vitrinite reflectance and in fluorescent light by measuring fluorescence spectral of alginite B. Results indicate low to intermediate maturation for all of the samples. Composition was determined by maceral analysis. Alginite B is the major organic component in all samples having significant oil potential. Oil yields obtained from the Fischer Assay process, and oil and gas potentials from Rock-Eval analyses correlate to themore » amounts of alginite B and bituminite determined in the samples. In some of the samples characterized by similar high concentrations of alginite B, decrease in Fischer Assay yields and oil and gas potentials is related to an increase in maturation, as expected by increase in the fluorescence parameter lambda/sub max/ and red/green quotient of alginite B. Incident light microscopy, particularly with fluorescent light, offers a valuable tool for the identification of the organic matter in oil shales and for the evaluation of their oil and gas potentials.« less

Light-responsive micelles of spiropyran initiated hyperbranched polyglycerol for smart drug delivery.

PubMed

Son, Suhyun; Shin, Eeseul; Kim, Byeong-Su

2014-02-10

Light-responsive polymeric micelles have emerged as site-specific and time-controlled systems for advanced drug delivery. Spiropyran (SP), a well-known photochromic molecule, was used to initiate the ring-opening multibranching polymerization of glycidol to afford a series of hyperbranched polyglycerols (SP-hb-PG). The micelle assembly and disassembly were induced by an external light source owing to the reversible photoisomerization of hydrophobic SP to hydrophilic merocyanine (MC). Transmission electron microscopy, atomic force microscopy, UV/vis spectroscopy, and dynamic light scattering demonstrated the successful assembly and disassembly of SP-hb-PG micelles. In addition, the critical micelle concentration (CMC) was determined through the fluorescence analysis of pyrene to confirm the amphiphilicity of respective SP-hb-PGn (n = 15, 29, and 36) micelles, with CMC values ranging from 13 to 20 mg/L, which is correlated to the length of the polar polyglycerol backbone. Moreover, the superior biocompatibility of the prepared SP-hb-PG was evaluated using WI-38 cells and HeLa cells, suggesting the prospective applicability of the micelles in smart drug delivery systems.

ZnO/Ag/CdO nanocomposite for visible light-induced photocatalytic degradation of industrial textile effluents.

PubMed

Saravanan, R; Mansoob Khan, M; Gupta, Vinod Kumar; Mosquera, E; Gracia, F; Narayanan, V; Stephen, A

2015-08-15

A ternary ZnO/Ag/CdO nanocomposite was synthesized using thermal decomposition method. The resulting nanocomposite was characterized by X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy, UV-Vis spectroscopy, and X-ray photoelectron spectroscopy. The ZnO/Ag/CdO nanocomposite exhibited enhanced photocatalytic activity under visible light irradiation for the degradation of methyl orange and methylene blue compared with binary ZnO/Ag and ZnO/CdO nanocomposites. The ZnO/Ag/CdO nanocomposite was also used for the degradation of the industrial textile effluent (real sample analysis) and degraded more than 90% in 210 min under visible light irradiation. The small size, high surface area and synergistic effect in the ZnO/Ag/CdO nanocomposite is responsible for high photocatalytic activity. These results also showed that the Ag nanoparticles induced visible light activity and facilitated efficient charge separation in the ZnO/Ag/CdO nanocomposite, thereby improving the photocatalytic performance. Copyright © 2015 Elsevier Inc. All rights reserved.

Atomic Force Microscopy of Photosystem II and Its Unit Cell Clustering Quantitatively Delineate the Mesoscale Variability in Arabidopsis Thylakoids

PubMed Central

Onoa, Bibiana; Schneider, Anna R.; Brooks, Matthew D.; Grob, Patricia; Nogales, Eva; Geissler, Phillip L.; Niyogi, Krishna K.; Bustamante, Carlos

2014-01-01

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arrays according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets. PMID:25007326

Correlative Instrumental Neutron Activation Analysis, Light Microscopy, Transmission Electron Microscopy, and X-ray Microanalysis for Qualitative and Quantitative Detection of Colloidal Gold Spheres in Biological Specimens

NASA Astrophysics Data System (ADS)

Hillyer, Julián F.; Albrecht, Ralph M.

1998-10-01

: Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.

Atomic Force Microscopy of Photosystem II and Its Unit Cell Clustering Quantitatively Delineate the Mesoscale Variability in Arabidopsis Thylakoids

DOE PAGES

Onoa, Bibiana; Schneider, Anna R.; Brooks, Matthew D.; ...

2014-07-09

Photoautotrophic organisms efficiently regulate absorption of light energy to sustain photochemistry while promoting photoprotection. Photoprotection is achieved in part by triggering a series of dissipative processes termed non-photochemical quenching (NPQ), which depend on the re-organization of photosystem (PS) II supercomplexes in thylakoid membranes. Using atomic force microscopy, we characterized the structural attributes of grana thylakoids from Arabidopsis thaliana to correlate differences in PSII organization with the role of SOQ1, a recently discovered thylakoid protein that prevents formation of a slowly reversible NPQ state. We developed a statistical image analysis suite to discriminate disordered from crystalline particles and classify crystalline arraysmore » according to their unit cell properties. Through detailed analysis of the local organization of PSII supercomplexes in ordered and disordered phases, we found evidence that interactions among light-harvesting antenna complexes are weakened in the absence of SOQ1, inducing protein rearrangements that favor larger separations between PSII complexes in the majority (disordered) phase and reshaping the PSII crystallization landscape. The features we observe are distinct from known protein rearrangements associated with NPQ, providing further support for a role of SOQ1 in a novel NPQ pathway. The particle clustering and unit cell methodology developed here is generalizable to multiple types of microscopy and will enable unbiased analysis and comparison of large data sets.« less

Evaluation of agave fiber delignification by means of microscopy techniques and image analysis.

PubMed

Hernández-Hernández, Hilda M; Chanona-Pérez, Jorge J; Calderón-Domínguez, Georgina; Perea-Flores, María J; Mendoza-Pérez, Jorge A; Vega, Alberto; Ligero, Pablo; Palacios-González, Eduardo; Farrera-Rebollo, Reynold R

2014-10-01

Recently, the use of different types of natural fibers to produce paper and textiles from agave plants has been proposed. Agave atrovirens can be a good source of cellulose and lignin; nevertheless, the microstructural changes that happen during delignification have scarcely been studied. The aim of this work was to study the microstructural changes that occur during the delignification of agave fibers by means of microscopy techniques and image analysis. The fibers of A. atrovirens were obtained from leaves using convective drying, milling, and sieving. Fibers were processed using the Acetosolv pulping method at different concentrations of acetic acid; increasing acid concentration promoted higher levels of delignification, structural damage, and the breakdown of fiber clumps. Delignification followed by spectrometric analysis and microstructural studies were carried out by light, confocal laser scanning and scanning electron microscopy and showed that the delignification process follows three stages: initial, bulk, and residual. Microscopy techniques and image analysis were efficient tools for microstructural characterization during delignification of agave fibers, allowing quantitative evaluation of the process and the development of linear prediction models. The data obtained integrated numerical and microstructural information that could be valuable for the study of pulping of lignocellulosic materials.

Advanced light microscopy core facilities: Balancing service, science and career.

PubMed

Ferrando-May, Elisa; Hartmann, Hella; Reymann, Jürgen; Ansari, Nariman; Utz, Nadine; Fried, Hans-Ulrich; Kukat, Christian; Peychl, Jan; Liebig, Christian; Terjung, Stefan; Laketa, Vibor; Sporbert, Anje; Weidtkamp-Peters, Stefanie; Schauss, Astrid; Zuschratter, Werner; Avilov, Sergiy

2016-06-01

Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463-479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC. © 2016 The Authors Microscopy Research and Technique Published by Wiley Periodicals, Inc.

In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

NASA Astrophysics Data System (ADS)

Paudel, Hari P.; Jung, Yookyung; Raphael, Anthony; Alt, Clemens; Wu, Juwell; Runnels, Judith; Lin, Charles P.

2018-02-01

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient's blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

Light Microscopy's New Jobs

NASA Astrophysics Data System (ADS)

Ritsch-Marte, Monika

2009-04-01

300 years since the first glimpse through the earliest microscopes, light microscopy is still an active field of research, breaking new frontiers in optical imaging and even becoming a means of mechanical manipulation of microparticles.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Analysis of thick brain sections by obverse-reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain.

PubMed

Glaser, E M; Van der Loos, H

1981-08-01

Exceptionally clear Golgi-Nissl sections of 300 micron thickness have been morphometrically studied by light microscopy using oil immersion objectives. The clarity results from a new variation of a staining procedure that combines Golgi and Nissl images in one section. A viewing technique has been developed that permits a histologic preparation to be examined from its obverse (or normally viewed) side and its reverse (or under) side. The technique was designed for use with a computer microscope but can be employed with any light microscope whose stage position can be measured within 100 micron. Sections thicker than 300 micron can be studied dependent on the working distance of the objective lens, provided that the clarity of the material permits it.

Comparisons of cotton maturity and fineness measurements (Cottonscope, AFIS, HVI™)

USDA-ARS?s Scientific Manuscript database

The Cottonscope, a new instrument for fiber maturity (MR) and fineness, utilizes polarized light microscopy and image analysis to measure longitudinal, weighted fiber snippets in water. Interest has been expressed by the Commercial Standardization of Instrument Testing of Cotton (CSITC) on the pote...

Green Synthesis and Characterization of SmVO4 Nanoparticles in the Presence of Carbohydrates As Capping Agents with Investigation of Visible-Light Photocatalytic Properties

NASA Astrophysics Data System (ADS)

Eghbali-Arani, Mohammad; Sobhani-Nasab, Ali; Rahimi-Nasrabadi, Mehdi; Pourmasoud, Saeid

2018-03-01

SmVO4 nanoparticles were synthesized through a fast and simple procedure (green method). The effects of three parameters including temperature, type of capping agent, and concentration on the size and morphology behavior of SmVO4 nanoparticles were explored. The analysis of SmVO4 nanoparticles was performed through some techniques including, Fourier transform infrared spectroscopy, x-ray diffraction, energy dispersive x-ray microanalysis, scanning electron microscopy, transmission electron microscopy, thermogravimetry, differential thermal analysis, ultraviolet-visible spectroscopy, and vibrating sample magnetometers. The study of photocatalytic behaviour of the SmVO4 nanoparticles in various conditions has been carried out. The impacts of different factors such as dosage, grain size, and kind of pollutant (methylene blue = MB and methyl orange = MO) on the photocatalytic property of SmVO4 nanoparticles were assessed. The photocatalytic activities of SmVO4 catalysts were studied for the degradation of dye under visible light (λ > 400 nm).

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

PubMed Central

Zhao, Ziqing W.; Roy, Rahul; Gebhardt, J. Christof M.; Suter, David M.; Chapman, Alec R.; Xie, X. Sunney

2014-01-01

Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells. PMID:24379392

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

NASA Astrophysics Data System (ADS)

Pirnstill, Casey W.; Coté, Gerard L.

2015-08-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform.

Malaria Diagnosis Using a Mobile Phone Polarized Microscope

PubMed Central

Pirnstill, Casey W.; Coté, Gerard L.

2015-01-01

Malaria remains a major global health burden, and new methods for low-cost, high-sensitivity, diagnosis are essential, particularly in remote areas with low-resource around the world. In this paper, a cost effective, optical cell-phone based transmission polarized light microscope system is presented for imaging the malaria pigment known as hemozoin. It can be difficult to determine the presence of the pigment from background and other artifacts, even for skilled microscopy technicians. The pigment is much easier to observe using polarized light microscopy. However, implementation of polarized light microscopy lacks widespread adoption because the existing commercial devices have complicated designs, require sophisticated maintenance, tend to be bulky, can be expensive, and would require re-training for existing microscopy technicians. To this end, a high fidelity and high optical resolution cell-phone based polarized light microscopy system is presented which is comparable to larger bench-top polarized microscopy systems but at much lower cost and complexity. The detection of malaria in fixed and stained blood smears is presented using both, a conventional polarized microscope and our cell-phone based system. The cell-phone based polarimetric microscopy design shows the potential to have both the resolution and specificity to detect malaria in a low-cost, easy-to-use, modular platform. PMID:26303238

Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

PubMed

Adnet, F A O; Anjos, D H S; Menezes-Oliveira, A; Lanfredi, R M

2009-04-01

Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique.

Using Stage- and Slit-Scanning to Improve Contrast and Optical Sectioning in Dual-View Inverted Light Sheet Microscopy (diSPIM)

PubMed Central

KUMAR, ABHISHEK; CHRISTENSEN, RYAN; GUO, MIN; CHANDRIS, PANOS; DUNCAN, WILLIAM; WU, YICONG; SANTELLA, ANTHONY; MOYLE, MARK; WINTER, PETER W.; COLÓN-RAMOS, DANIEL; BAO, ZHIRONG; SHROFF, HARI

2017-01-01

Dual-view inverted selective plane illumination microscopy (diSPIM) enables high-speed, long-term, fourdimensional (4D) imaging with isotropic spatial resolution. It is also compatible with conventional sample mounting on glass coverslips. However, broadening of the light sheet at distances far from the beam waist and sample-induced scattering degrades diSPIM contrast and optical sectioning. We describe two simple improvements that address both issues and entail no additional hardware modifications to the base diSPIM. First, we demonstrate improved diSPIM sectioning by keeping the light sheet and detection optics stationary, and scanning the sample through the stationary light sheet (rather than scanning the broadening light sheet and detection plane through the stationary sample, as in conventional diSPIM). This stage-scanning approach allows a thinner sheet to be used when imaging laterally extended samples, such as fixed microtubules or motile mitochondria in cell monolayers, and produces finer contrast than does conventional diSPIM. We also used stage-scanning diSPIM to obtain high-quality, 4D nuclear datasets derived from an uncompressed nematode embryo, and performed lineaging analysis to track 97% of cells until twitching. Second, we describe the improvement of contrast in thick, scattering specimens by synchronizing light-sheet synthesis with the rolling, electronic shutter of our scientific complementary metal-oxide-semiconductor (sCMOS) detector. This maneuver forms a virtual confocal slit in the detection path, partially removing out-of-focus light. We demonstrate the applicability of our combined stage- and slit-scanning-methods by imaging pollen grains and nuclear and neuronal structures in live nematode embryos. All acquisition and analysis code is freely available online. PMID:27638693

Rat animal model for preclinical testing of microparticle urethral bulking agents.

PubMed

Mann-Gow, Travis K; Blaivas, Jerry G; King, Benjamin J; El-Ghannam, Ahmed; Knabe, Christine; Lam, Michael K; Kida, Masatoshi; Sikavi, Cameron S; Plante, Mark K; Krhut, Jan; Zvara, Peter

2015-04-01

To develop an economic, practical and readily available animal model for preclinical testing of urethral bulking therapies, as well as to establish feasible experimental methods that allow for complete analysis of hard microparticle bulking agents. Alumina ceramic beads suspended in hyaluronic acid were injected into the proximal urethra of 15 female rats under an operating microscope. We assessed overall lower urinary tract function, bulking material intraurethral integrity and local host tissue response over time. Microphotographs were taken during injection and again 6 months postoperatively, before urethral harvest. Urinary flow rate and voiding frequency were assessed before and after injection. At 6 months, the urethra was removed and embedded in resin. Hard tissue sections were cut using a sawing microtome, and processed for histological analysis using scanning electron microscopy, light microscopy and immunohistochemistry. Microphotographs of the urethra showed complete volume retention of the bulking agent at 6 months. There was no significant difference between average urinary frequency and mean urinary flow rate at 1 and 3 months postinjection as compared with baseline. Scanning electron microscopy proved suitable for evaluation of microparticle size and integrity, as well as local tissue remodeling. Light microscopy and immunohistochemistry allowed for evaluation of an inflammatory host tissue reaction to the bulking agent. The microsurgical injection technique, in vivo physiology and novel hard tissue processing for histology, described in the present study, will allow for future comprehensive preclinical testing of urethral bulking therapy agents containing microparticles made of a hard material. © 2015 The Japanese Urological Association.

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

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.

Crawler Solids Unknown Analysis

NASA Technical Reports Server (NTRS)

Frandsen, Athela

2016-01-01

Crawler Transporter (CT) #2 has been undergoing refurbishment to carry the Space Launch System (SLS). After returning to normal operation, multiple filters of the gear box lubrication system failed/clogged and went on bypass during a test run to the launch pad. Analysis of the filters was done in large part with polarized light microscopy (PLM) to identify the filter contaminates and the source of origin.

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

Singh, Paviter; Kaur, Manpreet; Singh, Bikramjeet

Boron-carbon core shell structures have been synthesized by solvo-thermal synthesis route. The synthesized material is highly pure. X-ray diffraction analysis confirms the reduction of reactants in to boron and carbon. Scanning Electron Microscopy (SEM) analysis showed that the shell is uniform with average thickness of 340 nm. Photo luminescence studies showed that the material is blue light emitting with CIE color coordinates: x=0.16085, y=0.07554.

Elucidation of wear mechanisms by ferrographic analysis

NASA Technical Reports Server (NTRS)

Jones, W. R., Jr.

1981-01-01

The use of ferrographic analysis in conjunction with light and scanning electron microscopy is described for the elucidation of wear mechanisms taking place in operating equipment. Example of adhesive wear, abrasive wear, corrosive wear, rolling element fatigue, lubricant breakdown, and other wear modes are illustrated. In addition, the use of magnetic solutions to precipitate nonmagnetic debris from aqueous and nonaqueous fluids is described.

ONR (Office of Naval Research) Far East Scientific Information Bulletin. Volume 13, Number 4, October-December 1988

DTIC Science & Technology

1988-12-01

by light finger pressure; and surface shaded or rubbed by soft pencil, charcoal, or crayon. Anglers initiated this custom as a means of recording...related to the barium titanate or simple nects, and mixers. Some of these applica- perovskite unit cell (Figure 1). In this struc- tions can be...dispersive spectroscopy (EDS), Target-~ microprobe analysis, and x-ray diffraction R~orOC(XRD). MagnetsOptical microscopy with polarized light

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.

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

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.

Microscopic analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) embryonic development before and after treatment with azadirachtin, lufenuron, and deltamethrin.

PubMed

Correia, Alicely A; Wanderley-Teixeira, Valéria; Teixeira, Alvaro A C; Oliveira, José V; Gonçalves, Gabriel G A; Cavalcanti, MaríIia G S; Brayner, Fábio A; Alves, Luiz C

2013-04-01

The botanical insecticides, growth regulators, and pyrethroids have an effect on the biology of Spodoptera frugiperda (Smith). However, no emphasis has been given to the effect of these insecticides on embryonic development of insects, in histological level. Thus, this research aimed to examine by light and scanning electron microscopy S. frugiperda eggs and to describe the embryonic development, before and after immersion treatment, using commercial concentrations and lower concentrations than commercial ones, of the compounds lufenuron (Match), azadirachtin (AzaMax), and deltamethrin (Decis-positive control). For light microscopy semithin sections of eggs were used, and for scanning electron microscopy, images of the surface of eggs, treated and untreated with insecticides. The morphological characteristics of S. frugiperda eggs, in general, were similar to those described in the literature for most of the insects in the order Lepidoptera. Spherical eggs slightly flattened at the poles, with chorion, yolk, vitelline membrane, and embryo formation. In both microscopic analysis, we observed that insecticides acted immediately and independent of concentration, resulting absence, or incomplete embryo, presented yolk granules widely dispersed, without vitellophage formation, chorion disintegration, disorganized blastoderm, presenting vacuoles, yolk region with amorphous cells, and formation of completely uncharacterized appendages. Thus, we conclude that the compounds lufenuron and azadirachtin interfere on S. frugiperda embryonic development.

The Interior Analysis and 3-D Reconstruction of Internally-Mixed Light-Absorbing Atmospheric Particles

NASA Astrophysics Data System (ADS)

Conny, J. M.; Collins, S. M.; Anderson, I.; Herzing, A.

2010-12-01

Carbon-containing atmospheric particles may either absorb solar or outgoing long-wave radiation or scatter solar radiation, and thus, affect Earth’s radiative balance in multiple ways. Light-absorbing carbon that is common in urban air particles such as industrial coke dust, road dust, and diesel soot, often exists in the same particle with other phases that contain, for example, aluminum, calcium, iron, and sulfur. While the optical properties of atmospheric particles in general depend on overall particle size and shape, the inhomogeneity of chemical phases within internally-mixed particles may also greatly affect particle optical properties. In this study, a series of microscopic approaches were used to identify individual light-absorbing coarse-mode particles and to assess their interior structure and composition. Particle samples were collected in 2004 from one of the U.S. EPA’s Los Angeles Particulate Matter Supersites, and were likely affected substantially by road dust and construction dust. First, bright-field and dark-field light microscopy and computer-controlled scanning electron microscopy (SEM) with energy-dispersive x-ray spectroscopy (EDX) were used to distinguish predominantly light-absorbing carbonaceous particles from other particle types such as mineral dust, sea salt, and brake wear. Second, high-resolution SEM-EDX elemental mapping of individual carbonaceous particles was used to select particles with additional elemental phases that exhibited spatial inhomogeneity. Third, focused ion-beam SEM (FIB-SEM) with EDX was used to slice through selected particles to expose interior surfaces and to determine the spatial distribution of element phases throughout the particles. Fourth, study of the interior phases of a particle was augmented by the transmission electron microscopy (TEM) of a thin section of the particle prepared by FIB-SEM. Here, electron energy loss spectroscopy with TEM was used to study chemical bonding in the carbonaceous phase. Finally, automated serial slicing and imaging in the FIB-SEM generated a stack of secondary electron images of the particles’ interior surfaces that allowed for the 3-D reconstruction of the particles, a process known as FIB tomography. Interior surface of light-absorbing carbonaceous particle from FIB-SEM analysis.

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.

eduSPIM: Light Sheet Microscopy in the Museum

PubMed Central

Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light Sheet Microscopy in the Museum Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. Design Principles of an Educational Light Sheet Microscope To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The eduSPIM Design Is Tailored Easily to Fit Numerous Applications The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided. PMID:27560188

Electrochromic WO[subscript 3] Films: Nanotechnology Experiments in Instrumental Analysis and Physical Chemistry Laboratories

ERIC Educational Resources Information Center

Hepel, Maria

2008-01-01

This experiment teaches students the methodology of investigating novel properties of materials using new instrumental techniques: atomic force microscopy (AFM), electrochemical quartz crystal nanobalance (EQCN), voltammetric techniques (linear potential scan and chronoamperometry), and light reflectance measurements. The unique capabilities of…

Detection of internal fields in double-metal terahertz resonators

DOE PAGES

Mitrofanov, Oleg; Han, Zhanghua; Ding, Fei; ...

2017-02-06

(THz) plasmonic double-metal resonators enable enhanced light-matter coupling by utilizing strong localization of the resonant field. The closed resonator design however restricts investigations of the light-matter interaction effects. We propose and demonstrate a method for spatial mapping and spectroscopic analysis of the internal resonant THz fields in plasmonic double-metal THz resonators. We use the aperture-type scanning near-field THz time-domain microscopy and the concept of image charges to probe the THz fields confined within the resonator. The experimental method opens doors to studies of light-matter coupling in deeply sub-wavelength volumes at THz frequencies.

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis.

PubMed

Bonnell, B S; Larabell, C; Chandler, D E

1993-06-01

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a three-dimensional network of interconnected fibers extending nearly 50 microns from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a three-dimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50-60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix.

Rapid reconstruction of 3D neuronal morphology from light microscopy images with augmented rayburst sampling.

PubMed

Ming, Xing; Li, Anan; Wu, Jingpeng; Yan, Cheng; Ding, Wenxiang; Gong, Hui; Zeng, Shaoqun; Liu, Qian

2013-01-01

Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.

Conjugation of both on-axis and off-axis light in Nipkow disk confocal microscope to increase availability of incoherent light source.

PubMed

Saito, Kenta; Arai, Yoshiyuki; Zhang, Jize; Kobayashi, Kentaro; Tani, Tomomi; Nagai, Takeharu

2011-01-01

Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.

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.

Improving your four-dimensional image: traveling through a decade of light-sheet-based fluorescence microscopy research.

PubMed

Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

2017-06-01

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.

A Photomicrography Primer.

ERIC Educational Resources Information Center

Davidson, Michael W.

1991-01-01

Describes techniques and equipment which allows school microscopes to perform crossed-polarized light microscopy, reflected light microscopy, and photomicrography. Provides information on using chemicals from a high school stockroom to view crystals, viewing integrated circuits, and capturing images on film. Lists possible independent student…

In situ localization of nucleolin in the plant nucleolar matrix.

PubMed

Minguez, A; Moreno Diaz de la Espina, S

1996-01-10

The analysis of isolated nucleolar matrices from onion cells by light and electron microscopy, 2-D separation of proteins, and confocal microscopy has confirmed the existence of an organized nucleolar matrix with a complex protein composition to which are attached the insoluble processing complexes. In the present work, we present evidence from immunoblotting, immunofluorescence, immunogold labeling, and preferential cytochemical staining with bismuth salts that an insoluble fraction of the multifunctional protein nucleolin, is a component of the onion nucleolar matrix, and analyse its ultrastructural distribution in the described domains of the matrix.

A Comparative Study Between Smartphone-Based Microscopy and Conventional Light Microscopy in 1021 Dermatopathology Specimens.

PubMed

Jahan-Tigh, Richard R; Chinn, Garrett M; Rapini, Ronald P

2016-01-01

The incorporation of high-resolution cameras into smartphones has allowed for a variety of medical applications including the use of lens attachments that provide telescopic, macroscopic, and dermatoscopic data, but the feasibility and performance characteristics of such a platform for use in dermatopathology have not been described. To determine the diagnostic performance of a smartphone microscope compared to traditional light microscopy in dermatopathology specimens. A simple smartphone microscope constructed with a 3-mm ball lens was used to prospectively evaluate 1021 consecutive dermatopathology cases in a blinded fashion. Referred, consecutive specimens from the community were evaluated at a single university hospital. The performance characteristics of the smartphone platform were calculated by using conventional light microscopy as the gold standard. The sensitivity and specificity for the diagnosis of melanoma, nonmelanoma skin cancers, and other miscellaneous conditions by the phone microscopy platform, as compared with traditional light microscopy, were calculated. For basal cell carcinoma (n = 136), the sensitivity and specificity of smartphone microscopy were 95.6% and 98.1%, respectively. The sensitivity and specificity for squamous cell carcinoma (n = 94) were 89.4% and 97.3%, respectively. The lowest sensitivity was found in melanoma (n = 15) at 60%, although the specificity was high at 99.1%. The accuracy of diagnosis of inflammatory conditions and other neoplasms was variable. Mobile phone-based microscopy has excellent performance characteristics for the inexpensive diagnosis of nonmelanoma skin cancers in a setting where a traditional microscope is not available.

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy

PubMed Central

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). PMID:29568263

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

PubMed

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Stimulus-evoked outer segment changes in rod photoreceptors

NASA Astrophysics Data System (ADS)

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Lu, Yiming; Gai, Shaoyan; Yao, Xincheng

2016-06-01

Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.

Forensic species identification of elephant (Elephantidae) and giraffe (Giraffidae) tail hair using light microscopy.

PubMed

Yates, Bonnie C; Espinoza, Edgard O; Baker, Barry W

2010-09-01

Here we present methods for distinguishing tail hairs of African elephants (Loxodonta africana), Asian elephants (Elephas maximus), and giraffes (Giraffa camelopardalis) from forensic contexts. Such hairs are commonly used to manufacture jewelry artifacts that are often sold illegally in the international wildlife trade. Tail hairs from these three species are easily confused macroscopically, and morphological methods for distinguishing African and Asian tail hairs have not been published. We used cross section analysis and light microscopy to analyze the tail hair morphology of 18 individual African elephants, 18 Asian elephants, and 40 giraffes. We found that cross-sectional shape, pigment placement, and pigment density are useful morphological features for distinguishing the three species. These observations provide wildlife forensic scientists with an important analytical tool for enforcing legislation and international treaties regulating the trade in elephant parts.

Stimulus-evoked outer segment changes in rod photoreceptors

PubMed Central

Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Lu, Yiming; Gai, Shaoyan; Yao, Xincheng

2016-01-01

Abstract. Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation. PMID:27334933

Visible light photoactivity of Polypropylene coated Nano-TiO2 for dyes degradation in water

PubMed Central

Giovannetti, R.; Amato, C. A. D’; Zannotti, M.; Rommozzi, E.; Gunnella, R.; Minicucci, M.; Di Cicco, A.

2015-01-01

The use of Polypropylene as support material for nano-TiO2 photocatalyst in the photodegradation of Alizarin Red S in water solutions under the action of visible light was investigated. The optimization of TiO2 pastes preparation using two commercial TiO2, Aeroxide P-25 and Anatase, was performed and a green low-cost dip-coating procedure was developed. Scanning electron microscopy, Atomic Force Microscopy and X-Ray Diffraction analysis were used in order to obtain morphological and structural information of as-prepared TiO2 on support material. Equilibrium and kinetics aspects in the adsorption and successive photodegradation of Alizarin Red S, as reference dye, are described using polypropylene-TiO2 films in the Visible/TiO2/water reactor showing efficient dyes degradation. PMID:26627118

Rapid cotton maturity and fineness measurements using the Cottonscope®

USDA-ARS?s Scientific Manuscript database

Much interest has been shown in new and rapid measurements of fiber maturity and fineness. The Cottonscope is a new instrument for fiber maturity and fineness, using a longitudinal measurement of weighted fiber snippets in water by polarized light microscopy and image analysis. A program was implem...

Effect of ferroelastic domain pattern changes on the EPR spectra in TDM

NASA Astrophysics Data System (ADS)

Zapart, W.; Zapart, M. B.

2011-09-01

This article presents polarized light microscopy studies of the ferroelastic domain structure and the analysis of electron paramagnetic resonance spectra of Cr3+ admixture ions in trigonal double molybdates. The correlation has been found between abnormal EPR lineshape and domain structure in ferroelastic phases of these crystals.

Sub-microsecond-resolution probe microscopy

DOEpatents

Ginger, David; Giridharagopal, Rajiv; Moore, David; Rayermann, Glennis; Reid, Obadiah

2014-04-01

Methods and apparatus are provided herein for time-resolved analysis of the effect of a perturbation (e.g., a light or voltage pulse) on a sample. By operating in the time domain, the provided method enables sub-microsecond time-resolved measurement of transient, or time-varying, forces acting on a cantilever.

Trypanosoma janseni n. sp. (Trypanosomatida: Trypanosomatidae) isolated from Didelphis aurita (Mammalia: Didelphidae) in the Atlantic Rainforest of Rio de Janeiro, Brazil: integrative taxonomy and phylogeography within the Trypanosoma cruzi clade.

PubMed

Lopes, Camila Madeira Tavares; Menna-Barreto, Rubem Figueiredo Sadok; Pavan, Márcio Galvão; Pereira, Mirian Cláudia De Souza; Roque, André Luiz R

2018-01-01

Didelphis spp. are a South American marsupial species that are among the most ancient hosts for the Trypanosoma spp. We characterise a new species (Trypanosoma janseni n. sp.) isolated from the spleen and liver tissues of Didelphis aurita in the Atlantic Rainforest of Rio de Janeiro, Brazil. The parasites were isolated and a growth curve was performed in NNN and Schneider's media containing 10% foetal bovine serum. Parasite morphology was evaluated via light microscopy on Giemsa-stained culture smears, as well as scanning and transmission electron microscopy. Molecular taxonomy was based on a partial region (737-bp) of the small subunit (18S) ribosomal RNA gene and 708 bp of the nuclear marker, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. Maximum likelihood and Bayesian inference methods were used to perform a species coalescent analysis and to generate individual and concatenated gene trees. Divergence times among species that belong to the T. cruzi clade were also inferred. In vitro growth curves demonstrated a very short log phase, achieving a maximum growth rate at day 3 followed by a sharp decline. Only epimastigote forms were observed under light and scanning microscopy. Transmission electron microscopy analysis showed structures typical to Trypanosoma spp., except one structure that presented as single-membraned, usually grouped in stacks of three or four. Phylogeography analyses confirmed the distinct species status of T. janseni n. sp. within the T. cruzi clade. Trypanosoma janseni n. sp. clusters with T. wauwau in a well-supported clade, which is exclusive and monophyletic. The separation of the South American T. wauwau + T. janseni coincides with the separation of the Southern Super Continent. This clade is a sister group of the trypanosomes found in Australian marsupials and its discovery sheds light on the initial diversification process based on what we currently know about the T. cruzi clade.

Low cost light-sheet microscopy for whole brain imaging

NASA Astrophysics Data System (ADS)

Kumar, Manish; Nasenbeny, Jordan; Kozorovitskiy, Yevgenia

2018-02-01

Light-sheet microscopy has evolved as an indispensable tool in imaging biological samples. It can image 3D samples at fast speed, with high-resolution optical sectioning, and with reduced photobleaching effects. These properties make light-sheet microscopy ideal for imaging fluorophores in a variety of biological samples and organisms, e.g. zebrafish, drosophila, cleared mouse brains, etc. While most commercial turnkey light-sheet systems are expensive, the existing lower cost implementations, e.g. OpenSPIM, are focused on achieving high-resolution imaging of small samples or organisms like zebrafish. In this work, we substantially reduce the cost of light-sheet microscope system while targeting to image much larger samples, i.e. cleared mouse brains, at single-cell resolution. The expensive components of a lightsheet system - excitation laser, water-immersion objectives, and translation stage - are replaced with an incoherent laser diode, dry objectives, and a custom-built Arduino-controlled translation stage. A low-cost CUBIC protocol is used to clear fixed mouse brain samples. The open-source platforms of μManager and Fiji support image acquisition, processing, and visualization. Our system can easily be extended to multi-color light-sheet microscopy.

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

Timmermans, F. J.; Otto, C.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemicallymore » or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.« less

New approaches in renal microscopy: volumetric imaging and superresolution microscopy.

PubMed

Kim, Alfred H J; Suleiman, Hani; Shaw, Andrey S

2016-05-01

Histologic and electron microscopic analysis of the kidney has provided tremendous insight into structures such as the glomerulus and nephron. Recent advances in imaging, such as deep volumetric approaches and superresolution microscopy, have the capacity to dramatically enhance our current understanding of the structure and function of the kidney. Volumetric imaging can generate images millimeters below the surface of the intact kidney. Superresolution microscopy breaks the diffraction barrier inherent in traditional light microscopy, enabling the visualization of fine structures. Here, we describe new approaches to deep volumetric and superresolution microscopy of the kidney. Rapid advances in lasers, microscopic objectives, and tissue preparation have transformed our ability to deep volumetric image the kidney. Innovations in sample preparation have allowed for superresolution imaging with electron microscopy correlation, providing unprecedented insight into the structures within the glomerulus. Technological advances in imaging have revolutionized our capacity to image both large volumes of tissue and the finest structural details of a cell. These new advances have the potential to provide additional profound observations into the normal and pathologic functions of the kidney.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

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

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

DOE PAGES

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; ...

2017-08-29

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of thesemore » three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy.« less

Signal improvement in multiphoton microscopy by reflection with simple mirrors near the sample

NASA Astrophysics Data System (ADS)

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2010-03-01

In conventional fluorescence or confocal microscopy, emitted light is generated not only in the focal plane but also above and below. The situation is different in multiphoton-induced fluorescence and multiphoton-induced higher harmonic generation. Here, restriction of signal generation to a single focal point permits that all emitted photons can contribute to image formation if collected, regardless of their path through the specimen. Often, the intensity of the emitted light is rather low in biological specimens. We present a method to significantly increase the fraction of photons collected by an epi (backward) detector by placing a simple mirror, an aluminum-coated coverslip, directly under the sample. Samples investigated include fluorescent test slides, collagen gels, and thin-layered, intact mouse skeletal muscles. Quantitative analysis revealed an intensity increase of second- and third-harmonic generated signal in skeletal muscle of nine- and sevenfold respectively, and of fluorescent signal in test slides of up to twofold. Our approach thus allows significant signal improvement also for situations were a forward detection is impossible, e.g., due to the anatomy of animals in intravital microscopy.

A Comparative Study of Sample Preparation for Staining and Immunodetection of Plant Cell Walls by Light Microscopy

PubMed Central

Verhertbruggen, Yves; Walker, Jesse L.; Guillon, Fabienne; Scheller, Henrik V.

2017-01-01

Staining and immunodetection by light microscopy are methods widely used to investigate plant cell walls. The two techniques have been crucial to study the cell wall architecture in planta, its deconstruction by chemicals or cell wall-degrading enzymes. They have been instrumental in detecting the presence of cell types, in deciphering plant cell wall evolution and in characterizing plant mutants and transformants. The success of immunolabeling relies on how plant materials are embedded and sectioned. Agarose coating, wax and resin embedding are, respectively, associated with vibratome, microtome and ultramicrotome sectioning. Here, we have systematically carried out a comparative analysis of these three methods of sample preparation when they are applied for cell wall staining and cell wall immunomicroscopy. In order to help the plant community in understanding and selecting adequate methods of embedding and sectioning for cell wall immunodetection, we review in this article the advantages and limitations of these three methods. Moreover, we offer detailed protocols of embedding for studying plant materials through microscopy. PMID:28900439

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy.

PubMed

Takayama, Yuki; Yonekura, Koji

2016-03-01

Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

Summary of 2016 Light Microscopy Module (LMM) Physical Science Experiments on ISS. Update of LMM Science Experiments and Facility Capabilities

NASA Technical Reports Server (NTRS)

Sicker, Ronald J.; Meyer, William V.; Foster, William M.; Fletcher, William A.; Williams, Stuart J.; Lee, Chang-Soo

2016-01-01

This presentation will feature a series of short, entertaining, and informative videos that describe the current status and science support for the Light Microscopy Module (LMM) facility on the International Space Station. These interviews will focus on current experiments and provide an overview of future capabilities. The recently completed experiments include nano-particle haloing, 3-D self-assembly with Janus particles and a model system for nano-particle drug delivery. The videos will share perspectives from the scientists, engineers, and managers working with the NASA Light Microscopy program.

Optimizing low-light microscopy with back-illuminated electron multiplying charge-coupled device: enhanced sensitivity, speed, and resolution.

PubMed

Coates, Colin G; Denvir, Donal J; McHale, Noel G; Thornbury, Keith D; Hollywood, Mark A

2004-01-01

The back-illuminated electron multiplying charge-coupled device (EMCCD) camera is having a profound influence on the field of low-light dynamic cellular microscopy, combining highest possible photon collection efficiency with the ability to virtually eliminate the readout noise detection limit. We report here the use of this camera, in 512 x 512 frame-transfer chip format at 10-MHz pixel readout speed, in optimizing a demanding ultra-low-light intracellular calcium flux microscopy setup. The arrangement employed includes a spinning confocal Nipkow disk, which, while facilitating the need to both generate images at very rapid frame rates and minimize background photons, yields very weak signals. The challenge for the camera lies not just in detecting as many of these scarce photons as possible, but also in operating at a frame rate that meets the temporal resolution requirements of many low-light microscopy approaches, a particular demand of smooth muscle calcium flux microscopy. Results presented illustrate both the significant sensitivity improvement offered by this technology over the previous standard in ultra-low-light CCD detection, the GenIII+intensified charge-coupled device (ICCD), and also portray the advanced temporal and spatial resolution capabilities of the EMCCD. Copyright 2004 Society of Photo-Optical Instrumentation Engineers.

Probing neural tissue with airy light-sheet microscopy: investigation of imaging performance at depth within turbid media

NASA Astrophysics Data System (ADS)

Nylk, Jonathan; McCluskey, Kaley; Aggarwal, Sanya; Tello, Javier A.; Dholakia, Kishan

2017-02-01

Light-sheet microscopy (LSM) has received great interest for fluorescent imaging applications in biomedicine as it facilitates three-dimensional visualisation of large sample volumes with high spatiotemporal resolution whilst minimising irradiation of, and photo-damage to the specimen. Despite these advantages, LSM can only visualize superficial layers of turbid tissues, such as mammalian neural tissue. Propagation-invariant light modes have played a key role in the development of high-resolution LSM techniques as they overcome the natural divergence of a Gaussian beam, enabling uniform and thin light-sheets over large distances. Most notably, Bessel and Airy beam-based light-sheet imaging modalities have been demonstrated. In the single-photon excitation regime and in lightly scattering specimens, Airy-LSM has given competitive performance with advanced Bessel-LSM techniques. Airy and Bessel beams share the property of self-healing, the ability of the beam to regenerate its transverse beam profile after propagation around an obstacle. Bessel-LSM techniques have been shown to increase the penetration-depth of the illumination into turbid specimens but this effect has been understudied in biologically relevant tissues, particularly for Airy beams. It is expected that Airy-LSM will give a similar enhancement over Gaussian-LSM. In this paper, we report on the comparison of Airy-LSM and Gaussian-LSM imaging modalities within cleared and non-cleared mouse brain tissue. In particular, we examine image quality versus tissue depth by quantitative spatial Fourier analysis of neural structures in virally transduced fluorescent tissue sections, showing a three-fold enhancement at 50 μm depth into non-cleared tissue with Airy-LSM. Complimentary analysis is performed by resolution measurements in bead-injected tissue sections.

Cell segmentation in phase contrast microscopy images via semi-supervised classification over optics-related features.

PubMed

Su, Hang; Yin, Zhaozheng; Huh, Seungil; Kanade, Takeo

2013-10-01

Phase-contrast microscopy is one of the most common and convenient imaging modalities to observe long-term multi-cellular processes, which generates images by the interference of lights passing through transparent specimens and background medium with different retarded phases. Despite many years of study, computer-aided phase contrast microscopy analysis on cell behavior is challenged by image qualities and artifacts caused by phase contrast optics. Addressing the unsolved challenges, the authors propose (1) a phase contrast microscopy image restoration method that produces phase retardation features, which are intrinsic features of phase contrast microscopy, and (2) a semi-supervised learning based algorithm for cell segmentation, which is a fundamental task for various cell behavior analysis. Specifically, the image formation process of phase contrast microscopy images is first computationally modeled with a dictionary of diffraction patterns; as a result, each pixel of a phase contrast microscopy image is represented by a linear combination of the bases, which we call phase retardation features. Images are then partitioned into phase-homogeneous atoms by clustering neighboring pixels with similar phase retardation features. Consequently, cell segmentation is performed via a semi-supervised classification technique over the phase-homogeneous atoms. Experiments demonstrate that the proposed approach produces quality segmentation of individual cells and outperforms previous approaches. Copyright © 2013 Elsevier B.V. All rights reserved.

Isolation and characterization of nanocrystalline cellulose from roselle-derived microcrystalline cellulose.

PubMed

Kian, Lau Kia; Jawaid, Mohammad; Ariffin, Hidayah; Karim, Zoheb

2018-07-15

Roselle fiber is a renewable and sustainable agricultural waste enriched with cellulose polysaccharides. The isolation of Nanocrystalline cellulose (NCC) from roselle-derived microcrystalline cellulose (MCC) is an alternative approach to recover the agricultural roselle plant residue. In the present study, acid hydrolysis with different reaction time was carried out to degrade the roselle-derived MCC to form NCC. The characterizations of isolated NCC were conducted through Fourier Transform Infrared Ray (FTIR), Transmission Electron Microscopy (TEM), Field Emission Scanning Electron Microscopy (FESEM), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS), Energy Dispersive Spectroscopy (EDS), X-ray Diffraction (XRD), Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). As evaluated from the performed morphological investigations, the needle-like shape NCC nanostructures were observed under TEM and AFM microscopy studies, while irregular rod-like shape of NCC was observed under FESEM analysis. With 60min hydrolysis time, XRD analysis demonstrated the highest NCC crystallinity degree with 79.5%. In thermal analysis by TGA and DSC, the shorter hydrolysis time tended to produce NCC with higher thermal stability. Thus, the isolated NCC from roselle-derived MCC has high potential to be used in application of pharmaceutical and biomedical fields for nanocomposite fabrication. Copyright © 2018 Elsevier B.V. All rights reserved.

Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Parton, Robert G

2017-01-01

The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

Imaging galectin-3 dependent endocytosis with lattice light-sheet microscopy

NASA Astrophysics Data System (ADS)

Baek, Jongho; Lou, Jieqiong; Coelho, Simao; Lim, Yean Jin; Seidlitz, Silvia; Nicovich, Philip R.; Wunder, Christian; Johannes, Ludger; Gaus, Katharina

2017-04-01

Lattice light-sheet (LLS) microscopy provides ultrathin light sheets of a two-dimensional optical lattice that allows us imaging three-dimensional (3D) objects for hundreds of time points at sub-second intervals and at or below the diffraction limit. Galectin-3 (Gal3), a carbohydrate-binding protein, triggers glycosphingolipid (GSL)-dependent biogenesis of morphologically distinct endocytic vesicles that are cargo specific and clathrin independent. In this study, we apply LLS microscopy to study the dynamics of Gal3 dependent endocytosis in live T cells. This will allow us to observe Gal3-mediated endocytosis at high temporal and excellent 3D spatial resolution, which may shed light on our understanding of the mechanism and physiological function of Gal3-induced endocytosis.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

[THE CHARACTERISTICS OF MORPHOLOGY OF BIOFILM OF PERIODONTIUM UNDER INFLAMMATORY DISEASES OF GUMS (CHRONIC CATARRHAL GINGIVITIS, CHRONIC PERIODONTITIS, CANDIDA-ASSOCIATED PERIODONTITIS) ACCORDING RESULTS OF ELECTRONIC MICROSCOPY].

PubMed

Ippolitov, E V; Didenko, L V; Tzarev, V N

2015-12-01

The study was carried out to analyze morphology of biofilm of periodontium and to develop electronic microscopic criteria of differentiated diagnostic of inflammatory diseases of gums. The scanning electronic microscopy was applied to analyze samples of bioflm of periodont from 70 patients. Including ten patients with every nosologic form of groups with chronic catarrhal periodontitis. of light, mean and severe degree, chronic catarrhal gingivitis, Candida-associated paroperiodontitis and 20 healthy persons with intact periodontium. The analysis was implemented using dual-beam scanning electronic microscope Quanta 200 3D (FEI company, USA) and walk-through electronic micJEM 100B (JEOL, Japan). To detect marker DNA of periodont pathogenic bacteria in analyzed samples the kit of reagentsfor polymerase chain reaction "MultiDent-5" ("GenLab", Russia). The scanning electronic microscopy in combination with transmission electronic microscopy and polymerase chain reaction permits analyzing structure, composition and degree of development of biofilm of periodontium and to apply differentiated diagnostic of different nosologic forms of inflammatory diseases of periodontium, including light form of chronic periodontitis and gingivitis. The electronic microscopical indications of diseases ofperiodontium of inflammatory character are established: catarrhal gingivitis, (coccal morphological alternate), chronic periodontitis (bacillary morphological alternate), Candida-associated periodontitis (Candida morphological alternate of biofilm ofperiodontium).

Reduction of parasitic interferences in digital holographic microscopy by numerically decreased coherence length

NASA Astrophysics Data System (ADS)

Kosmeier, S.; Langehanenberg, P.; von Bally, G.; Kemper, B.

2012-01-01

Due to the large coherence length of laser light, optical path length (OPL) resolution in laser based digital holographic microscopy suffers from parasitic interferences caused by multiple reflections within the experimental setup. Use of partially coherent light reduces this drawback but requires precise and stable matching of object and reference arm's OPLs and limits the spatial frequency of the interference pattern in off-axis holography. Here, we investigate if the noise properties of spectrally broadened light sources can be generated numerically. Therefore, holograms are coherently captured at different laser wavelengths and the corresponding reconstructed wave fields are numerically superimposed utilizing variable weightings. Gaussian and rectangular spectral shapes of the so synthesized field are analyzed with respect to the resulting noise level, which is quantified in OPL distributions of a reflective test target. Utilizing a Gaussian weighting, the noise level is found to be similar to the one obtained with the partially coherent light of a superluminescent diode. With a rectangular shaped synthesized spectrum, noise is reduced more efficient than with a Gaussian one. The applicability of the method in label-free cell analysis is demonstrated by quantitative phase contrast images obtained from living cancer cells.

Longitudinal spatial coherence gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination

NASA Astrophysics Data System (ADS)

Mehta, Dalip Singh; Ahmad, Azeem; Dubey, Vishesh; Singh, Veena; Butola, Ankit; Mohanty, Tonmoy; Nandi, Sreyankar

2018-02-01

We report longitudinal spatial coherence (LSC) gated high-resolution tomography and quantitative phase microscopy of biological cells and tissues with uniform illumination using laser as a light source. To accomplish this a pseudo thermal light source was synthesized by passing laser beams through an optical system, which is basically a speckle reduction system with combined effect of spatial, temporal, angular and polarisation diversity. The longitudinal spatial coherence length of such light was significantly reduced by synthesizing a pseudo thermal source with the combined effect of spatial, angular and temporal diversity. This results in a low spatially coherent (i.e., broad angular frequency spectrum) light source with narrow temporal frequency spectrum. Light from such a pseudo thermal light source was passed through an interference microscope with varying magnification, such as, 10X and 50X. The interference microscope was used for full-field OCT imaging of multilayer objects and topography of industrial objects. Experimental results of optical sectioning of multilayer biological objects with high axial-resolution less than 10μm was achieved which is comparable to broadband white light source. The synthesized light source with reduced speckles having uniform illumination on the sample, which can be very useful for fluorescence microscopy as well as quantitative phase microscopy with less phase noise. The present system does not require any dispersion compensation optical system for biological samples as a highly monochromatic light source is used.

Evaluation of the Surface Characteristics of Various Implant Abutment Materials Using Confocal Microscopy and White Light Interferometry.

PubMed

Park, Jun-Beom; Yang, Seung-Min; Ko, Youngkyung

2015-12-01

The purpose of this study was to evaluate the surface characteristics of various implant abutment materials, such as of titanium alloy (Ti6Al4V; Ma), machined cobalt-chrome-molybdenum alloy (CCM), titanium nitride coating on a titanium alloy disc (TiN), anodic oxidized titanium alloy disc (AO), composite resin coating on a titanium alloy disc (Res), and zirconia disc (Zr), using confocal microscopy and white light interferometry. Measurements from the 2 methods were evaluated to see if these methods would give equivalent results. The precision of measurements were evaluated by the coefficient of variation. Five discs each of Ma, CCM, TiN, AO, Res, and Zr were used. The surface roughness was evaluated by confocal laser microscopy and white light interferometry. Confocal microscopy showed that the Res group showed significantly greater Ra, Rq, Rz, Sa, Sq, and Sz values compared with those of the Ma group (P < 0.05). The white light interferometry results showed that the Res group had significantly higher Ra, Rq, Rz, Rt, Sa, Sq, Sz, and Sdr values compared with the Ma group (P < 0.05). All the roughness parameters obtained from the 2 methods differed, and the Sa values of the Zr group from confocal microscopy were greater by 0.163 μm than those obtained by white light interferometry. Least difference was seen in the TiN group where the difference was 0.058 μm. Roughness parameters of different abutment materials varied significantly. Precision of measurement differed according to the characteristics of the material used. White light interferometry could be recommended for measurement of TiN and AO. Confocal microscopy gave more precise measurements for Ma and CCM groups. The optical characteristics of the surface should be considered before choosing the examination method.

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.

Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy.

PubMed

Carlson, David B; Evans, James E

2011-06-05

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

Designing Image Analysis Pipelines in Light Microscopy: A Rational Approach.

PubMed

Arganda-Carreras, Ignacio; Andrey, Philippe

2017-01-01

With the progress of microscopy techniques and the rapidly growing amounts of acquired imaging data, there is an increased need for automated image processing and analysis solutions in biological studies. Each new application requires the design of a specific image analysis pipeline, by assembling a series of image processing operations. Many commercial or free bioimage analysis software are now available and several textbooks and reviews have presented the mathematical and computational fundamentals of image processing and analysis. Tens, if not hundreds, of algorithms and methods have been developed and integrated into image analysis software, resulting in a combinatorial explosion of possible image processing sequences. This paper presents a general guideline methodology to rationally address the design of image processing and analysis pipelines. The originality of the proposed approach is to follow an iterative, backwards procedure from the target objectives of analysis. The proposed goal-oriented strategy should help biologists to better apprehend image analysis in the context of their research and should allow them to efficiently interact with image processing specialists.

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.

Structured Illumination Microscopy for the Investigation of Synaptic Structure and Function.

PubMed

Hong, Soyon; Wilton, Daniel K; Stevens, Beth; Richardson, Douglas S

2017-01-01

The neuronal synapse is a primary building block of the nervous system to which alterations in structure or function can result in numerous pathologies. Studying its formation and elimination is the key to understanding how brains are wired during development, maintained throughout adulthood plasticity, and disrupted during disease. However, due to its diffraction-limited size, investigations of the synaptic junction at the structural level have primarily relied on labor-intensive electron microscopy or ultra-thin section array tomography. Recent advances in the field of super-resolution light microscopy now allow researchers to image synapses and associated molecules with high-spatial resolution, while taking advantage of the key characteristics of light microscopy, such as easy sample preparation and the ability to detect multiple targets with molecular specificity. One such super-resolution technique, Structured Illumination Microscopy (SIM), has emerged as an attractive method to examine synapse structure and function. SIM requires little change in standard light microscopy sample preparation steps, but results in a twofold improvement in both lateral and axial resolutions compared to widefield microscopy. The following protocol outlines a method for imaging synaptic structures at resolutions capable of resolving the intricacies of these neuronal connections.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

PubMed

Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

2015-01-01

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Near-infrared branding efficiently correlates light and electron microscopy.

PubMed

Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

2011-06-05

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

Hyperspectral stimulated emission depletion microscopy and methods of use thereof

DOEpatents

Timlin, Jerilyn A; Aaron, Jesse S

2014-04-01

A hyperspectral stimulated emission depletion ("STED") microscope system for high-resolution imaging of samples labeled with multiple fluorophores (e.g., two to ten fluorophores). The hyperspectral STED microscope includes a light source, optical systems configured for generating an excitation light beam and a depletion light beam, optical systems configured for focusing the excitation and depletion light beams on a sample, and systems for collecting and processing data generated by interaction of the excitation and depletion light beams with the sample. Hyperspectral STED data may be analyzed using multivariate curve resolution analysis techniques to deconvolute emission from the multiple fluorophores. The hyperspectral STED microscope described herein can be used for multi-color, subdiffraction imaging of samples (e.g., materials and biological materials) and for analyzing a tissue by Forster Resonance Energy Transfer ("FRET").

Silver stain for electron microscopy

NASA Technical Reports Server (NTRS)

Corbett, R. L.

1972-01-01

Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

Whole-animal imaging with high spatio-temporal resolution

NASA Astrophysics Data System (ADS)

Chhetri, Raghav; Amat, Fernando; Wan, Yinan; Höckendorf, Burkhard; Lemon, William C.; Keller, Philipp J.

2016-03-01

We developed isotropic multiview (IsoView) light-sheet microscopy in order to image fast cellular dynamics, such as cell movements in an entire developing embryo or neuronal activity throughput an entire brain or nervous system, with high resolution in all dimensions, high imaging speeds, good physical coverage and low photo-damage. To achieve high temporal resolution and high spatial resolution at the same time, IsoView microscopy rapidly images large specimens via simultaneous light-sheet illumination and fluorescence detection along four orthogonal directions. In a post-processing step, these four views are then combined by means of high-throughput multiview deconvolution to yield images with a system resolution of ≤ 450 nm in all three dimensions. Using IsoView microscopy, we performed whole-animal functional imaging of Drosophila embryos and larvae at a spatial resolution of 1.1-2.5 μm and at a temporal resolution of 2 Hz for up to 9 hours. We also performed whole-brain functional imaging in larval zebrafish and multicolor imaging of fast cellular dynamics across entire, gastrulating Drosophila embryos with isotropic, sub-cellular resolution. Compared with conventional (spatially anisotropic) light-sheet microscopy, IsoView microscopy improves spatial resolution at least sevenfold and decreases resolution anisotropy at least threefold. Compared with existing high-resolution light-sheet techniques, such as lattice lightsheet microscopy or diSPIM, IsoView microscopy effectively doubles the penetration depth and provides subsecond temporal resolution for specimens 400-fold larger than could previously be imaged.

Facile one-pot synthesis of hexagons of NaSrB5O9:Tb3+ phosphor for solid-state lighting

NASA Astrophysics Data System (ADS)

Ramesh, B.; Dillip, G. R.; Deva Prasad Raju, B.; Somasundaram, K.; Prasad Peddi, Siva; de Carvalho dos Anjos, Virgilio; Joo, S. W.

2017-04-01

NaSrB5O9:Tb3+ hexagons were synthesized by a facile solid-state reaction method. The synthesized powders were structurally examined by x-ray diffraction analysis (XRD), and Rietveld refinement was performed using the XRD data and Fullprof software. Hexagon-like morphology was observed using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The elemental composition of the phosphors was investigated qualitatively by energy dispersive x-ray analysis (EDS) and quantitatively by x-ray photoelectron spectroscopy (XPS). The phosphor has a strong green emission at 545 nm under excitation of 379 nm, which is due to the 5{{\\text{D}}4}{{\\to}7}{{\\text{F}}5} transition of the Tb3+ ion. A lifetime of 3.48 ms was obtained for the phosphor. The important parameters of the light source were determined, such as the thermal quenching, critical distance, the nature of the dopant ion interaction, color coordinates, and quantum yield values. Other reported properties include the site occupancy of the dopant, surface properties, morphological properties, and optical properties.

Surface plasmon resonance microscopy: achieving a quantitative optical response

PubMed Central

Peterson, Alexander W.; Halter, Michael; Plant, Anne L.; Elliott, John T.

2016-01-01

Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction, and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based configuration. We carry out SPR imaging on a microscope by launching light into a sample, and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit, and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data, and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy. PMID:27782542

Laser scanning saturated structured illumination microscopy based on phase modulation

NASA Astrophysics Data System (ADS)

Huang, Yujia; Zhu, Dazhao; Jin, Luhong; Kuang, Cuifang; Xu, Yingke; Liu, Xu

2017-08-01

Wide-field saturated structured illumination microscopy has not been widely used due to the requirement of high laser power. We propose a novel method called laser scanning saturated structured illumination microscopy (LS-SSIM), which introduces high order of harmonics frequency and greatly reduces the required laser power for SSIM imaging. To accomplish that, an excitation PSF with two peaks is generated and scanned along different directions on the sample. Raw images are recorded cumulatively by a CCD detector and then reconstructed to form a high-resolution image with extended optical transfer function (OTF). Our theoretical analysis and simulation results show that LS-SSIM method reaches a resolution of 0.16 λ, equivalent to 2.7-fold resolution than conventional wide-field microscopy. In addition, LS-SSIM greatly improves the optical sectioning capability of conventional wide-field illumination system by diminishing our-of-focus light. Furthermore, this modality has the advantage of implementation in multi-photon microscopy with point scanning excitation to image samples in greater depths.

Microwave Processing of Crowns from Winter Cereals for Light Microscopy.

USDA-ARS?s Scientific Manuscript database

Microwave processing of tissue considerably shortens the time it takes to prepare samples for light and electron microscopy. However, plant tissues from different species and different regions of the plant respond differently making it impossible to use a single protocol for all plant tissue. The ...

Assessment of the dorsal fin spine for chimaeroid (Holocephali: Chimaeriformes) age estimation.

PubMed

Barnett, L A K; Ebert, D A; Cailliet, G M

2009-10-01

Previous attempts to age chimaeroids have not rigorously tested assumptions of dorsal fin spine growth dynamics. Here, novel imaging and data-analysis techniques revealed that the dorsal fin spine of the spotted ratfish Hydrolagus colliei is an unreliable structure for age estimation. Variation among individuals in the relationship between spine width and distance from the spine tip indicated that the technique of transverse sectioning may impart imprecision and bias to age estimates. The number of growth-band pairs observed by light microscopy in the inner dentine layer was not a good predictor of body size. Mineral density gradients, indicative of growth zones, were absent in the dorsal fin spine of H. colliei, decreasing the likelihood that the bands observed by light microscopy represent a record of growth with consistent periodicity. These results indicate that the hypothesis of aseasonal growth remains plausible and it should not be assumed that chimaeroid age is quantifiable by standard techniques.

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

Hyperspectral interferometry: Sizing microscale surface features in the pine bark beetle.

PubMed

Beach, James M; Uertz, James L; Eckhardt, Lori G

2015-10-01

A new method of interferometry employing a Fabry-Perot etalon model was used to locate and size microscale features on the surface of the pine bark beetle. Oscillations in the reflected light spectrum, caused by self-interference of light reflecting from surfaces of foreleg setae and spores on the elytrum, were recorded using white light hyperspectral microscopy. By making the assumption that pairs of reflecting surfaces produce an etalon effect, the distance between surfaces could be determined from the oscillation frequency. Low frequencies of less than 0.08 nm(-1) were observed in the spectrum below 700 nm while higher frequencies generally occupied wavelengths from 600 to 850 nm. In many cases, two frequencies appeared separately or in combination across the spectrum. The etalon model gave a mean spore size of 3.04 ± 1.27 μm and a seta diameter of 5.44 ± 2.88 μm. The tapering near the setae tip was detected as a lowering of frequency. Spatial fringes were observed together with spectral oscillations from surfaces on the exoskeleton at higher magnification. These signals were consistent with embedded multi-layer reflecting surfaces. Possible applications for hyperspectral interferometry include medical imaging, detection of spore loads in insects and other fungal carriers, wafer surface and subsurface inspection, nanoscale materials, biological surface analysis, and spectroscopy calibration. This is, to our knowledge, the first report of oscillations directly observed by microscopy in the reflected light spectra from Coleoptera, and the first demonstration of broadband hyperspectral interferometry using microscopy that does not employ an internal interferometer. © 2015 Wiley Periodicals, Inc.

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.

Wide-field imaging through scattering media by scattered light fluorescence microscopy

NASA Astrophysics Data System (ADS)

Zhou, Yulan; Li, Xun

2017-08-01

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

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

Three-dimensional fluorescent microscopy via simultaneous illumination and detection at multiple planes.

PubMed

Ma, Qian; Khademhosseinieh, Bahar; Huang, Eric; Qian, Haoliang; Bakowski, Malina A; Troemel, Emily R; Liu, Zhaowei

2016-08-16

The conventional optical microscope is an inherently two-dimensional (2D) imaging tool. The objective lens, eyepiece and image sensor are all designed to capture light emitted from a 2D 'object plane'. Existing technologies, such as confocal or light sheet fluorescence microscopy have to utilize mechanical scanning, a time-multiplexing process, to capture a 3D image. In this paper, we present a 3D optical microscopy method based upon simultaneously illuminating and detecting multiple focal planes. This is implemented by adding two diffractive optical elements to modify the illumination and detection optics. We demonstrate that the image quality of this technique is comparable to conventional light sheet fluorescent microscopy with the advantage of the simultaneous imaging of multiple axial planes and reduced number of scans required to image the whole sample volume.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Validation of Digital Microscopy Compared With Light Microscopy for the Diagnosis of Canine Cutaneous Tumors.

PubMed

Bertram, Christof A; Gurtner, Corinne; Dettwiler, Martina; Kershaw, Olivia; Dietert, Kristina; Pieper, Laura; Pischon, Hannah; Gruber, Achim D; Klopfleisch, Robert

2018-07-01

Integration of new technologies, such as digital microscopy, into a highly standardized laboratory routine requires the validation of its performance in terms of reliability, specificity, and sensitivity. However, a validation study of digital microscopy is currently lacking in veterinary pathology. The aim of the current study was to validate the usability of digital microscopy in terms of diagnostic accuracy, speed, and confidence for diagnosing and differentiating common canine cutaneous tumor types and to compare it to classical light microscopy. Therefore, 80 histologic sections including 17 different skin tumor types were examined twice as glass slides and twice as digital whole-slide images by 6 pathologists with different levels of experience at 4 time points. Comparison of both methods found digital microscopy to be noninferior for differentiating individual tumor types within the category epithelial and mesenchymal tumors, but diagnostic concordance was slightly lower for differentiating individual round cell tumor types by digital microscopy. In addition, digital microscopy was associated with significantly shorter diagnostic time, but diagnostic confidence was lower and technical quality was considered inferior for whole-slide images compared with glass slides. Of note, diagnostic performance for whole-slide images scanned at 200× magnification was noninferior in diagnostic performance for slides scanned at 400×. In conclusion, digital microscopy differs only minimally from light microscopy in few aspects of diagnostic performance and overall appears adequate for the diagnosis of individual canine cutaneous tumors with minor limitations for differentiating individual round cell tumor types and grading of mast cell tumors.

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022460 (9 Nov. 2009) --- Canadian Space Agency astronaut Robert Thirsk, Expedition 21 flight engineer, installs the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. NASA astronaut Nicole Stott (out of frame), flight engineer, assisted Thirsk.

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022459 (9 Nov. 2009) --- NASA astronaut Nicole Stott, Expedition 21 flight engineer, installs the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. Canadian Space Agency astronaut Robert Thirsk (out of frame) assisted Stott.

A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

PubMed Central

2013-01-01

Background In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. Results We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system. Conclusions The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations. PMID:23915384

Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations.

PubMed

Taylor, Michael A; Vanwalleghem, Gilles C; Favre-Bulle, Itia A; Scott, Ethan K

2018-06-19

Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy (SPIM) in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Analysis of Sheng-Mai-San, a Ginseng-Containing Multiple Components Traditional Chinese Herbal Medicine Using Liquid Chromatography Tandem Mass Spectrometry and Physical Examination by Electron and Light Microscopies.

PubMed

Cheng, Yung-Yi; Tsai, Tung-Hu

2016-09-01

Sheng-Mai-San is a multi-component traditional Chinese herbal preparation. Due to the fact granulated additives, such as starch, carboxymethyl cellulose, lactose and raw herbal powder may alter the content of the bioactive markers in the herbal products, a developed ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was used to measure the herbal biomarkers of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rg₁, Rh₁, compound K, ophiopogonin D and schizandrin from the Sheng-Mai-San herbal formulation. Besides, scanning electron microscopy (SEM) was used to observe the morphology of the herbal granular powders. Light microscopy with Congo red and iodine-KI reagent staining was used to identify the cellulose fiber and cornstarch added to pharmaceutical herbal products. The swelling power (SP), water solubility index (WSI), and crude fiber analysis were used to determine the contents of cellulose fiber and cornstarch in pharmaceutical herbal products. In this study, we developed a novel skill to assess the quantification of appended cornstarch in pharmaceutical herbal products using Aperio ImageScope software. Compared with the traditional cornstarch analysis, our analysis method is a rapid, simple and conversion process which could be applied to detect the percentage of added cornstarch in unknown powder products. The various range of the herbal content for the five pharmaceutical manufacturers varied by up to several hundreds-fold. The physical examination reveals that the morphology of the herbal pharmaceutical products is rough and irregular with sharp layers. This study provides a reference standard operating procedure guide for the quality control of the Chinese herbal pharmaceutical products of Sheng-Mai-San.

To boldly glow ... applications of laser scanning confocal microscopy in developmental biology.

PubMed

Paddock, S W

1994-05-01

The laser scanning confocal microscope (LSCM) is now established as an invaluable tool in developmental biology for improved light microscope imaging of fluorescently labelled eggs, embryos and developing tissues. The universal application of the LSCM in biomedical research has stimulated improvements to the microscopes themselves and the synthesis of novel probes for imaging biological structures and physiological processes. Moreover the ability of the LSCM to produce an optical series in perfect register has made computer 3-D reconstruction and analysis of light microscope images a practical option.

High-fidelity large area nano-patterning of silicon with femtosecond light sheet

NASA Astrophysics Data System (ADS)

Sidhu, Mehra S.; Munjal, Pooja; Singh, Kamal P.

2018-01-01

We employ a femtosecond light sheet generated by a cylindrical lens to rapidly produce high-fidelity nano-structures over large area on silicon surface. The Fourier analysis of electron microscopy images of the laser-induced surface structures reveals sharp peaks indicating good homogeneity. We observed an emergence of second-order spatial periodicity on increasing the scan speed. Our reliable approach may rapidly nano-pattern curved solid surfaces and tiny objects for diverse potential applications in optical devices, structural coloring, plasmonic substrates and in high-harmonic generation.

Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations

PubMed Central

Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W.; Mai, Sabine

2017-01-01

Abstract Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. PMID:28082388

An introduction to optical super-resolution microscopy for the adventurous biologist

NASA Astrophysics Data System (ADS)

Vangindertael, J.; Camacho, R.; Sempels, W.; Mizuno, H.; Dedecker, P.; Janssen, K. P. F.

2018-04-01

Ever since the inception of light microscopy, the laws of physics have seemingly thwarted every attempt to visualize the processes of life at its most fundamental, sub-cellular, level. The diffraction limit has restricted our view to length scales well above 250 nm and in doing so, severely compromised our ability to gain true insights into many biological systems. Fortunately, continuous advancements in optics, electronics and mathematics have since provided the means to once again make physics work to our advantage. Even though some of the fundamental concepts enabling super-resolution light microscopy have been known for quite some time, practically feasible implementations have long remained elusive. It should therefore not come as a surprise that the 2014 Nobel Prize in Chemistry was awarded to the scientists who, each in their own way, contributed to transforming super-resolution microscopy from a technological tour de force to a staple of the biologist’s toolkit. By overcoming the diffraction barrier, light microscopy could once again be established as an indispensable tool in an age where the importance of understanding life at the molecular level cannot be overstated. This review strives to provide the aspiring life science researcher with an introduction to optical microscopy, starting from the fundamental concepts governing compound and fluorescent confocal microscopy to the current state-of-the-art of super-resolution microscopy techniques and their applications.

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

PubMed

Castejon, O J; Castejon, H V; Diaz, M; Castellano, A

2001-10-01

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

Microscopic study of dental hard tissues in primary teeth with Dentinogenesis Imperfecta Type II: Correlation of 3D imaging using X-ray microtomography and polarising microscopy.

PubMed

Davis, Graham R; Fearne, Janice M; Sabel, Nina; Norén, Jörgen G

2015-07-01

The aim of this study was to examine the histological appearance of dental hard tissues in primary teeth from children with DI using conventional polarised light microscopy and correlate that with 3D imaging using X-ray microtomograpy (XMT) to gain a further understanding of the dentine structure of teeth diagnosed with dentinogenesis imperfecta. Undecalcified sections of primary teeth from patients diagnosed with Dentinogenesis Imperfecta Type II were examined using polarised light microscopy. XMT was employed for 3D-imaging and analysis of the dentine. The polarised light microscopy and XMT revealed tubular structures in the dentine seen as vacuoles coinciding with the path of normal dentinal tubules but not continuous tubules. The size of the tubules was close to that of capillaries. The largest tubular structures had a direction corresponding to where the pulp tissue would have been located during primary dentine formation. The dysfunctional mineralisation of the dentine and obliteration of the pulp evidently leaves blood vessels in the dentine which have in the main been tied off and, in the undecalcified sections, appear as vacuoles. Although from radiographs, the pulp in teeth affected by Dentinogenesis Imperfect type II appears to be completely obliterated, a network of interconnected vessels may remain. The presence of large dentinal tubules and blood vessels, or the remnants of blood vessels, could provide a pathway for bacteria from the oral cavity. This might account for why some of these teeth develop periapical abscesses in spite of apparently having no pulp. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microstructure of milk

USDA-ARS?s Scientific Manuscript database

The fat and protein in milk may be examined by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy, and any bacteria present may be viewed by light microscopy. The fat exists as globules, the bulk of the protein is in the form of casein micelles, a...

Faster and less phototoxic 3D fluorescence microscopy using a versatile compressed sensing scheme

PubMed Central

Woringer, Maxime; Darzacq, Xavier; Zimmer, Christophe

2017-01-01

Three-dimensional fluorescence microscopy based on Nyquist sampling of focal planes faces harsh trade-offs between acquisition time, light exposure, and signal-to-noise. We propose a 3D compressed sensing approach that uses temporal modulation of the excitation intensity during axial stage sweeping and can be adapted to fluorescence microscopes without hardware modification. We describe implementations on a lattice light sheet microscope and an epifluorescence microscope, and show that images of beads and biological samples can be reconstructed with a 5-10 fold reduction of light exposure and acquisition time. Our scheme opens a new door towards faster and less damaging 3D fluorescence microscopy. PMID:28788909

Camera array based light field microscopy

PubMed Central

Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

2015-01-01

This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

PubMed Central

Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

2016-01-01

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

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

The Light Microscopy Module: An On-Orbit Multi-User Microscope Facility

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Snead, John H.

2002-01-01

The Light Microscopy Module (LMM) is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and operation of fluids and biology experiments within the Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM will be the first integrated payload with the FIR to conduct four fluid physics experiments. A description of the LMM diagnostic capabilities, including video microscopy, interferometry, laser tweezers, confocal, and spectrophotometry, will be provided.

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.

Dynamic longitudinal investigation of individual nerve endings in the skin of anesthetized mice using in vivo two-photon microscopy

NASA Astrophysics Data System (ADS)

Yuryev, Mikhail; Khiroug, Leonard

2012-04-01

Visualization of individual cutaneous nerve endings has previously relied on laborious procedures of tissue excision, fixation, sectioning and staining for light or electron microscopy. We present a method for non-invasive, longitudinal two-photon microscopy of single nerve endings within the skin of anesthetized transgenic mice. Besides excellent signal-to-background ratio and nanometer-scale spatial resolution, this method offers time-lapse ``movies'' of pathophysiological changes in nerve fine structure over minutes, hours, days or weeks. Structure of keratinocytes and dermal matrix is visualized simultaneously with nerve endings, providing clear landmarks for longitudinal analysis. We further demonstrate feasibility of dissecting individual nerve fibers with infra-red laser and monitoring their degradation and regeneration. In summary, our excision-free optical biopsy technique is ideal for longitudinal microscopic analysis of animal skin and skin innervations in vivo and can be applied widely in preclinical models of chronic pain, allergies, skin cancers and a variety of dermatological disorders.

Application of SEM and EDX in studying biomineralization in plant tissues.

PubMed

He, Honghua; Kirilak, Yaowanuj

2014-01-01

This chapter describes protocols using formalin-acetic acid-alcohol (FAA) to fix plant tissues for studying biomineralization by means of scanning electron microscopy (SEM) and qualitative energy-dispersive X-ray microanalysis (EDX). Specimen preparation protocols for SEM and EDX mainly include fixation, dehydration, critical point drying (CPD), mounting, and coating. Gold-coated specimens are used for SEM imaging, while gold- and carbon-coated specimens are prepared for qualitative X-ray microanalyses separately to obtain complementary information on the elemental compositions of biominerals. During the specimen preparation procedure for SEM, some biominerals may be dislodged or scattered, making it difficult to determine their accurate locations, and light microscopy is used to complement SEM studies. Specimen preparation protocols for light microscopy generally include fixation, dehydration, infiltration and embedding with resin, microtome sectioning, and staining. In addition, microwave processing methods are adopted here to speed up the specimen preparation process for both SEM and light microscopy.

Preparation strategy and illumination of three-dimensional cell cultures in light sheet-based fluorescence microscopy

NASA Astrophysics Data System (ADS)

Bruns, Thomas; Schickinger, Sarah; Wittig, Rainer; Schneckenburger, Herbert

2012-10-01

A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

PubMed

Gualda, Emilio J; Simão, Daniel; Pinto, Catarina; Alves, Paula M; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

Impact of New Camera Technologies on Discoveries in Cell Biology.

PubMed

Stuurman, Nico; Vale, Ronald D

2016-08-01

New technologies can make previously invisible phenomena visible. Nowhere is this more obvious than in the field of light microscopy. Beginning with the observation of "animalcules" by Antonie van Leeuwenhoek, when he figured out how to achieve high magnification by shaping lenses, microscopy has advanced to this day by a continued march of discoveries driven by technical innovations. Recent advances in single-molecule-based technologies have achieved unprecedented resolution, and were the basis of the Nobel prize in Chemistry in 2014. In this article, we focus on developments in camera technologies and associated image processing that have been a major driver of technical innovations in light microscopy. We describe five types of developments in camera technology: video-based analog contrast enhancement, charge-coupled devices (CCDs), intensified sensors, electron multiplying gain, and scientific complementary metal-oxide-semiconductor cameras, which, together, have had major impacts in light microscopy. © 2016 Marine Biological Laboratory.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

PubMed Central

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

Dual-view inverted selective plane illumination microscopy (diSPIM) with improved background rejection for accurate 3D digital pathology

NASA Astrophysics Data System (ADS)

Hu, Bihe; Bolus, Daniel; Brown, J. Quincy

2018-02-01

Current gold-standard histopathology for cancerous biopsies is destructive, time consuming, and limited to 2D slices, which do not faithfully represent true 3D tumor micro-morphology. Light sheet microscopy has emerged as a powerful tool for 3D imaging of cancer biospecimens. Here, we utilize the versatile dual-view inverted selective plane illumination microscopy (diSPIM) to render digital histological images of cancer biopsies. Dual-view architecture enabled more isotropic resolution in X, Y, and Z; and different imaging modes, such as adding electronic confocal slit detection (eCSD) or structured illumination (SI), can be used to improve degraded image quality caused by background signal of large, scattering samples. To obtain traditional H&E-like images, we used DRAQ5 and eosin (D&E) staining, with 488nm and 647nm laser illumination, and multi-band filter sets. Here, phantom beads and a D&E stained buccal cell sample have been used to verify our dual-view method. We also show that via dual view imaging and deconvolution, more isotropic resolution has been achieved for optical cleared human prostate sample, providing more accurate quantitation of 3D tumor architecture than was possible with single-view SPIM methods. We demonstrate that the optimized diSPIM delivers more precise analysis of 3D cancer microarchitecture in human prostate biopsy than simpler light sheet microscopy arrangements.

Advanced light microscopy core facilities: Balancing service, science and career

PubMed Central

Hartmann, Hella; Reymann, Jürgen; Ansari, Nariman; Utz, Nadine; Fried, Hans‐Ulrich; Kukat, Christian; Peychl, Jan; Liebig, Christian; Terjung, Stefan; Laketa, Vibor; Sporbert, Anje; Weidtkamp‐Peters, Stefanie; Schauss, Astrid; Zuschratter, Werner; Avilov, Sergiy

2016-01-01

ABSTRACT Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463–479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC. PMID:27040755

FIR Light Microscopy Module Set Up

NASA Image and Video Library

2009-11-09

ISS021-E-022457 (9 Nov. 2009) --- NASA astronaut Nicole Stott, Expedition 21 flight engineer, uses a communication system while installing the Light Microscopy Module (LMM) Spindle Bracket Assembly in the Fluids Integrated Rack (FIR) in the Destiny laboratory of the International Space Station. Canadian Space Agency astronaut Robert Thirsk (out of frame) assisted Stott.

Facile fabrication of CuO-Pb2O3 nanophotocatalyst for efficient degradation of Rose Bengal dye under visible light irradiation

NASA Astrophysics Data System (ADS)

Kamaraj, Eswaran; Somasundaram, Sivaraman; Balasubramani, Kavitha; Eswaran, Muthu Prema; Muthuramalingam, Rajarajan; Park, Sanghyuk

2018-03-01

A p-type CuO/n-type Pb2O3 heterojunction photocatalyst was prepared by a simple wet chemical process and the photocatalytic ability was evaluated for the degradation of Rose Bengal (RB) under visible light irradiation. Synthesized nanocatalysts were characterized by X-ray diffraction (XRD), UV-vis diffuse reflectance spectroscopy (DRS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Energy-dispersive X-ray spectroscopy (EDS), Brunauer-Emmett-Teller (BET) surface area analysis, and X-ray photoelectron spectroscopy (XPS). The p-n heterojunction of CuO-Pb2O3 nanostructures can promote the light absorption capability of photocatalyst and charge separation of electron-hole pairs. Photodegradation assays showed that the addition of CuO effectively enhanced the photocatalytic activity of CuO-Pb2O3 under visible light irradiation (λmax > 420 nm). Compared with pure Pb2O3 and CuO, the CuO-Pb2O3 exhibited significantly enhanced photocatalytic degradation activity. The reaction rate constant of CuO-Pb2O3 is 0.092 min-1, which is much higher than those of CuO (0.073 min-1) and Pb2O3 (0.045 min-1).

Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas.

PubMed

Dhaliwal, Jasmeet S; Kaufman, Stephen C

2009-01-01

The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy. The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy. Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology. Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

eduSPIM: Light Sheet Microscopy in the Museum.

PubMed

Jahr, Wiebke; Schmid, Benjamin; Weber, Michael; Huisken, Jan

2016-01-01

Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.

Photoactivation of curcumin and sodium hypochlorite to enhance antibiofilm efficacy in root canal dentin.

PubMed

Neelakantan, Prasanna; Cheng, Cheng Qing; Ravichandran, Vinoddhine; Mao, Teresa; Sriraman, Priyanka; Sridharan, Swetha; Subbarao, Chandana; Sharma, Subash; Kishen, Anil

2015-03-01

To test the effect of ultrasonic or light activated curcumin and sodium hypochlorite against Enterococcus faecalis biofilms in vitro. E. faecalis biofilms were grown within root canals (n=175) and divided into 7 groups (n=25). Group 1, sterile saline; group 2, 3% sodium hypochlorite; group 3, 3% sodium hypochlorite activated with ultrasonic files (30s cycles for 4min); group 4, 3% sodium hypochlorite irradiated with blue light (1200mw/cm(2) for 4min); group 5, curcumin (2.5mg/mL); group 6, curcumin (2.5mg/mL) activated with ultrasonic files (30s cycles for 4min); group 7, curcumin (2.5mg/mL) irradiated with blue light. The biofilms' ultrastructure was examined using scanning electron microscopy. Bacterial viability was assessed by confocal microscopy. Data were analyzed by one-way ANOVA and Student-Newman-Keuls test (P=0.05). The quantitative analysis of the colony-forming units was carried out from dentinal shaving and analyzed by One-way ANOVA and Tukey multiple comparison test (P=0.05). All treatment groups showed a significantly higher percentage of dead bacteria than the saline control (P<0.05). The percentage of dead bacteria was significantly higher when light activated curcumin was used (P<0.05). At both depths (200 and 400 microns), light activated curcumin showed no growth of bacteria. Light activation produced significantly higher antibacterial efficacy than ultrasonic agitation, with light activated curcumin producing the maximum elimination of biofilm bacteria within the root canal lumen and dentinal tubules. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

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.

Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification.

PubMed

Schipke, Julia; Brandenberger, Christina; Rajces, Alexandra; Manninger, Martin; Alogna, Alessio; Post, Heiner; Mühlfeld, Christian

2017-04-01

Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson's trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining. NEW & NOTEWORTHY Direct comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes. Copyright © 2017 the American Physiological Society.

Effect of superoxidized water and sodium hypochlorite, associated or not with EDTA, on organic and inorganic components of bovine root dentin.

PubMed

Ghisi, Alexandre Corrêa; Kopper, Patrícia Maria Poli; Baldasso, Flávia E R; Stürmer, Caroline P; Rossi-Fedele, Giampiero; Steier, Liviu; de Figueiredo, José Antonio Poli; Morgental, Renata Dornelles; Vier-Pelisser, Fabiana Vieira

2015-06-01

This study aimed to evaluate the effects of Sterilox (Sx), a superoxidized water, 5% and 2% sodium hypochlorite (5NaOCl and 2NaOCl), and 17% EDTA (E) on the organic and inorganic components of bovine dentin. Eighty bovine incisors were randomly divided into 8 groups (n = 10): 5NaOCl, 5NaOCl + E, 2NaOCl, 2NaOCl + E, Sx, Sx + E, E alone, and distilled water (H2O). Root canal instrumentation was performed by using the corresponding irrigant. The apical 15 mm was longitudinally sectioned into 2 fragments, one for light microscopy analysis in slides stained with picrosirius red (organic component) and the other for scanning electron microscopy analysis (inorganic component). Scores data obtained in the light microscopy analysis were submitted to the Kruskal-Wallis test, followed by multiple comparisons test (P < .05). Scanning electron microscopy images were analyzed descriptively. The chemical solution 5NaOCl had a greater effect on the organic component of dentin in area and depth than 2NaOCl. The chemical solutions 5NaOCl + E, 5NaOCl and 2NaOCl + E caused the greatest change in the collagenous organic matrix near the root canal lumen. The chemical solution 2NaOCl showed similar behavior to Sx, associated or not with E, promoting more superficial disorganization of collagen in a smaller area. Demineralization was observed in all groups in which E was used. However, areas of erosion and open dentinal tubules were detected only when it was combined with NaOCl. Five percent NaOCl promoted the most extensive damage to the organic component of dentin, and when associated to EDTA, dentinal erosion could be seen. Considering these specific aspects, 2% NaOCl and Sx had milder effects on bovine root dentin. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Holographic microscopy for in situ studies of microorganism motility

NASA Astrophysics Data System (ADS)

Nadeau, J.; Hu, S.; Jericho, S.; Lindensmith, C.

2011-12-01

Robust technologies for the detection and identification of microorganisms at low concentrations in complex liquid media are needed for numerous applications: environmental and medical microbiology, food safety, and for the search for microbial life elsewhere in the Solar System. The best current method for microbial enumeration is specific labeling with fluorescent dyes followed by high-resolution light microscopy. However, fluorescent techniques are difficult to use in situ in extreme environments (such as the Arctic and Antarctic or the open ocean) due to the fragility of the instruments and their high power demands. In addition, light microscopic techniques rarely provide insight into microbial motility behaviors. Tracking single cells would provide important insight into the physics of micron-scale motility as well as into key microbial phenomena such as surface attachment and invasiveness. An alternative to traditional light microscopy that is attracting increasing attention is holographic microscopy. Holographic microscopy works by illuminating the object of interest with coherent light from a laser. The light reflected from (or transmitted through) the object is then combined with a coherent reference beam to create an interference pattern that contains the phase and intensity information required to reconstruct a three dimensional image of the object. The interference pattern is recorded on a high resolution detector and can be used to computationally reconstruct a 3D image of the object. The lateral resolution of the image depends upon the wavelength of the light used, the laser power, camera quality, and external noise sources (vibration, stray light, and so forth). Although the principle is simple, technological barriers have prevented wider use of holographic microscopy. Laser sources and CCD cameras with the appropriate properties have only very recently become affordable. In addition, holographic microscopy leads to large data sets that are computationally intensive to reconstruct images from, so the technology to store and process large amounts of data are required. We have successfully deployed a digital in-line holographic microscope in lakes of the Canadian High Arctic and the open ocean. We present characteristic data sets from these experiments, as well as discussing how data acquisition and instrumentation can be improved. A design for a new type of autonomous, submersible holographic microscope incorporating an off-axis reference beam is presented, and future plans for controlled microbe-polymer studies are detailed.

Performance comparison of CareStart™ HRP2/pLDH combo rapid malaria test with light microscopy in north-western Tigray, Ethiopia: a cross-sectional study.

PubMed

Feleke, Daniel Getacher; Tarko, Shambel; Hadush, Haftom

2017-06-06

Rapid diagnostic tests (RDTs) are alternative methods for microscopy in the diagnosis of malaria in resource limited settings. Among commercially available RDTs, CareStart™ Malaria test was found to show reliable results. This study evaluated the performance of CareStart™ Malaria Combo test kit in Northwestern Tigray in Ethiopia. Blood samples were collected from 320 malaria-suspected patients at Mayani Hospital in Northwestern Tigray from December 2015 to March 2016. All blood samples were examined using both light microscopy and CareStart™ Malaria HRP2/pLDH Combo Test kit. Statistical analyses were performed using SPSS version 20. The overall parasite positivity using light microscopy and CareStart™ RDT was 41 (12.8%) and 43 (13.4%), respectively. The sensitivity and specificity of CareStart™ RDT, regardless of species, were found to be 95.4 and 99.3%, respectively. Furthermore, the sensitivity of CareStart™ RDT for Plasmodium falciparum or mixed infection and non-falciparum malaria parasites was 94.4 and 85.0%, respectively while the specificity was found to be 98.9 and 99.7%, respectively. The agreement between the two test methods was "excellent" with a kappa value of 0.92. CareStart™ RDT has very good sensitivity and specificity for malaria diagnosis. The test kit also has an excellent agreement with light microscopy. It is therefore useful in resource-limited areas where microscopy is not available.

Image analysis applied to luminescence microscopy

NASA Astrophysics Data System (ADS)

Maire, Eric; Lelievre-Berna, Eddy; Fafeur, Veronique; Vandenbunder, Bernard

1998-04-01

We have developed a novel approach to study luminescent light emission during migration of living cells by low-light imaging techniques. The equipment consists in an anti-vibration table with a hole for a direct output under the frame of an inverted microscope. The image is directly captured by an ultra low- light level photon-counting camera equipped with an image intensifier coupled by an optical fiber to a CCD sensor. This installation is dedicated to measure in a dynamic manner the effect of SF/HGF (Scatter Factor/Hepatocyte Growth Factor) both on activation of gene promoter elements and on cell motility. Epithelial cells were stably transfected with promoter elements containing Ets transcription factor-binding sites driving a luciferase reporter gene. Luminescent light emitted by individual cells was measured by image analysis. Images of luminescent spots were acquired with a high aperture objective and time exposure of 10 - 30 min in photon-counting mode. The sensitivity of the camera was adjusted to a high value which required the use of a segmentation algorithm dedicated to eliminate the background noise. Hence, image segmentation and treatments by mathematical morphology were particularly indicated in these experimental conditions. In order to estimate the orientation of cells during their migration, we used a dedicated skeleton algorithm applied to the oblong spots of variable intensities emitted by the cells. Kinetic changes of luminescent sources, distance and speed of migration were recorded and then correlated with cellular morphological changes for each spot. Our results highlight the usefulness of the mathematical morphology to quantify kinetic changes in luminescence microscopy.

Form follows function: ultrastructure of different morphotypes of Physarum polycephalum

NASA Astrophysics Data System (ADS)

Oettmeier, Christina; Lee, Jonghyun; Döbereiner, Hans-Günther

2018-04-01

The multinucleate, unicellular slime mold Physarum polycephalum is a highly motile and morphologically diverse giant amoeba. Despite being brainless and lacking neurons, it exhibits ‘smart’ behavior. There is considerable interest in describing such traits and to investigate the underlying mechanochemical patterns which may hint at universal principles of behavior and decision-making. Furthermore, the slime mold’s mechanism of locomotion is unique. It resembles amoeboid movement, but differs from the locomotion of other amoebae in many ways, e.g. in their much larger size and lack of lobopodia. These two aspects, behavior and locomotion, are linked by the cytoskeleton and the overall morphology of P. polycephalum. In this paper, we present a structural analysis of different growth forms (micro-, meso- and macroplasmodia) by transmission electron microscopy (TEM), scanning electron microscopy (SEM), light microscopy, and fluorescence microscopy of F-actin. With these detailed investigations of cellular ultrastructure and morphology, we provide the basis for the analysis of, e.g. viscoelastic and rheological measurements. Our data also provide structural details for the many models that have been constructed for the understanding of locomotion. We conclude that morphological information is vital for the assessment and measurement of material properties.

Combined SERS and Raman analysis for the identification of red pigments in cross-sections from historic oil paintings.

PubMed

Frano, Kristen A; Mayhew, Hannah E; Svoboda, Shelley A; Wustholz, Kristin L

2014-12-21

The analysis of paint cross-sections can reveal a remarkable amount of information about the layers and materials in a painting without visibly altering the artwork. Although a variety of analytical approaches are used to detect inorganic pigments as well as organic binders, proteins, and lipids in cross-sections, they do not provide for the unambiguous identification of natural, organic colorants. Here, we develop a novel combined surface-enhanced Raman scattering (SERS), light microscopy, and normal Raman scattering (NRS) approach for the identification of red organic and inorganic pigments in paint cross-sections obtained from historic 18th and 19th century oil paintings. In particular, Ag nanoparticles are directly applied to localized areas of paint cross-sections mounted in polyester resin for SERS analysis of the organic pigments. This combined extractionless non-hydrolysis SERS and NRS approach provides for the definitive identification of carmine lake, madder lake, and vermilion in multiple paint layers. To our knowledge, this study represents the first in situ identification of natural, organic pigments within paint cross-sections from oil paintings. Furthermore, the combination of SERS and normal Raman, with light microscopy provides conservators with a more comprehensive understanding of a painting from a single sample and without the need for sample pretreatment.

The influence of filling technique on depth of tubule penetration by root canal sealer: a study using light microscopy and digital image processing.

PubMed

De Deus, Gustavo A; Gurgel-Filho, Eduardo Diogo; Maniglia-Ferreira, Cláudio; Coutinho-Filho, Tauby

2004-04-01

The purpose of this study was to compare the depth of sealer penetration into dentinal tubules by three root-filling techniques using light microscopy and digital image processing. Thirty-two maxillary central incisors were prepared. Two teeth were separated for the control group. The rest were divided into three equal groups and obturated as following--G1: lateral condensation; G2: warm vertical compaction of gutta-percha and G3: Thermafil system. Each sample was sectioned longitudinally and prepared for microscopic analysis. A sequence of photomicrographs with magnifications of X50, X200 and X500 were taken. Through digital image analysis and processing, measurements for each field were obtained. A non-parametric ANOVA Kruskal-Wallis analysis was used to determine whether there were significant differences among the groups. Significant differences between G2 and G1 (p = 0.034) and between G3 and G1 (p = 0.021) were identified. There were no significant differences between G2 and G3 (p > 0.05). The results of this research suggest that samples root-filled by thermoplasticised gutta-percha techniques lead to deeper penetration of the root canal sealer into the dentinal tubules.

A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis.

PubMed

Novas, Romulo Bourget; Fazan, Valeria Paula Sassoli; Felipe, Joaquim Cezar

2016-02-01

Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and the vestibulocochlear nerve of adult guinea pigs were used herein. The proposed pipeline for fiber segmentation is based on the techniques of competitive clustering and concavity analysis. The evaluation of the proposed method for segmentation of images was done by comparing the automatic segmentation with the manual segmentation. To further evaluate the proposed method considering morphometric features extracted from the segmented images, the distributions of these features were tested for statistical significant difference. The method achieved a high overall sensitivity and very low false-positive rates per image. We detect no statistical difference between the distribution of the features extracted from the manual and the pipeline segmentations. The method presented a good overall performance, showing widespread potential in experimental and clinical settings allowing large-scale image analysis and, thus, leading to more reliable results.

Lead free CH{sub 3}NH{sub 3}SnI{sub 3} perovskite thin-film with p-type semiconducting nature and metal-like conductivity

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

Iefanova, Anastasiia; Adhikari, Nirmal; Dubey, Ashish

Lead free CH{sub 3}NH{sub 3}SnI{sub 3} perovskite thin film was prepared by low temperature solution processing and characterized using current sensing atomic force microscopy (CS-AFM). Analysis of electrical, optical, and optoelectrical properties reveals unique p-type semiconducting nature and metal like conductivity of this material. CH{sub 3}NH{sub 3}SnI{sub 3} film also showed a strong absorption in visible and near infrared spectrum with absorption onset of 1.3 eV. X-ray Diffraction analysis and scanning electron microscopy (SEM) confirmed a structure of this compound and uniform film formation. The morphology, film uniformity, light harvesting and electrical properties strongly depend on preparation method and precursormore » solution. CH{sub 3}NH{sub 3}SnI{sub 3} films prepared based on dimethylformamide (DMF) showed higher crystallinity and light harvesting capability compared to the film based on combination of dimethyl sulfoxide (DMSO) with gamma-butyrolactone (GBL). Local photocurrent mapping analysis showed that CH{sub 3}NH{sub 3}SnI{sub 3} can be used as an active layer and have a potential to fabricate lead free photovoltaic devices.« less

Microwave-accelerated cytochemical stains for the image analysis and the electron microscopic examination of light microscopy diagnostic slides.

PubMed

Hanker, J; Giammara, B

1993-01-01

Recent studies in our laboratories have shown how microwave (MW) irradiation can accelerate a number of tissue-processing techniques, especially staining, to aid in the preparation of single specimens on glass microscope slides or coverslips for examination by light microscopy (and electron microscopy, if required) for diagnostic purposes. Techniques have been developed, which give permanently stained preparations, that can be studied initially by light microscopy, their areas of interest mapped, and computer-automated image analysis performed to obtain quantitative information. This is readily performed after MW-accelerated staining with silver methenamine by the Giammara-Hanker PATS or PATS-TS reaction. This variation of the PAS reaction gives excellent markers for specific infectious agents such as lipopolysaccharides for gram-negative bacteria or mannans for fungi. It is also an excellent stain for glycogen and basement membranes and an excellent marker for type III collagen or reticulin in the endoneurium or perineurium of peripheral nerve or in the capillary walls. Our improved MW-accelerated Feulgen reaction with silver methenamine for nuclear DNA is useful to show the nuclei of bacteria and fungi as well as of cells they are infecting. Improved coating and penetration of tissue surfaces by thiocarbohydrazide bridging of ruthenium red, applied under MW-acceleration, render biologic specimens sufficiently conductive for SEM so that sputter coating with gold is unnecessary. The specimens treated with these highly visible electron-opaque stains can be screened with the light microscope after mounting in polyethylene glycol (PEG) and the structures or areas selected for EM study are mapped with a Micro-Locator slide. After removal of the water soluble PEG the specimens are remounted in the usual EM media for scanning electron microscopy (SEM) or transmission electron microscopy (TEM) study of the mapped areas. By comparing duplicate smears from areas of infection, such as two coverslips of buffy coat smears of blood from a patient with septicemia, the microorganisms responsible can occasionally be classified for antimicrobial therapy long before culture results are available; gram-negative bacteria are positive with the Giammara-Hanker PATS-TS stain, and gram-positive bacteria are positive with the SIGMA HT40 Gram stain. The gram-positive as well as gram-negative bacteria are both initially stained by the crystal violet component of the Gram stain. The crystal violet stain is readily removed from the gram-negative (but not the gram-positive) bacteria when the specimens are rinsed with alcohol/acetone. If this rinse step is omitted, the crystal violet remains attached to both gram-negative and gram-positive bacteria. It can then be rendered insoluble, electron-opaque, and conductive by treatment with silver methenamine solution under MW-irradiation. This metallized crystal violet is a more effective silver stain than the PATS-TS stain for a number of gram-negative spirochetes such as Treponema pallidum, the microbe that causes syphilis.

Noise reduction in digital lensless holographic microscopy by engineering the light from a light-emitting diode.

PubMed

Garcia-Sucerquia, Jorge

2013-01-01

By engineering the light from a light-emitting diode (LED) the noises present in digital lensless holographic microscopy (DLHM) are reduced. The partially coherent light from an LED is tailored to produce a spherical wavefront with limited coherence time and the spatial coherence needed by DLHM to work. DLHM with this engineered light source is used to image biological samples that cover areas of the order of mm(2). The ratio between the diameter of the area that is almost coherently illuminated to the diameter of the illumination area is utilized as parameter to quantify the performance of the DLHM with the engineered LED light source. Experimental results show that while the noises can be reduced effectively the spatial resolution can be kept in the micrometer range.

Microscopic and ultrastructural changes of Müller's muscle in patients with simple congenital ptosis.

PubMed

Alshehri, Mohammed D; Al-Fakey, Yasser H; Alkhalidi, Hisham M; Mubark, Mohamed A; Alsuhaibani, Adel H

2014-01-01

To study microscopic and ultrastructural changes of Müller's muscle in patients with isolated congenital ptosis. In this prospective, observational case-control study, Müller's muscle specimens were collected during ptosis surgical correction for 18 consecutive patients. Each specimen was divided into 2 parts. One part was embedded in formalin for light microscopy, and the other one was fixed in 3% glutaraldehyde for electron microscopy. A neuropathologist, serving as a masked evaluator, blindly reviewed all the different features for every case and counted the number of myocytes showing distinct myofilaments in the whole grid for every case. Statistical analysis using compare means and correlation tests was conducted to investigate potential associations and/or differences within and across groups. Twelve Müller's muscle specimens from patients with simple congenital ptosis of various severities and 6 specimens from patients with aponeurotic ptosis (controls) were collected and studied. Under light microscopy, congenital ptosis slides showed a small number of dispersed myocytes in a fibrotic background, whereas acquired ptosis slides showed a greater number of well-defined myocytes. Under electron microscopy, all congenital ptosis specimens had only a very small number of myocytes with clear, distinct myofilaments. Most myocytes in the aponeurotic ptosis group showed clear, distinct myofilaments, indicating a well-preserved muscle. No relationship existed between the number of clear, distinct myofilaments observed in the congenital ptosis group by transmission electron microscopy and patient age or ptosis severity. Substantial Müller's muscle atrophy was observed in patients with different severities of isolated congenital ptosis.

A novel fibrous duct structure discovered in the brain meninges by using polarized light microscopy

NASA Astrophysics Data System (ADS)

Nam, Min-Ho; Jung, Sharon Jiyoon; Soh, Kwang-Sup; Lim, Jaekwan; Seo, Eunseok; Lim, Jun; Baek, Miok; Lee, Sang Joon

2016-05-01

We have previously reported the discovery of a novel fibrous structure (NFS) consisting of unidirectionally arranged collagen fibers in the spinal pia mater. Due to its unique structure, it was easily detected using polarized light microscopy. In the current study, we describe the discovery of a similar NFS in the brain meninges of rats by using polarized light microscopy. This NFS is located beneath the superior sagittal sinus. Initially, we systemically analyzed the polarization properties of the NFS. The change in the light intensity of the NFS, with respect to the polarization angle, was eight times greater than that of blood vessels, showing that the collagen fibers are oriented in a particular direction with almost perfect parallelism (0.99). The orientation angle of the polarization ellipse confirmed the orientation of the collagen fibers in the NFS. Histological studies further confirmed that the unidirectionally arranged collagen fibers were responsible for this distinct polarization property. Surprisingly, X-ray microtomography and 3D confocal imaging revealed that the NFS contains within it a duct structure, a putative primo vessel. In conclusion, we report a NFS in the brain meninges, detected by using polarized light microscopy, that provides space for a putative primo vessel, not a blood vessel.

The evolution of structured illumination microscopy in studies of HIV.

PubMed

Marno, Kelly; Al'Zoubi, Lara; Pearson, Matthew; Posch, Markus; McKnight, Áine; Wheeler, Ann P

2015-10-15

The resolution limit of conventional light microscopy has proven to be limiting for many biological structures such as viruses including Human immunodeficiency virus (HIV). Individual HIV virions are impossible to study using confocal microscopy as they are well below the 200 nm resolution limit of conventional light microscopes. Structured illumination microscopy (SIM) allows a twofold enhancement in image resolution compared to standard widefield illumination and so provides an excellent tool for study of HIV. Viral capsids (CAs) vary between 110 and 146 nm so this study challenges the performance of SIM microscopes. SIM microscopy was first developed in 2000, commercialised in 2007 and rapidly developed. Here we present the changes in capabilities of the SIM microscopes for study of HIV localisation as the instrumentation for structured illumination microscopy has evolved over the past 8 years. Copyright © 2015. Published by Elsevier Inc.

Laser Light-field Fusion for Wide-field Lensfree On-chip Phase Contrast Microscopy of Nanoparticles

NASA Astrophysics Data System (ADS)

Kazemzadeh, Farnoud; Wong, Alexander

2016-12-01

Wide-field lensfree on-chip microscopy, which leverages holography principles to capture interferometric light-field encodings without lenses, is an emerging imaging modality with widespread interest given the large field-of-view compared to lens-based techniques. In this study, we introduce the idea of laser light-field fusion for lensfree on-chip phase contrast microscopy for detecting nanoparticles, where interferometric laser light-field encodings acquired using a lensfree, on-chip setup with laser pulsations at different wavelengths are fused to produce marker-free phase contrast images of particles at the nanometer scale. As a proof of concept, we demonstrate, for the first time, a wide-field lensfree on-chip instrument successfully detecting 300 nm particles across a large field-of-view of ~30 mm2 without any specialized or intricate sample preparation, or the use of synthetic aperture- or shift-based techniques.

Laser Light-field Fusion for Wide-field Lensfree On-chip Phase Contrast Microscopy of Nanoparticles.

PubMed

Kazemzadeh, Farnoud; Wong, Alexander

2016-12-13

Wide-field lensfree on-chip microscopy, which leverages holography principles to capture interferometric light-field encodings without lenses, is an emerging imaging modality with widespread interest given the large field-of-view compared to lens-based techniques. In this study, we introduce the idea of laser light-field fusion for lensfree on-chip phase contrast microscopy for detecting nanoparticles, where interferometric laser light-field encodings acquired using a lensfree, on-chip setup with laser pulsations at different wavelengths are fused to produce marker-free phase contrast images of particles at the nanometer scale. As a proof of concept, we demonstrate, for the first time, a wide-field lensfree on-chip instrument successfully detecting 300 nm particles across a large field-of-view of ~30 mm 2 without any specialized or intricate sample preparation, or the use of synthetic aperture- or shift-based techniques.

Methods and apparatus of spatially resolved electroluminescence of operating organic light-emitting diodes using conductive atomic force microscopy

NASA Technical Reports Server (NTRS)

Hersam, Mark C. (Inventor); Pingree, Liam S. C. (Inventor)

2008-01-01

A conductive atomic force microscopy (cAFM) technique which can concurrently monitor topography, charge transport, and electroluminescence with nanometer spatial resolution. This cAFM approach is particularly well suited for probing the electroluminescent response characteristics of operating organic light-emitting diodes (OLEDs) over short length scales.

Light Microscopy Module (LMM)-Emulator

NASA Technical Reports Server (NTRS)

Levine, Howard G.; Smith, Trent M.; Richards, Stephanie E.

2016-01-01

The Light Microscopy Module (LMM) is a microscope facility developed at Glenn Research Center (GRC) that provides researchers with powerful imaging capability onboard the International Space Station (ISS). LMM has the ability to have its hardware recongured on-orbit to accommodate a wide variety of investigations, with the capability of remotely acquiring and downloading digital images across multiple levels of magnication.

Light Microscopy of the Hair: A Simple Tool to “Untangle” Hair Disorders

PubMed Central

Adya, Keshavmurthy A; Inamadar, Arun C; Palit, Aparna; Shivanna, Ragunatha; Deshmukh, Niranjan S

2011-01-01

Light microscopy of the hair forms an important bedside clinical tool for the diagnosis of various disorders affecting the hair. Hair abnormalities can be seen in the primary diseases affecting the hair or as a secondary involvement of hair in diseases affecting the scalp. Hair abnormalities also form a part of various genodermatoses and syndromes. In this review, we have briefly highlighted the light microscopic appearance of various infectious and non-infectious conditions affecting the hair. PMID:21769242

Characterization of Polymer Blends: Optical Microscopy (*Polarized, Interference and Phase Contrast Microscopy*) and Confocal Microscopy

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

Ramanathan, Nathan Muruganathan; Darling, Seth B.

2015-01-01

Chapter 15 surveys the characterization of macro, micro and meso morphologies of polymer blends by optical microscopy. Confocal Microscopy offers the ability to view the three dimensional morphology of polymer blends, popular in characterization of biological systems. Confocal microscopy uses point illumination and a spatial pinhole to eliminate out-of focus light in samples that are thicker than the focal plane.

Characteristics of angular cross correlations studied by light scattering from two-dimensional microsphere films

NASA Astrophysics Data System (ADS)

Schroer, M. A.; Gutt, C.; Grübel, G.

2014-07-01

Recently the analysis of scattering patterns by angular cross-correlation analysis (CCA) was introduced to reveal the orientational order in disordered samples with special focus to future applications on x-ray free-electron laser facilities. We apply this CCA approach to ultra-small-angle light-scattering data obtained from two-dimensional monolayers of microspheres. The films were studied in addition by optical microscopy. This combined approach allows to calculate the cross-correlations of the scattering patterns, characterized by the orientational correlation function Ψl(q), as well as to obtain the real-space structure of the monolayers. We show that CCA is sensitive to the orientational order of monolayers formed by the microspheres which are not directly visible from the scattering patterns. By mixing microspheres of different radii the sizes of ordered monolayer domains is reduced. For these samples it is shown that Ψl(q) quantitatively describes the degree of hexagonal order of the two-dimensional films. The experimental CCA results are compared with calculations based on the microscopy images. Both techniques show qualitatively similar features. Differences can be attributed to the wave-front distortion of the laser beam in the experiment. This effect is discussed by investigating the effect of different wave fronts on the cross-correlation analysis results. The so-determined characteristics of the cross-correlation analysis will be also relevant for future x-ray-based studies.

Synthesis of a ternary Ag/RGO/ZnO nanocomposite via microwave irradiation and its application for the degradation of Rhodamine B under visible light.

PubMed

Surendran, Divya Kollikkara; Xavier, Marilyn Mary; Viswanathan, Vandana Parakkal; Mathew, Suresh

2017-06-01

Reduced graphene oxide supporting plasmonic photocatalyst (Ag) on ZnO has been synthesized via a facile two-step microwave synthesis using RGO/ZnO and AgNO 3 . First step involves fabrication of RGO/ZnO via microwave irradiation. The nanocomposites were characterized by X-ray diffraction analysis, transmission electron microscopy, Fourier transform infrared spectroscopy, and Raman spectroscopy. Ag/RGO/ZnO shows enhanced photoactivity under visible light for the degradation of Rhodamine B. Enhanced charge separation and migration have been assigned using UV-vis diffuse reflectance spectra, photoluminescence spectra, electrochemical impedance spectra, and TCSPC analysis. The improved photoactivity of Ag/RGO/ZnO can be ascribed to the prolonged lifetime of photogenerated electron-hole pairs and effective interfacial hybridization between RGO and Ag with ZnO nanoparticles. Ag nanoparticles can absorb visible light via surface plasmon resonance to enhance photocatalytic activity.

Colorimetric analysis of pigmented skin lesions: a pilot study with the Visi-Chroma VC-100 device.

PubMed

Vereecken, P; Mommaerts, M; Duez, C; Petein, M; Laporte, M; Hubinon, J-L; Heenen, M

2006-01-01

Definition of the colour of pigmented skin lesions (PSLs) with the naked eye remains subjective and may be influenced by lighting. This problem underlines the usefulness of instrumental assessments such as epiluminescence microscopy and colorimetric devices. We describe here a new method of colour analysis of PSLs with the Visi-Chroma VC-100 device, which illuminates the surface of the skin with white light-emitting diodes (LEDs) and analyses the reflected light by a red-green-blue (RGB) charge-coupled device (CCD) colour camera. Twenty-one PSLs to be excised for cosmetic or medical reasons were analysed by this device with clinicopathological correlation. This method is feasible and might be useful to assess the colour of PSLs and allow comparisons for changes over time. Further studies are needed to determine the usefulness of this device in clinical practice.

Application of principal component analysis to multispectral-multimodal optical image analysis for malaria diagnostics.

PubMed

Omucheni, Dickson L; Kaduki, Kenneth A; Bulimo, Wallace D; Angeyo, Hudson K

2014-12-11

Multispectral imaging microscopy is a novel microscopic technique that integrates spectroscopy with optical imaging to record both spectral and spatial information of a specimen. This enables acquisition of a large and more informative dataset than is achievable in conventional optical microscopy. However, such data are characterized by high signal correlation and are difficult to interpret using univariate data analysis techniques. In this work, the development and application of a novel method which uses principal component analysis (PCA) in the processing of spectral images obtained from a simple multispectral-multimodal imaging microscope to detect Plasmodium parasites in unstained thin blood smear for malaria diagnostics is reported. The optical microscope used in this work has been modified by replacing the broadband light source (tungsten halogen lamp) with a set of light emitting diodes (LEDs) emitting thirteen different wavelengths of monochromatic light in the UV-vis-NIR range. The LEDs are activated sequentially to illuminate same spot of the unstained thin blood smears on glass slides, and grey level images are recorded at each wavelength. PCA was used to perform data dimensionality reduction and to enhance score images for visualization as well as for feature extraction through clusters in score space. Using this approach, haemozoin was uniquely distinguished from haemoglobin in unstained thin blood smears on glass slides and the 590-700 spectral range identified as an important band for optical imaging of haemozoin as a biomarker for malaria diagnosis. This work is of great significance in reducing the time spent on staining malaria specimens and thus drastically reducing diagnosis time duration. The approach has the potential of replacing a trained human eye with a trained computerized vision system for malaria parasite blood screening.

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.

Imaging three-dimensional light propagation through periodic nanohole arrays using scanning aperture microscopy

PubMed Central

Chowdhury, Mustafa H.; Catchmark, Jeffrey M.; Lakowicz, Joseph R.

2009-01-01

The authors introduce a technique for three-dimensional (3D) imaging of the light transmitted through periodic nanoapertures using a scanning probe to perform optical sectioning microscopy. For a 4×4 nanohole array, the transmitted light displays intensity modulations along the propagation axis, with the maximum intensity occurring at 450 μm above the surface. The propagating fields show low divergence, suggesting a beaming effect induced by the array. At distances within 25 μm from the surface, they observe subwavelength confinement of light propagating from the individual nanoholes. Hence, this technique can potentially be used to map the 3D distribution of propagating light, with high spatial resolution. PMID:19696912

Rapid diagnosis of tinea incognito using handheld reflectance confocal microscopy: a paradigm shift in dermatology?

PubMed

Navarrete-Dechent, Cristián; Bajaj, Shirin; Marghoob, Ashfaq A; Marchetti, Michael A

2015-06-01

Dermatophytoses are common skin infections. Traditional diagnostic tests such as skin scrapings for light microscopy examination, fungal cultures and biopsies remain imperfect due to false-negative test results, cost, time required to perform the procedure, time delays in test results and/or a requirement for an invasive procedure. Herein, we present a case of an 80-year-old female whose tinea incognito was non-invasively diagnosed within seconds using handheld reflectance confocal microscopy (RCM). As non-invasive skin imaging continues to improve, we expect light-based office microscopy to be replaced with technologies such as RCM, which has multiple and continually expanding diagnostic applications. © 2015 Blackwell Verlag GmbH.

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899).

PubMed

Asensio, L; Lopez-Llorca, L V; López-Jiménez, J A

2005-01-01

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.

X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

PubMed

Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

2015-02-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

PubMed Central

Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

2015-01-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

Visible light driven photocatalysis and antibacterial activity of AgVO{sub 3} and Ag/AgVO{sub 3} nanowires

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

Singh, Anamika; Dutta, Dimple P., E-mail: dimpled@barc.gov.in; Ballal, A.

Graphical abstract: - Highlights: • Ag/AgVO{sub 3} and pure AgVO{sub 3} nanowires synthesized by sonochemical process. • Characterization done using XRD, SEM, TEM, EDX and BET analysis. • Visible light degradation of RhB by Ag/AgVO{sub 3} within 45 min. • Antibacterial activity of Ag/AgVO{sub 3} demonstrated. - Abstract: Ag/AgVO{sub 3} nanowires and AgVO{sub 3} nanorods were synthesized in aqueous media via a facile sonochemical route. The as-synthesized products were characterized by X-ray diffraction, Brunauer–Emmett–Teller surface area analysis, scanning electron microscopy together with an energy dispersion X-ray spectrum analysis, transmission electron microscopy and UV–vis diffuse reflectance spectroscopy. The results revealed thatmore » inert atmosphere promotes the formation of Ag/AgVO{sub 3} nanowires. The photocatalytic studies revealed that the Ag/AgVO{sub 3} nanowires exhibited complete photocatalytic degradation of Rhodamine B within 45 min under visible light irradiation. The antibacterial activity of Ag/AgVO{sub 3} nanowires was tested against Escherechia coli and Bacillus subtilis. The minimum growth inhibitory concentration value was found to be 50 and 10 folds lower than for the antibiotic ciprofloxacin for E. coli and B. subtilis, respectively. The antibacterial properties of the β-AgVO{sub 3} nanorods prove that in case of the Ag dispersed Ag/AgVO{sub 3} nanowires, the enhanced antibacterial action is also due to contribution from the AgVO{sub 3} support.« less

Neonatal lines in the enamel of primary teeth--a morphological and scanning electron microscopic investigation.

PubMed

Sabel, Nina; Johansson, Carina; Kühnisch, Jan; Robertson, Agneta; Steiniger, Frank; Norén, Jörgen G; Klingberg, Gunilla; Nietzsche, Sandor

2008-10-01

The neonatal line (NNL) is in principle found in all primary teeth and the line represents the time of birth. Earlier findings of the appearance of the NNL in light microscope and in microradiographs have shown not only changes in the prism direction of the enamel, but that the NNL has a hypomineralized character. The neonatal line was analyzed in un-decalcified sections of primary lower and central incisors, collected from individuals of different ages utilizing polarized light microscopy, microradiography, scanning electron microscopy (SEM) and X-ray analysis (XRMA). In polarized light the NNL appeared to have a more porous structure than the enamel in general. The appearance of the NNL as a dark line in microradiographs is interpreted as the NNL being less mineralized than neighbouring enamel. Analysis with ImageJ visualized the reduction of the amount of grey value, indicating that the NNL is less mineralized. Analysis of the NNL in SEM showed a reduction of the diameter of enamel prisms, the more narrow diameters continued through the postnatal enamel. A change of the growth direction of the prisms was also observed at the NNL. In a three-dimensional image the NNL appeared as a grove, however, in non-etched enamel no grove was seen. The elemental analyses with XRMA showed no marked changes in the content of C, Ca, P, N, O or S in the area around the NNL. The NNL is an optical phenomenon due to alterations in height, and degree of mineralization of the enamel prisms.

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images

PubMed Central

Afshar, Yaser; Sbalzarini, Ivo F.

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 1010 pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments. PMID:27046144

A Parallel Distributed-Memory Particle Method Enables Acquisition-Rate Segmentation of Large Fluorescence Microscopy Images.

PubMed

Afshar, Yaser; Sbalzarini, Ivo F

2016-01-01

Modern fluorescence microscopy modalities, such as light-sheet microscopy, are capable of acquiring large three-dimensional images at high data rate. This creates a bottleneck in computational processing and analysis of the acquired images, as the rate of acquisition outpaces the speed of processing. Moreover, images can be so large that they do not fit the main memory of a single computer. We address both issues by developing a distributed parallel algorithm for segmentation of large fluorescence microscopy images. The method is based on the versatile Discrete Region Competition algorithm, which has previously proven useful in microscopy image segmentation. The present distributed implementation decomposes the input image into smaller sub-images that are distributed across multiple computers. Using network communication, the computers orchestrate the collectively solving of the global segmentation problem. This not only enables segmentation of large images (we test images of up to 10(10) pixels), but also accelerates segmentation to match the time scale of image acquisition. Such acquisition-rate image segmentation is a prerequisite for the smart microscopes of the future and enables online data compression and interactive experiments.

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome.

PubMed

Markert, Sebastian Matthias; Britz, Sebastian; Proppert, Sven; Lang, Marietta; Witvliet, Daniel; Mulcahy, Ben; Sauer, Markus; Zhen, Mei; Bessereau, Jean-Louis; Stigloher, Christian

2016-10-01

Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.

Facile sonochemical synthesis of Ag modified Bi{sub 4}Ti{sub 3}O{sub 12} nanoparticles with enhanced photocatalytic activity under visible light

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

Dutta, Dimple P., E-mail: dimpled@barc.gov.in; Tyagi, A.K.

2016-02-15

Highlights: • Ag doped/dispersed Bi{sub 4}Ti{sub 3}O{sub 12} nanoparticles synthesized sonochemically. • Undoped Bi{sub 4}Ti{sub 3}O{sub 12} exhibited 100% RhB degradation in 45 min under UV light. • Under visible light 100% RhB degradation occured with Bi{sub 4}Ti{sub 3}O{sub 12} in 150 min. • Improved photodegradation of RhB by Ag doped Bi{sub 4}Ti{sub 3}O{sub 12} in visible light. • The Bi{sub 4}Ti{sub 3}O{sub 12}:Ag(5%) sample photodegrades RhB from wastewater under sunlight. - Abstract: Unmodified and Ag modified Bi{sub 4}Ti{sub 3}O{sub 12}:Ag(x%) (x = 2 and 5) nanoparticles have been synthesized sonochemically and characterized using X-ray diffraction, X-ray photoelectron spectroscopy (XPS),more » Brunauer–Emmett–Teller (BET) surface area analysis, scanning electron microscopy (SEM), energy dispersion X-ray spectrum (EDS) analysis, transmission electron microscopy (TEM) and UV–vis diffuse reflectance spectroscopy. In the presence of unmodified Bi{sub 4}Ti{sub 3}O{sub 12} nanoparticles, complete photocatalytic degradation of Rhodamine B (RhB) was observed under UV light within 45 min. However, the response of this material for photodegradation of RhB under visible light was poor and could be greatly enhanced with Ag modification. A possible mechanism for this observation has been discussed in detail. The reusability of the material has also been tested and it has been found to have favorable recycling capability. Moreover, the Ag- modified Bi{sub 4}Ti{sub 3}O{sub 12} has been tested for RhB degradation from a wastewater sample under sunlight and promising results have been obtained.« less

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.

Accessible microscopy workstation for students and scientists with mobility impairments.

PubMed

Duerstock, Bradley S

2006-01-01

An integrated accessible microscopy workstation was designed and developed to allow persons with mobility impairments to control all aspects of light microscopy with minimal human assistance. This system, named AccessScope, is capable of performing brightfield and fluorescence microscopy, image analysis, and tissue morphometry requisite for undergraduate science courses to graduate-level research. An accessible microscope is necessary for students and scientists with mobility impairments to be able to use a microscope independently to better understand microscopical imaging concepts and cell biology. This knowledge is not always apparent by simply viewing a catalog of histological images. The ability to operate a microscope independently eliminates the need to hire an assistant or rely on a classmate and permits one to take practical laboratory examinations by oneself. Independent microscope handling is also crucial for graduate students and scientists with disabilities to perform scientific research. By making a personal computer as the user interface for controlling AccessScope functions, different upper limb mobility impairments could be accommodated by using various computer input devices and assistive technology software. Participants with a range of upper limb mobility impairments evaluated the prototype microscopy workstation. They were able to control all microscopy functions including loading different slides without assistance.

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

NASA Astrophysics Data System (ADS)

Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

2016-03-01

Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

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.

One-step growth of nanosheet-assembled BiOCl/BiOBr microspheres for highly efficient visible photocatalytic performance

NASA Astrophysics Data System (ADS)

Zhang, Jinfeng; Lv, Jiali; Dai, Kai; Liang, Changhao; Liu, Qi

2018-02-01

In this work, we have developed a simple synthetic approach of nanosheet-assembled BiOCl/BiOBr microspheres by an ethylene glycol (EG)-assisted hydrothermal method. The crystalline form, morphology, chemical composition, optical performance and surface area of BiOCl/BiOBr microspheres were identified using X-ray diffraction (XRD), transmission electron microscopy (TEM), high resolution TEM (HRTEM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy spectra (EDX), UV-vis diffuse reflectance spectroscopy (DRS) analysis, high resolution X-ray photoelectron spectra (XPS) and N2 adsorption-desorption isotherms. BiOCl/BiOBr microspheres were nanosheet-assembled particles, which possessed visible light absorption under LED light irridation. Additionally, the methylene blue (MB) photodegradation performance of different BiOCl/BiOBr microspheres irradiated under 410 nm LED light arrays were investigated, the results exhibited that as-prepared BiOCl/BiOBr products showed higher catalytic effiency than pure BiOCl or BiOBr. By optimizing the composition ration of the BiOCl and BiOBr, up to 93% degradation rate can be obtained in the 40%BiOCl/BiOBr microspheres. Finally, the photocatalytic mechanism of BiOCl/BiOBr microspheres had been proposed.

Potential of ultraviolet wide-field imaging and multiphoton microscopy for analysis of dehydroergosterol in cellular membranes.

PubMed

Wüstner, Daniel; Brewer, Jonathan R; Bagatolli, Luis; Sage, Daniel

2011-01-01

Dehydroergosterol (DHE) is an intrinsically fluorescent sterol with absorption/emission in the ultraviolet (UV) region and biophysical properties similar to those of cholesterol. We compared the potential of UV-sensitive low-light-level wide-field (UV-WF) imaging with that of multiphoton (MP) excitation microscopy to monitor DHE in living cells. Significantly reduced photobleaching in MP microscopy of DHE enabled us to acquire three-dimensional z-stacks of DHE-stained cells and to obtain high-resolution maps of DHE in surface ruffles, nanotubes, and the apical membrane of epithelial cells. We found that the lateral resolution of MP microscopy is ∼1.5-fold higher than that of UV-WF deconvolution microscopy, allowing for improved spatiotemporal analysis of plasma membrane sterol distribution. Surface intensity patterns of DHE with a diameter of 0.2 μm persisting over several minutes could be resolved by MP time-lapse microscopy. Diffusion coefficients of 0.25-μm-diameter endocytic vesicles containing DHE were determined by MP spatiotemporal image correlation spectroscopy. The requirement of extremely high laser power for visualization of DHE by MP microscopy made this method less potent for multicolor applications with organelle markers like green fluorescent protein-tagged proteins. The signal-to-noise ratio obtainable by UV-WF imaging could be significantly improved by pixelwise bleach rate fitting and calculation of an amplitude image from the decay model and by frame averaging after pixelwise bleaching correction of the image stacks. We conclude that UV-WF imaging and MP microscopy of DHE provide complementary information regarding membrane distribution and intracellular targeting of sterols. © 2010 Wiley-Liss, Inc.

Opto-electrochemical In Situ Monitoring of the Cathodic Formation of Single Cobalt Nanoparticles.

PubMed

Brasiliense, Vitor; Clausmeyer, Jan; Dauphin, Alice L; Noël, Jean-Marc; Berto, Pascal; Tessier, Gilles; Schuhmann, Wolfgang; Kanoufi, Fréderic

2017-08-21

Single-particle electrochemistry at a nanoelectrode is explored by dark-field optical microscopy. The analysis of the scattered light allows in situ dynamic monitoring of the electrodeposition of single cobalt nanoparticles down to a radius of 65 nm. Larger sub-micrometer particles are directly sized optically by super-localization of the edges and the scattered light contains complementary information concerning the particle redox chemistry. This opto-electrochemical approach is used to derive mechanistic insights about electrocatalysis that are not accessible from single-particle electrochemistry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acquired Fanconi syndrome with proximal tubular cytoplasmic fibrillary inclusions of λ light chain restriction.

PubMed

Yao, Ying; Wang, Su-Xia; Zhang, You-Kang; Wang, Yan; Liu, Li; Liu, Gang

2014-01-01

Light chain proximal tubulopathy is a rarely reported entity associated with plasma cell dyscrasia that classically manifests as acquired Fanconi syndrome and is characterized by the presence of κ-restricted crystals in the proximal tubular cytoplasm. We herein present a case of multiple myeloma with Fanconi syndrome and acute kidney injury due to light chain proximal tubulopathy with light chain cast nephropathy. Prominent phagolysosomes and numerous irregularly shaped inclusions with a fibrillary matrix in the cytoplasm of the proximal tubules were identified on electron microscopy. A monotypic light chain of the λ type was detected in the distal tubular casts, proximal tubular cytoplasmic lysosomes and fibrillary inclusions on immunofluorescence and immune electron microscopy. This case underscores the importance of conducting careful ultrastructural investigations and immunocytologic examinations of light chains for detecting and diagnosing light chain proximal tubulopathy.

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

PubMed Central

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

2016-01-01

We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

[Current approaches to evaluating the anatomic and functional status of the cornea].

PubMed

Avetisov, S E; Borodina, N V; Kobzova, M V; Musaeva, G M

2010-01-01

The review provides data on current methods for evaluating the anatomic and functional status of the cornea (light refraction, light transmission, and biomechanical properties, in particular). It analyzes the main advantages and disadvantages of basic (biomicroscopy, endothelial microscopy, ophthalmometry, topography, and pachymetry) and special (confocal microscopy, optical coherence tomography, ultrasound biomicroscopy, aberrometry, bidirectional corneal applanation, and keratoesthesiometry) studies.

Label-free, multi-scale imaging of ex-vivo mouse brain using spatial light interference microscopy

NASA Astrophysics Data System (ADS)

Min, Eunjung; Kandel, Mikhail E.; Ko, Chemyong J.; Popescu, Gabriel; Jung, Woonggyu; Best-Popescu, Catherine

2016-12-01

Brain connectivity spans over broad spatial scales, from nanometers to centimeters. In order to understand the brain at multi-scale, the neural network in wide-field has been visualized in detail by taking advantage of light microscopy. However, the process of staining or addition of fluorescent tags is commonly required, and the image contrast is insufficient for delineation of cytoarchitecture. To overcome this barrier, we use spatial light interference microscopy to investigate brain structure with high-resolution, sub-nanometer pathlength sensitivity without the use of exogenous contrast agents. Combining wide-field imaging and a mosaic algorithm developed in-house, we show the detailed architecture of cells and myelin, within coronal olfactory bulb and cortical sections, and from sagittal sections of the hippocampus and cerebellum. Our technique is well suited to identify laminar characteristics of fiber tract orientation within white matter, e.g. the corpus callosum. To further improve the macro-scale contrast of anatomical structures, and to better differentiate axons and dendrites from cell bodies, we mapped the tissue in terms of its scattering property. Based on our results, we anticipate that spatial light interference microscopy can potentially provide multiscale and multicontrast perspectives of gross and microscopic brain anatomy.

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

PubMed

Migliori, Bianca; Datta, Malika S; Dupre, Christophe; Apak, Mehmet C; Asano, Shoh; Gao, Ruixuan; Boyden, Edward S; Hermanson, Ola; Yuste, Rafael; Tomer, Raju

2018-05-29

Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Imaging a seizure model in zebrafish with structured illumination light sheet microscopy

NASA Astrophysics Data System (ADS)

Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Baraban, Scott; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

Studies on hydrothermal synthesis of photolumniscent rare earth (Eu3+ & Tb3+) doped NG@FeMoO4 for enhanced visible light photodegradation of methylene blue dye

NASA Astrophysics Data System (ADS)

Singh, R.; Kumar, M.; Khajuria, H.; Sharma, S.; Sheikh, H. Nawaz

2018-02-01

FeMoO4 nanorods and their rare earth (Eu3+ and Tb3+) doped composites with nitrogen doped graphene (NG) were synthesized by facile hydrothermal method in aqueous medium. X-ray diffraction (XRD) analysis of the as-synthesized samples was done to study the phase purity and crystalline nature. FTIR and Raman Spectroscopy have been studied for investigating the bonding in nanostructures. The surface morphology of the samples was investigated with field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The photolumniscent nature of the samples was investigated by the using the fluorescence spectrophotometer. The photocatalytic degradation efficiency of the prepared pure FeMoO4 and its rare earth doped composites with nitrogen doped graphene was evaluated as function of visible light irradiation versus concentration of methylene blue (MB dye). The prepared nanocomposites show enhanced photocatalytic efficiency as compared to the bare FeMoO4 nanorods.

Further description of Aspidodera raillieti (Nematoda: Aspidoderidae) from Didelphis marsupialis (Mammalia: Didelphidae) by light and scanning electron microscopy.

PubMed

Chagas-Moutinho, V A; Oliveira-Menezes, A; Cárdenas, M Q; Lanfredi, R M

2007-10-01

Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Freitas 1956 are widely distributed from Americas. The species of the genus Aspidodera Railliet and Henry 1912 are parasites of mammals of the orders Edentata, Marsupialia, and Rodentia. In the present work, Aspidodera raillieti (L. Travassos, Mem Inst Oswaldo Cruz 5(3):271-318, 1913), collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Valle del Cauca, Colombia, is redescribed. The association of light and scanning electron microscopy (SEM) allowed a detailed analysis of the morphology and ultrastructure of this nematode. Some taxonomic features, such as cephalic region, topography of the cuticle, sucker, spicules, posterior end of males, localization of vulva, the anus, and posterior end of females were observed. Important structures such as amphid, details of cephalic region, phasmid, and number and localization of caudal papillae are documented by SEM, for the first time adding characters to identify this species. Colombia is a new geographical record for A. raillieti.

Investigating Bacterial-Animal Symbioses with Light Sheet Microscopy

PubMed Central

Taormina, Michael J.; Jemielita, Matthew; Stephens, W. Zac; Burns, Adam R.; Troll, Joshua V.; Parthasarathy, Raghuveer; Guillemin, Karen

2014-01-01

SUMMARY Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high resolution imaging of bacterial colonization of the zebrafish intestine. The methodology allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional datasets generated by these imaging approaches require new strategies for image analysis. When integrated with other “omics” datasets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts. PMID:22983029

Femtosecond digital lensless holographic microscopy to image biological samples.

PubMed

Mendoza-Yero, Omel; Calabuig, Alejandro; Tajahuerce, Enrique; Lancis, Jesús; Andrés, Pedro; Garcia-Sucerquia, Jorge

2013-09-01

The use of femtosecond laser radiation in digital lensless holographic microscopy (DLHM) to image biological samples is presented. A mode-locked Ti:Sa laser that emits ultrashort pulses of 12 fs intensity FWHM, with 800 nm mean wavelength, at 75 MHz repetition rate is used as a light source. For comparison purposes, the light from a light-emitting diode is also used. A section of the head of a drosophila melanogaster fly is studied with both light sources. The experimental results show very different effects of the pinhole size on the spatial resolution with DLHM. Unaware phenomena on the field of the DLHM are analyzed.

Brain tumor classification using AFM in combination with data mining techniques.

PubMed

Huml, Marlene; Silye, René; Zauner, Gerald; Hutterer, Stephan; Schilcher, Kurt

2013-01-01

Although classification of astrocytic tumors is standardized by the WHO grading system, which is mainly based on microscopy-derived, histomorphological features, there is great interobserver variability. The main causes are thought to be the complexity of morphological details varying from tumor to tumor and from patient to patient, variations in the technical histopathological procedures like staining protocols, and finally the individual experience of the diagnosing pathologist. Thus, to raise astrocytoma grading to a more objective standard, this paper proposes a methodology based on atomic force microscopy (AFM) derived images made from histopathological samples in combination with data mining techniques. By comparing AFM images with corresponding light microscopy images of the same area, the progressive formation of cavities due to cell necrosis was identified as a typical morphological marker for a computer-assisted analysis. Using genetic programming as a tool for feature analysis, a best model was created that achieved 94.74% classification accuracy in distinguishing grade II tumors from grade IV ones. While utilizing modern image analysis techniques, AFM may become an important tool in astrocytic tumor diagnosis. By this way patients suffering from grade II tumors are identified unambiguously, having a less risk for malignant transformation. They would benefit from early adjuvant therapies.

White-light diffraction phase microscopy at doubled space-bandwidth product.

PubMed

Shan, Mingguang; Kandel, Mikhail E; Majeed, Hassaan; Nastasa, Viorel; Popescu, Gabriel

2016-12-12

White light diffraction microscopy (wDPM) is a quantitative phase imaging method that benefits from both temporal and spatial phase sensitivity, granted, respectively, by the common-path geometry and white light illumination. However, like all off-axis quantitative phase imaging methods, wDPM is characterized by a reduced space-bandwidth product compared to phase shifting approaches. This happens essentially because the ultimate resolution of the image is governed by the period of the interferogram and not just the diffraction limit. As a result, off-axis techniques generates single-shot, i.e., high time-bandwidth, phase measurements, at the expense of either spatial resolution or field of view. Here, we show that combining phase-shifting and off-axis, the original space-bandwidth is preserved. Specifically, we developed phase-shifting diffraction phase microscopy with white light, in which we measure and combine two phase shifted interferograms. Due to the white light illumination, the phase images are characterized by low spatial noise, i.e., <1nm pathlength. We illustrate the operation of the instrument with test samples, blood cells, and unlabeled prostate tissue biopsy.

Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p.

PubMed

Bodvard, Kristofer; Wrangborg, David; Tapani, Sofia; Logg, Katarina; Sliwa, Piotr; Blomberg, Anders; Kvarnström, Mats; Käll, Mikael

2011-02-01

Light exposure is a potentially powerful stress factor during in vivo optical microscopy studies. In yeast, the general transcription factor Msn2p translocates from the cytoplasm to the nucleus in response to illumination. However, previous time-lapse fluorescence microscopy studies of Msn2p have utilized a variety of discrete exposure settings, which makes it difficult to correlate stress levels and illumination parameters. We here investigate how continuous illumination with blue light, corresponding to GFP excitation wavelengths, affects the localization pattern of Msn2p-GFP in budding yeast. The localization pattern was analyzed using a novel approach that combines wavelet decomposition and change point analysis. It was found that the Msn2p nucleocytoplasmic localization trajectories for individual cells exhibit up to three distinct and successive states; i) Msn2p localizes to the cytoplasm; ii) Msn2p rapidly shuttles between the cytoplasm and the nucleus; iii) Msn2p localizes to the nucleus. Many cells pass through all states consecutively at high light intensities, while at lower light intensities most cells only reach states i) or ii). This behaviour strongly indicates that continuous light exposure gradually increases the stress level over time, presumably through continuous accumulation of toxic photoproducts, thereby forcing the cell through a bistable region corresponding to nucleocytoplasmic oscillations. We also show that the localization patterns are dependent on protein kinase A (PKA) activity, i.e. yeast cells with constantly low PKA activity showed a stronger stress response. In particular, the nucleocytoplasmic oscillation frequency was found to be significantly higher for cells with low PKA activity for all light intensities. 2010 Elsevier B.V. All rights reserved.

UV-visible light-activated Ag-decorated, monodisperse TiO2 aggregates for treatment of the pharmaceutical oxytetracycline.

PubMed

Han, Changseok; Likodimos, Vlassis; Khan, Javed Ali; Nadagouda, Mallikarjuna N; Andersen, Joel; Falaras, Polycarpos; Rosales-Lombardi, Pablo; Dionysiou, Dionysios D

2014-10-01

Noble metal Ag-decorated, monodisperse TiO2 aggregates were successfully synthesized by an ionic strength-assisted, simple sol-gel method and were used for the photocatalytic degradation of the antibiotic oxytetracycline (OTC) under both UV and visible light (UV-visible light) irradiation. The synthesized samples were characterized by X-ray diffraction analysis (XRD); UV-vis diffuse reflectance spectroscopy; environmental scanning electron microscopy (ESEM); transmission electron microscopy (TEM); high-resolution TEM (HR-TEM); micro-Raman, energy-dispersive X-ray spectroscopy (EDS); and inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that the uniformity of TiO2 aggregates was finely tuned by the sol-gel method, and Ag was well decorated on the monodisperse TiO2 aggregates. The absorption of the samples in the visible light region increased with increasing Ag loading that was proportional to the amount of Ag precursor added in the solution over the tested concentration range. The Brunauer, Emmett, and Teller (The BET) surface area slightly decreased with increasing Ag loading on the TiO2 aggregates. Ag-decorated TiO2 samples demonstrated enhanced photocatalytic activity for the degradation of OTC under UV-visible light illumination compared to that of pure TiO2. The sample containing 1.9 wt% Ag showed the highest photocatalytic activity for the degradation of OTC under both UV-visible light and visible light illumination. During the experiments, the detected Ag leaching for the best TiO2-Ag photocatalyst was much lower than the National Secondary Drinking Water Regulation for Ag limit (0.1 mg L(-1)) issued by the US Environmental Protection Agency.

Spectrally resolved laser interference microscopy

NASA Astrophysics Data System (ADS)

Butola, Ankit; Ahmad, Azeem; Dubey, Vishesh; Senthilkumaran, P.; Singh Mehta, Dalip

2018-07-01

We developed a new quantitative phase microscopy technique, namely, spectrally resolved laser interference microscopy (SR-LIM), with which it is possible to quantify multi-spectral phase information related to biological specimens without color crosstalk using a color CCD camera. It is a single shot technique where sequential switched on/off of red, green, and blue (RGB) wavelength light sources are not required. The method is implemented using a three-wavelength interference microscope and a customized compact grating based imaging spectrometer fitted at the output port. The results of the USAF resolution chart while employing three different light sources, namely, a halogen lamp, light emitting diodes, and lasers, are discussed and compared. The broadband light sources like the halogen lamp and light emitting diodes lead to stretching in the spectrally decomposed images, whereas it is not observed in the case of narrow-band light sources, i.e. lasers. The proposed technique is further successfully employed for single-shot quantitative phase imaging of human red blood cells at three wavelengths simultaneously without color crosstalk. Using the present technique, one can also use a monochrome camera, even though the experiments are performed using multi-color light sources. Finally, SR-LIM is not only limited to RGB wavelengths, it can be further extended to red, near infra-red, and infra-red wavelengths, which are suitable for various biological applications.

3D nanoscale imaging of biological samples with laboratory-based soft X-ray sources

NASA Astrophysics Data System (ADS)

Dehlinger, Aurélie; Blechschmidt, Anne; Grötzsch, Daniel; Jung, Robert; Kanngießer, Birgit; Seim, Christian; Stiel, Holger

2015-09-01

In microscopy, where the theoretical resolution limit depends on the wavelength of the probing light, radiation in the soft X-ray regime can be used to analyze samples that cannot be resolved with visible light microscopes. In the case of soft X-ray microscopy in the water-window, the energy range of the radiation lies between the absorption edges of carbon (at 284 eV, 4.36 nm) and oxygen (543 eV, 2.34 nm). As a result, carbon-based structures, such as biological samples, posses a strong absorption, whereas e.g. water is more transparent to this radiation. Microscopy in the water-window, therefore, allows the structural investigation of aqueous samples with resolutions of a few tens of nanometers and a penetration depth of up to 10μm. The development of highly brilliant laser-produced plasma-sources has enabled the transfer of Xray microscopy, that was formerly bound to synchrotron sources, to the laboratory, which opens the access of this method to a broader scientific community. The Laboratory Transmission X-ray Microscope at the Berlin Laboratory for innovative X-ray technologies (BLiX) runs with a laser produced nitrogen plasma that emits radiation in the soft X-ray regime. The mentioned high penetration depth can be exploited to analyze biological samples in their natural state and with several projection angles. The obtained tomogram is the key to a more precise and global analysis of samples originating from various fields of life science.

Posterior vitreous detachment induced by nattokinase (subtilisin NAT): a novel enzyme for pharmacologic vitreolysis.

PubMed

Takano, Akiomi; Hirata, Akira; Ogasawara, Kazuya; Sagara, Nina; Inomata, Yasuya; Kawaji, Takahiro; Tanihara, Hidenobu

2006-05-01

To investigate the effects of intravitreal injection of nattokinase (subtilisin NAT), a serine protease that is produced by Bacillus subtilis (natto), for induction of posterior vitreous detachment (PVD). Different doses of nattokinase (1, 0.1, or 0.01 fibrin-degradation units [FU]) or physiologic saline as a control were injected into the vitreous cavity of rabbit eyes. Scanning electron microscopy was used to observe the retinal surfaces of four rabbit eyes per concentration. Histologic alterations were assessed by light microscopy, using four eyes from each group. Electroretinography (ERG) was performed to observe retinal function, ranging from 1 hour to 1 week after the nattokinase (1 or 0.1 FU) or saline solution administration, using four eyes from each group at each time point. Also, findings in all rabbits were monitored by slit lamp examination and by indirect ophthalmoscopy with a 20-D lens. Scanning electron microscopy showed smooth retinal surfaces, indicating the occurrence of PVD at 30 minutes after intervention in all the experimental eyes injected with 0.1 or 1.0 FU nattokinase, but none of the control eyes. Light microscopy and ERG analysis showed no critical change even after the use of 0.1 FU nattokinase, an amount sufficient to induce PVD. However, toxicity in the forms of preretinal hemorrhage and ERG changes was noted with the higher dose (1 FU) of nattokinase. The results suggested that nattokinase is a useful enzyme for pharmacologic vitreolysis because of its efficacy in inducing PVD.

Asymmetrical flow field-flow fractionation with multi-angle light scattering and quasi-elastic light scattering for characterization of polymersomes: comparison with classical techniques.

PubMed

Till, Ugo; Gaucher-Delmas, Mireille; Saint-Aguet, Pascale; Hamon, Glenn; Marty, Jean-Daniel; Chassenieux, Christophe; Payré, Bruno; Goudounèche, Dominique; Mingotaud, Anne-Françoise; Violleau, Frédéric

2014-12-01

Polymersomes formed from amphiphilic block copolymers, such as poly(ethyleneoxide-b-ε-caprolactone) (PEO-b-PCL) or poly(ethyleneoxide-b-methylmethacrylate), were characterized by asymmetrical flow field-flow fractionation coupled with quasi-elastic light scattering (QELS), multi-angle light scattering (MALS), and refractive index detection, leading to the determination of their size, shape, and molecular weight. The method was cross-examined with more classical ones, like batch dynamic and static light scattering, electron microscopy, and atomic force microscopy. The results show good complementarities between all the techniques; asymmetrical flow field-flow fractionation being the most pertinent one when the sample exhibits several different types of population.

Interferometric temporal focusing microscopy using three-photon excitation fluorescence.

PubMed

Toda, Keisuke; Isobe, Keisuke; Namiki, Kana; Kawano, Hiroyuki; Miyawaki, Atsushi; Midorikawa, Katsumi

2018-04-01

Super-resolution microscopy has become a powerful tool for biological research. However, its spatial resolution and imaging depth are limited, largely due to background light. Interferometric temporal focusing (ITF) microscopy, which combines structured illumination microscopy and three-photon excitation fluorescence microscopy, can overcome these limitations. Here, we demonstrate ITF microscopy using three-photon excitation fluorescence, which has a spatial resolution of 106 nm at an imaging depth of 100 µm with an excitation wavelength of 1060 nm.

Pump-probe optical microscopy for imaging nonfluorescent chromophores.

PubMed

Wei, Lu; Min, Wei

2012-06-01

Many chromophores absorb light intensely but have undetectable fluorescence. Hence microscopy techniques other than fluorescence are highly desirable for imaging these chromophores inside live cells, tissues, and organisms. The recently developed pump-probe optical microscopy techniques provide fluorescence-free contrast mechanisms by employing several fundamental light-molecule interactions including excited state absorption, stimulated emission, ground state depletion, and the photothermal effect. By using the pump pulse to excite molecules and the subsequent probe pulse to interrogate the created transient states on a laser scanning microscope, pump-probe microscopy offers imaging capability with high sensitivity and specificity toward nonfluorescent chromophores. Single-molecule sensitivity has even been demonstrated. Here we review and summarize the underlying principles of this emerging class of molecular imaging techniques.

Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens

PubMed Central

Glaser, Adam K.; Reder, Nicholas P.; Chen, Ye; McCarty, Erin F.; Yin, Chengbo; Wei, Linpeng; Wang, Yu; True, Lawrence D.; Liu, Jonathan T.C.

2017-01-01

For the 1.7 million patients per year in the U.S. who receive a new cancer diagnosis, treatment decisions are largely made after a histopathology exam. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 μm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 sec/cm2), and volumetric assessment of optically cleared core–needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens. PMID:29750130

Quadriplegic areflexic ICU illness: selective thick filament loss and normal nerve histology.

PubMed

Sander, Howard W; Golden, Marianna; Danon, Moris J

2002-10-01

Areflexic quadriplegia that occurs in the intensive care unit (ICU) is commonly ascribed to critical illness polyneuropathy based upon electrophysiology or muscle light microscopy. However, electron microscopy often documents a selective thick filament loss myopathy. Eight ICU patients who developed areflexic quadriplegia underwent biopsy. Seven patients had received steroids, and 2 had also received paralytic agents. Electrodiagnostic studies revealed absent or low-amplitude motor responses in 7. Sensory responses were normal in 5 of 6 and absent in 1. Initial electromyography revealed absent (n = 3), small (n = 3), or polyphasic (n = 1) motor unit potentials, and diffuse fibrillation potentials (n = 5). In all 8, light microscopy of muscle revealed numerous atrophic-angulated fibers and corelike lesions, and electron microscopy revealed extensive thick filament loss. Morphology of sural and intramuscular nerves, and, in one autopsied case, of the obturator nerve and multiple nerve roots, was normal. Although clinical, electrodiagnostic, and light microscopic features mimicked denervating disease, muscle electron microscopy revealed thick filament loss, and nerve histology was normal. This suggests that areflexic ICU quadriplegia is a primary myopathy and not an axonal polyneuropathy. Copyright 2002 Wiley Periodicals, Inc. Muscle Nerve 26: 499-505, 2002

The Fluids Integrated Rack and Light Microscopy Module Integrated Capabilities

NASA Technical Reports Server (NTRS)

Motil, Susan M.; Gati, Frank; Snead, John H.; Hill, Myron E.; Griffin, DeVon W.

2003-01-01

The Fluids Integrated Rack (FIR), a facility class payload, and the Light Microscopy Module (LMM), a subrack payload, are scheduled to be launched in 2005. The LMM integrated into the FIR will provide a unique platform for conducting fluids and biological experiments on ISS. The FIR is a modular, multi-user scientific research facility that will fly in the U.S. laboratory module, Destiny, of the International Space Station (ISS). The first payload in the FIR will be the Light Microscopy Module (LMM). The LMM is planned as a remotely controllable, automated, on-orbit microscope subrack facility, allowing flexible scheduling and control of fluids and biology experiments within the FIR. Key diagnostic capabilities for meeting science requirements include video microscopy to observe microscopic phenomena and dynamic interactions, interferometry to make thin film measurements with nanometer resolution, laser tweezers for particle manipulation, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure photonic properties of materials. The LMM also provides experiment sample containment for frangibles and fluids. This paper will provide a description of the current FIR and LMM designs, planned capabilities and key features. In addition a brief description of the initial five experiments planned for LMM/FIR will be provided.

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

Cooke, Gary A.; Pestovich, John A.; Huber, Heinz J.

This report presents the results for solid phase characterization (SPC) of solid samples removed from tank 241-C-108 (C-108) on August 12-13,2012, using the off-riser sampler. Samples were received at the 222-S Laboratory on August 13 and were described and photographed. The SPC analyses that were performed include scanning electron microscopy (SEM) using the ASPEX(R)l scanning electron microscope, X-ray diffraction (XRD) using the Rigaku(R) 2 MiniFlex X-ray diffractometer, and polarized light microscopy (PLM) using the Nikon(R) 3 Eclipse Pol optical microscope. The SEM is equipped with an energy dispersive X-ray spectrometer (EDS) to provide chemical information. Gary A. Cooke conducted themore » SEM analysis, John A. Pestovich performed the XRD analysis, and Dr. Heinz J. Huber performed the PLM examination. The results of these analyses are presented here.« less

Balantidium grimi n. sp. (Ciliophora, Litostomatea), a new species inhabiting the rectum of the frog Quasipaa spinosa from Lishui, China.

PubMed

Zhao, Weishan; Li, Can; Zhang, Dong; Wang, Runqiu; Zheng, Yingzhen; Zou, Hong; Li, Wenxiang; Wu, Shangong; Wang, Guitang; Li, Ming

2018-01-01

Balantidium grimi n. sp. is described from the rectum of the frog Quasipaa spinosa (Amphibia, Dicroglossidae) from Lishui, Zhejiang Province, China. The new species is described by both light microscopy (LM) and scanning electron microscopy (SEM), and a molecular phylogenetic analysis is also presented. This species has unique morphological features in that the body shape is somewhat flattened and the vestibulum is "V"-shaped, occupying nearly 3/8 to 4/7 of the body length. Only one contractile vacuole, situated at the posterior body, was observed. The phylogenetic analysis based on SSU-rDNA indicates that B. grimi groups together with B. duodeni and B. entozoon. In addition, the genus Balantidium is clearly polyphyletic. © W. Zhao et al., published by EDP Sciences, 2018.

A field trial of a PCR-based Mansonella ozzardi diagnosis assay detects high-levels of submicroscopic M. ozzardi infections in both venous blood samples and FTA card dried blood spots.

PubMed

Medeiros, Jansen Fernandes; Almeida, Tatiana Amaral Pires; Silva, Lucyane Bastos Tavares; Rubio, Jose Miguel; Crainey, James Lee; Pessoa, Felipe Arley Costa; Luz, Sergio Luiz Bessa

2015-05-20

Mansonella ozzardi is a poorly understood human filarial parasite with a broad distribution throughout Latin America. Most of what is known about its parasitism has come from epidemiological studies that have estimated parasite incidence using light microscopy. Light microscopy can, however, miss lighter, submicroscopic, infections. In this study we have compared M. ozzardi incidence estimates made using light microscopy, with estimates made using PCR. 214 DNA extracts made from Large Volume Venous Blood Samples (LVVBS) were taken from volunteers from two study sites in the Rio Solimões region: Codajás [n = 109] and Tefé [n = 105] and were subsequently assayed for M. ozzardi parasitism using a diagnostic PCR (Mo-dPCR). Peripheral finger-prick blood samples were taken from the same individuals and used for microscopic examination. Finger-prick blood, taken from individuals from Tefé, was also used for the creation of FTAcard dried blood spots (DBS) that were subsequently subjected to Mo-dPCR. Overall M. ozzardi incidence estimates made with LVVBS PCRs were 1.8 times higher than those made using microscopy (44.9% [96/214] compared with 24.3% [52/214]) and 1.5 times higher than the PCR estimates made from FTAcard DBS (48/105 versus 31/105). PCR-based detection of FTAcard DBS proved 1.3 times more sensitive at diagnosing infections from peripheral blood samples than light microscopy did: detecting 24/105 compared with 31/105. PCR of LVVBS reported the fewest number of false negatives, detecting: 44 of 52 (84.6%) individuals diagnosed by microscopy; 27 of 31 (87.1%) of those diagnosed positive from DBSs and 17 out of 18 (94.4%) of those diagnosed as positive by both alternative methodologies. In this study, Mo-dPCR of LVVBS was by far the most sensitive method of detecting M. ozzardi infections and detected submicroscopic infections. Mo-dPCR FTAcard DBS also provided a more sensitive test for M. ozzardi diagnosis than light microscopy based diagnosis did and thus in settings where only finger-prick assays can be carried-out, it may be a more reliable method of detection. Most existing M. ozzardi incidence estimates, which are often based on light microscope diagnosis, are likely to dramatically underestimate true M. ozzardi parasitism incidence levels.

Light Microscopy Microscope Experiment

NASA Image and Video Library

2016-02-04

Ground testing for the first confocal Light Microscopy Microscope (LMM) Experiment. Procter and Gamble is working with NASA Glenn scientists to prepare for a study that examines product stabilizers in a microgravity environment. The particles in the tube glow orange because they have been fluorescently tagged with a dye that reacts to green laser lights to allow construction of a 3D image point by point. The experiment, which will be sent to the ISS later this year, will help P&G develop improved product stabilizers to extend shelf life and develop more environmentally friendly packaging.

Evaluation of a new soft tipped injector for the implantation of foldable intraocular lenses.

PubMed

Kleinmann, Guy; Apple, David J

2007-08-01

To evaluate the R-INJ-04 soft-tipped injector, a new injector with an integral round nozzle manufactured by Rayner Intraocular Lenses, England. 16 Rayner C-flex intraocular lenses (IOLs; Rayner Intraocular lenses, England) ranging between +10 and +30 D (2 for each power) were tested. An ophthalmic viscoelastic device (Healon, AMO, Santa Ana, California, USA) was applied to the injectors. The IOLs were loaded according to the company injector's instructions for use and were injected into a Petri dish. After the injection, all the IOLs and nozzles were evaluated by gross (macroscopic) and microscopic analyses and photographed under a light microscope. One lens of each power and the cartridge used for the implantation were then sent for further analysis by scanning electron microscopy (SEM). The rest of the IOLs were tested for power and modulation transfer function (MTF). All the injections were successful. No damage to the IOLs or to the injectors was found by gross examination, light microscopy and SEM. No deposits were found on the IOL optical surfaces or haptics. Power and MTF analysis showed a close match with the original measurements. Our results suggest that the R-INJ-04 soft-tipped injector is safe for the implantation of the C-flex IOL with power range from 10 to 30 D. No structural damage to the IOLs or to the injectors was found, and the lens power and light transmission properties were not damaged in any way by the injection process.

Two-dimensional molybdenum disulphide nanoflakes synthesized by liquid-solid phase reaction method: regenerative photocatalytic performance under UV-visible light irradiation by advance oxidation process

NASA Astrophysics Data System (ADS)

Afsar, M. F.; Rafiq, M. A.; Siddique, Fizza; Saira, F.; Chaudhary, M. M.; Hasan, M. M.; Tok, A. I. Y.

2018-05-01

Molybdenum disulphide (MoS2) nanoflakes were prepared through liquid-solid phase reaction technique. X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM) analysis revealed the formation of pure, polycrystalline, hexagonal phase of MoS2 nanoflakes. The texture coefficient (T{c}hkl) analysis showed that (100) plane was preferentially oriented. The specific surface area of the nanoflakes was 21 m2 g‑1 as determined using Brunaure-Emmett-Teller (BET) technique. A band gap of ∼2.05 eV for MoS2 nanoflakes was estimated from UV-visible spectrum. Regenerative photocatalytic activity of MoS2 nanoflakes was assessed by degrading methylene blue (MB) and safranin-o (SO) dyes under UV-visible light irradiation. Under light irradiation, degradation efficiency for MB was ∼99.58% in 100 min while for SO it was ∼99.89% in 70 min. The MoS2 nanoflakes exhibited excellent photocatalytic performance and good stability in a wide pH range (3–11). MoS2 nanoflakes showed a high reaction rate constant (k app ) for SO ∼ 0.104 49 min‑1 and MB ∼ 0.092 18 min‑1 as compared to other MoS2 nanostructures. The obtained exceptional photocatalytic performance of MoS2 nanoflakes offers potential applications for the treatment of polluted water as well as in other correlated fields.

Development of an ultralow-light-level luminescence image analysis system for dynamic measurements of transcriptional activity in living and migrating cells.

PubMed

Maire, E; Lelièvre, E; Brau, D; Lyons, A; Woodward, M; Fafeur, V; Vandenbunder, B

2000-04-10

We have developed an approach to study in single living epithelial cells both cell migration and transcriptional activation, which was evidenced by the detection of luminescence emission from cells transfected with luciferase reporter vectors. The image acquisition chain consists of an epifluorescence inverted microscope, connected to an ultralow-light-level photon-counting camera and an image-acquisition card associated to specialized image analysis software running on a PC computer. Using a simple method based on a thin calibrated light source, the image acquisition chain has been optimized following comparisons of the performance of microscopy objectives and photon-counting cameras designed to observe luminescence. This setup allows us to measure by image analysis the luminescent light emitted by individual cells stably expressing a luciferase reporter vector. The sensitivity of the camera was adjusted to a high value, which required the use of a segmentation algorithm to eliminate the background noise. Following mathematical morphology treatments, kinetic changes of luminescent sources were analyzed and then correlated with the distance and speed of migration. Our results highlight the usefulness of our image acquisition chain and mathematical morphology software to quantify the kinetics of luminescence changes in migrating cells.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Crystal morphology of sunflower wax in soybean oil organogel

USDA-ARS?s Scientific Manuscript database

While sunflower wax has been recognized as an excellent organogelator for edible oil, the detailed morphology of sunflower wax crystals formed in an edible oil organogel has not been fully understood. In this study, polarized light microscopy, phase contrast microscopy, scanning electron microscopy ...

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

Adding an Extra Dimension to What Students See through the Light Microscope: A Lab Exercise Demonstrating Critical Analysis for Microscopy Students

ERIC Educational Resources Information Center

Garrill, Ashley

2011-01-01

This article describes an undergraduate lab exercise that demonstrates the importance of students thinking critically about what they see through a microscope. The students are given growth data from tip-growing organisms that suggest the cells grow in a pulsatile manner. The students then critique this data in several exercises that incorporate…

Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

DOE PAGES

Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; ...

2016-01-01

Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection

PubMed Central

Zhi, Yanan; Wang, Benquan; Yao, Xincheng

2016-01-01

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches—including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy—have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact–free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina. PMID:27480461

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase. II. Light-microscopic application.

PubMed

Beier, K; Fahimi, H D

1987-01-01

The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semithin (0.25 and 1 micron) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for light-microscopic analysis may account for the differences. The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.

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.

Reproducibility in light microscopy: Maintenance, standards and SOPs.

PubMed

Deagle, Rebecca C; Wee, Tse-Luen Erika; Brown, Claire M

2017-08-01

Light microscopy has grown to be a valuable asset in both the physical and life sciences. It is a highly quantitative method available in individual research laboratories and often centralized in core facilities. However, although quantitative microscopy is becoming a customary tool in research, it is rarely standardized. To achieve accurate quantitative microscopy data and reproducible results, three levels of standardization must be considered: (1) aspects of the microscope, (2) the sample, and (3) the detector. The accuracy of the data is only as reliable as the imaging system itself, thereby imposing the need for routine standard performance testing. Depending on the task some maintenance procedures should be performed once a month, some before each imaging session, while others conducted annually. This text should be implemented as a resource for researchers to integrate with their own standard operating procedures to ensure the highest quality quantitative microscopy data. Copyright © 2017. Published by Elsevier Ltd.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection.

PubMed

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

NASA Astrophysics Data System (ADS)

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

PubMed

Yuan, Yongming; Li, Mingyou; Hong, Yunhan

2014-07-15

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. Copyright © 2014 Elsevier B.V. All rights reserved.

Reevaluation of Physaloptera bispiculata (Nematoda: Spiruroidaea) by light and scanning electron microscopy.

PubMed

Mafra, A C; Lanfredi, R M

1998-06-01

This study was undertaken to clarify several aspects of morphological and taxonomic characters of Physaloptera bispiculata Vaz and Pereira, 1935, a parasite of the water rat, Nectomys squamipes. The cephalic structures (including lips, papillae, teeth, amphids, and porous areas) and details of the posterior end of male and female adult worms were examined by scanning electron microscopy, leading to the addition of new taxonomic characters for this species. We consider P. bispiculata a valid species, based on a comparative analysis of the specific characters for P. bispiculata and P. getula Seurat, 1917, including the morphology and morphometry of body structures as well as number and disposition of caudal papillae of the males.

Quadratic grating apodized photon sieves for simultaneous multiplane microscopy

NASA Astrophysics Data System (ADS)

Cheng, Yiguang; Zhu, Jiangping; He, Yu; Tang, Yan; Hu, Song; Zhao, Lixin

2017-10-01

We present a new type of imaging device, named quadratic grating apodized photon sieve (QGPS), used as the objective for simultaneous multiplane imaging in X-rays. The proposed QGPS is structured based on the combination of two concepts: photon sieves and quadratic gratings. Its design principles are also expounded in detail. Analysis of imaging properties of QGPS in terms of point-spread function shows that QGPS can image multiple layers within an object field onto a single image plane. Simulated and experimental results in visible light both demonstrate the feasibility of QGPS for simultaneous multiplane imaging, which is extremely promising to detect dynamic specimens by X-ray microscopy in the physical and life sciences.

Light sheet microscopy reveals more gradual light attenuation in light green versus dark green soybean leaves

USDA-ARS?s Scientific Manuscript database

Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This oversaturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light, causing a steep gradient in carbon fixation. Reducing chl content could create a mor...

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

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Web-based virtual microscopy at the RWTH Aachen University: didactic concept, methods and analysis of acceptance by the students.

PubMed

Merk, Magdalene; Knuechel, Ruth; Perez-Bouza, Alberto

2010-12-20

Fundamental knowledge of microscopic anatomy and pathology has always been an essential part in medical education. The traditional didactic concept comprises theoretical and practical lessons using a light microscope and glass slides. High-speed Internet connections and technical improvement in whole-slide digital microscopy (commonly termed "virtual microscopy") provide a new and attractive approach for both teachers and students. High picture quality and unlimited temporal and spatial availability of histology samples from different fields are key advantages of web-based digital microscopy. In this report we discuss the technical requirements, system efficiency, optical resolution and didactic concept. Furthermore, we present a review of the experience gained in the course of one year based on an analysis of student acceptance. Three groups with a total of 192 students between the 3rd and 5th year of medical studies attending the practical courses of general and advanced histopathology had access to both glass-mounted and digitalized slides. Prior to exams, students were asked to answer an anonymous questionnaire. The results of the study reflect the high acceptance and intensive use of the web-based digital histology by students, thus encouraging the development of further Web-based learning strategies for the teaching of histology and pathology. 2010 Elsevier GmbH. All rights reserved.

Analysis of Four Automated Urinalysis Systems Compared to Reference Methods.

PubMed

Bartosova, Kamila; Kubicek, Zdenek; Franekova, Janka; Louzensky, Gustav; Lavrikova, Petra; Jabor, Antonin

2016-11-01

The aim of this study was to compare four automated urinalysis systems: the Iris iQ200 Sprint (Iris Diagnostics, U.S.A.) combined with the Arkray AUTION MAX AX 4030, Iris + AUTION, Arkray AU 4050 (Arkray Global Business, Inc., Japan), Dirui FUS 2000 (Dirui Industrial Co., P.R.C.), and Menarini sediMAX (Menarini, Italy). Urine concentrations of protein and glucose (Iris, Dirui) were compared using reference quantitative analysis on an Abbott Architect c16000. Leukocytes, erythrocytes, epithelia, and casts (Iris, Arkray, Diuri, Menarini) were compared to urine sediment under reference light microscopy, Leica DM2000 (Leica Microsystems GmbH, Germany) with calibrated FastRead plates (Biosigma S.r.l., Italy), using both native and stained preparations. Total protein and glucose levels were measured using the Iris + AUTION system with borderline trueness, while the Dirui analysis revealed worse performances for the protein and glucose measurements. True classifications of leukocytes and erythrocytes were above 85% and 72%, respectively. Kappa statistics revealed a nearly perfect evaluation of leukocytes for all tested systems; the erythrocyte evaluation was nearly perfect for the Iris, Dirui and Arkray analyzers and substantial for the Menarini analyzer. The epithelia identification was connected to high false negativity (above 15%) in the Iris, Arkray, and Menarini analyses. False-negative casts were above 70% for all tested systems. The use of automated urinalysis demonstrated some weaknesses and should be checked by experienced laboratory staff using light microscopy.

Development of software and modification of Q-FISH protocol for estimation of individual telomere length in immunopathology.

PubMed

Barkovskaya, M Sh; Bogomolov, A G; Knauer, N Yu; Rubtsov, N B; Kozlov, V A

2017-04-01

Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (C 3 TA[Formula: see text] peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated.

Evaluation of laser ablation microtomy for correlative microscopy of hard tissues.

PubMed

Boyde, A

2018-02-27

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 μm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.

PubMed

Amat, Fernando; Keller, Philipp J

2013-05-01

Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

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

Mechanical behavior and failure analysis of prosthetic retaining screws after long-term use in vivo. Part 4: Failure analysis of 10 fractured retaining screws retrieved from three patients.

PubMed

Al Jabbari, Youssef S; Fournelle, Raymond; Ziebert, Gerald; Toth, Jeffrey; Iacopino, Anthony M

2008-04-01

The aim of this study was to perform a failure analysis on fractured prosthetic retaining screws after long-term use in vivo. Additionally, the study addresses the commonly asked question regarding whether complex repeated functional occlusal forces initiate fatigue-type cracks in prosthetic retaining screws. Ten fractured prosthetic retaining screws retrieved from three patients treated with fixed detachable hybrid prostheses were subjected to a failure analysis. In patients 1 and 2, the middle three retaining screws of the prostheses were found fractured at retrieval time after they had been in service for 20 and 19 months, respectively. In patient 3, the middle three and one of the posterior retaining screws were found to be fractured at retrieval after they had been in service for 18 months. Low power stereomicroscopy and high-power scanning electron microscopy (SEM) were performed to analyze the fractured surfaces of the retaining screws examining fatigue cracks in greater detail. Typical fatigue failure characterized by ratchet mark formation was revealed by light microscopy and SEM for all examined screws. Using low magnification light microscopy, ratchet marks were visible on the fracture surfaces of only two screws. SEM examination revealed all three classical stages of fatigue failure, and it was possible to see the ratchet marks on the fracture surfaces of all specimens, indicating a fatigue zone. The final catastrophic overload fracture appeared fibrous, indicating ductile fracture. The final overload ductile fracture surfaces showed equiaxed dimples, suggesting tensile overload in all examined screws except in two specimens that showed an elongated dimple pattern indicating shear/tearing overload forces. Fracture of prosthetic retaining screws in hybrid prostheses occurs mainly through a typical fatigue mode involving mostly the middle anterior three screws. Fatigue cracks can grow in more than one prosthetic retaining screw, leading to fracture before the patient or clinician determines that any problem exists.

In situ fabrication of the Bi2O3-V2O5 hybrid embedded with graphitic carbon nitride nanosheets: Oxygen vacancies mediated enhanced visible-light-driven photocatalytic degradation of organic pollutants and hydrogen evolution

NASA Astrophysics Data System (ADS)

Vattikuti, S. V. Prabhakar; Police, Anil Kumar Reddy; Shim, Jaesool; Byon, Chan

2018-07-01

Novel mesoporous ternary hybrids comprising Bi2O3/V2O5 photocatalysts anchored on graphitic carbon nitride (g-C3N4) nanosheets were synthesized via an in situ co-pyrolysis approach and characterized by a series of techniques, including X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy (TEM), high-resolution TEM, X-ray photoelectron spectroscopy, Brunauer-Emmett-Teller analysis, thermogravimetric-differential thermal analysis, Fourier transform infrared spectroscopy, ultraviolet-visible spectrometry, photoluminescence and electron paramagnetic resonance (EPR). The hybrids were subsequently tested as photocatalysts for the degradation of the phenol red (PR) pollutant under visible light irradiation. The well-designed ternary hybrids showed pure and randomly distributed Bi2O3/V2O5 (denoted as BiV) nanoparticles on monodispersed g-C3N4 nanosheets. The as-prepared ternary Bi2O3/V2O5@g-C3N4 (i.e., BiV@g-C3N4) hybrids demonstrated high specific surface areas with remarkable mesoporous characteristics. The photodegradation efficiencies of the ternary hybrids for PR were 1.2 and 1.8 times higher than those of binary BiV and pristine Bi2O3, respectively, at 50 min irradiation time under simulated solar light irradiation. At the end of the phototreatment, the amount of PR pollutant was reduced to 98.1% in 50 min by using the BiV@g-C3N4 nanocomposites under simulated solar light irradiation and more efficient for photocatalytic H2 production. Based on an electrochemical analysis, we propose a photocatalytic degradation pathway for PR under visible light irradiation. In addition, the BiV@g-C3N4 nanocomposite photocatalysts exhibited both long-term stability and photocatalytic efficiency for the degradation of the PR dye. The excellent photoelectrochemical performance of the BiV@g-C3N4 photocatalysts can be ascribed to their highly dispersed V2O5 and Bi2O3 nanoparticles, mesoporous structure, and high specific surface area (83.75 m2 g-1).

Gold-FISH: A correlative approach to microscopic imaging of single microbial cells in environmental samples

NASA Astrophysics Data System (ADS)

Schmidt, Hannes; Seki, David; Woebken, Dagmar; Eickhorst, Thilo

2017-04-01

Fluorescence in situ hybridization (FISH) is routinely used for the phylogenetic identification, detection, and quantification of single microbial cells environmental microbiology. Oligonucleotide probes that match the 16S rRNA sequence of target organisms are generally applied and the resulting signals are visualized via fluorescence microscopy. Consequently, the detection of the microbial cells of interest is limited by the resolution and the sensitivity of light microscopy where objects smaller than 0.2 µm can hardly be represented. Visualizing microbial cells at magnifications beyond light microscopy, however, can provide information on the composition and potential complexity of microbial habitats - the actual sites of nutrient cycling in soil and sediments. We present a recently developed technique that combines (1) the phylogenetic identification and detection of individual microorganisms by epifluorescence microscopy, with (2) the in situ localization of gold-labelled target cells on an ultrastructural level by SEM. Based on 16S rRNA targeted in situ hybridization combined with catalyzed reporter deposition, a streptavidin conjugate labeled with a fluorescent dye and nanogold particles is introduced into whole microbial cells. A two-step visualization process including an autometallographic enhancement of nanogold particles then allows for either fluorescence or electron microscopy, or a correlative application thereof. We will present applications of the Gold-FISH protocol to samples of marine sediments, agricultural soils, and plant roots. The detection and enumeration of bacterial cells in soil and sediment samples was comparable to CARD-FISH applications via fluorescence microscopy. Examples of microbe-surface interaction analysis will be presented on the basis of bacteria colonizing the rhizoplane of rice roots. In principle, Gold-FISH can be performed on any material to give a snapshot of microbe-surface interactions and provides a promising tool for the acquisition of correlative information on microorganisms within their respective habitats.

[Polarized light microscopy for evaluation of oocytes as a prognostic factor in the evolution of a cycle in assisted reproduction].

PubMed

González-Ortega, C; Cancino-Villarreal, P; Alonzo-Torres, V E; Martínez-Robles, I; Pérez-Peña, E; Gutiérrez-Gutiérrez, A M

2016-04-01

Identification of the best embryos to transfer is a key element for success in assisted reproduction. In the last decade, several morphological criteria of oocytes and embryos were evaluated with regard to their potential for predicting embryo viability. The introduction of polarization light microscopy systems has allowed the visualization of the meiotic spindle and the different layers of the zona pellucida in human oocytes on the basis of birefringence in a non-destructive way. Conflicting results have been reported regarding the predictive value in ICSI cycles. To assess the predictive ability of meiotic spindle and zona pellucida of human oocytes to implant by polarized microscopy in ICSI cycles. Prospective and observational clinical study. 903 oocytes from 94 ICSI cycles were analyzed with polarized microscopy. Meiotic spindle visualization and zona pellucida birefringence values by polarized microscopy were correlated with ICSI cycles results. Meiotic spindle visualization and birefringence values of zona pellucida decreased in a direct basis with increasing age. In patients aged over the 35 years, the percentage of a visible spindle and mean zona pellucida birefringence was lower than in younger patients. Fertilization rate were higher in oocytes with visible meiotic spindle (81.3% vs. 64%; p < 0.0001), as well as embryo quality (47.4% vs. 39%; p=0.01). Fertilization rate was higher in oocytes with positive values of birefringence (77.5 % vs. 68.5% p=0.005) with similar embryo quality. Conception cycles showed oocytes with higher mean value of zona birefringence and visible spindle vs. no-conception cycles (p<0.05). Polarized light microscopy improves oocyte selection, which significantly impacts in the development of embryos with greater implantation potential. The use of polarized light microscopy with sperm selection methods, blastocyst culture and deferred embryo transfers will contribute to transfer fewer embryos without diminishing rates of live birth and single embryo transfer will be more feasible.

Role of Eu{sup 2+} on the blue‐green photoluminescence of In{sub 2}O{sub 3}:Eu{sup 2+} nanocrystals

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

Devi, Konsam Reenabati, E-mail: reena.kay14@manipuruniv.ac.in; Meetei, Sanoujam Dhiren, E-mail: sdmdhiren@gmail.com; Department of Physics, North Eastern Regional Institute of Science & Technology, Nirjuli, Itanagar 791109, Arunachal Pradesh

Blue‐green light emitting undoped and europium doped indium oxide nanocrystal were synthesized by simple precipitation method. X-ray diffraction (XRD) pattern confirmed the cubic phase of undoped and europium doped samples. Further, transmission electron microscopy (TEM), scanning electron microscopy (SEM) , energy dispersive analysis of X-rays (EDAX), Fourier transform infra-red (FT-IR), photoluminescence (PL), electron paramagnetic resonance (EPR) studies were performed to characterise the samples. PL analysis of the samples is the core of the present research. It includes excitation, emission and CIE (Commission Internationale de l’e´ clairage) studies of the samples. On doping europium to In{sub 2}O{sub 3} lattice, ln{sup 3+}more » site is substituted by Eu{sup 2+} thereby increasing the concentration of singly ionized oxygen vacancy and hence blue–green emission from the host is found to increase. Further, this increase in blue–green emission after doping may also be attributed to 4f → 5d transitions of Eu{sup 2+}. However, the blue–green PL emission is found to decrease after an optimum dopant concentration (Eu{sup 2+} = 4%) due to luminescence and size quenching. CIE co-ordinates of the samples are calculated to know colour of light emitted from the samples. It suggests that this blue–green light emitting In{sub 2}O{sub 3}: Eu{sup 2+} nanocrystals may find application in lighting such as in generation of white light. - Highlight: • XRD and TEM study confirms the synthesis of cubic doped and europium doped nanocrystals. • EPR study reveals the doped europium is in + 2 oxidation state. • Enhance PL emission intensity of host material due to increase in singly ionized oxygen vacancy and 4f–5d transitions of Eu{sup 2+} • CIE co-ordinates suggest the blue–green colour of the samples.« less

SearchLight: a freely available web-based quantitative spectral analysis tool (Conference Presentation)

NASA Astrophysics Data System (ADS)

Prabhat, Prashant; Peet, Michael; Erdogan, Turan

2016-03-01

In order to design a fluorescence experiment, typically the spectra of a fluorophore and of a filter set are overlaid on a single graph and the spectral overlap is evaluated intuitively. However, in a typical fluorescence imaging system the fluorophores and optical filters are not the only wavelength dependent variables - even the excitation light sources have been changing. For example, LED Light Engines may have a significantly different spectral response compared to the traditional metal-halide lamps. Therefore, for a more accurate assessment of fluorophore-to-filter-set compatibility, all sources of spectral variation should be taken into account simultaneously. Additionally, intuitive or qualitative evaluation of many spectra does not necessarily provide a realistic assessment of the system performance. "SearchLight" is a freely available web-based spectral plotting and analysis tool that can be used to address the need for accurate, quantitative spectral evaluation of fluorescence measurement systems. This tool is available at: http://searchlight.semrock.com/. Based on a detailed mathematical framework [1], SearchLight calculates signal, noise, and signal-to-noise ratio for multiple combinations of fluorophores, filter sets, light sources and detectors. SearchLight allows for qualitative and quantitative evaluation of the compatibility of filter sets with fluorophores, analysis of bleed-through, identification of optimized spectral edge locations for a set of filters under specific experimental conditions, and guidance regarding labeling protocols in multiplexing imaging assays. Entire SearchLight sessions can be shared with colleagues and collaborators and saved for future reference. [1] Anderson, N., Prabhat, P. and Erdogan, T., Spectral Modeling in Fluorescence Microscopy, http://www.semrock.com (2010).

Spectral analysis of scattered light from flowers' petals

NASA Astrophysics Data System (ADS)

Ozawa, Atsumi; Uehara, Tomomi; Sekiguchi, Fumihiko; Imai, Hajime

2009-07-01

A new method was developed for studying absorption characteristics of opaque samples based on the light scattering spectroscopy. Measurements were made in white, red and violet petals of Petunia hybrida, and gave the absorption spectra in a non-destructive manner without damaging the cell structures of the petal. The red petal has absorption peak at 550 nm and the violet has three absorption peaks: at 450, 670, and 550 nm. The results were discussed in correlation with the microscopic cell structures of the petal observed with optical microscope and transmission electron microscopy (TEM). Only the cells placed in the surface have the pigments giving the color of the petal.

A national facility for biological cryo-electron microscopy

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

Saibil, Helen R., E-mail: h.saibil@mail.cryst.bbk.ac.uk; Grünewald, Kay; Stuart, David I.

2015-01-01

This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron. Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided ofmore » the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.« less

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

Direct correlations of structural and optical properties of three-dimensional GaN/InGaN core/shell micro-light emitting diodes

NASA Astrophysics Data System (ADS)

Sadat Mohajerani, Matin; Müller, Marcus; Hartmann, Jana; Zhou, Hao; Wehmann, Hergo-H.; Veit, Peter; Bertram, Frank; Christen, Jürgen; Waag, Andreas

2016-05-01

Three-dimensional (3D) InGaN/GaN quantum-well (QW) core-shell light emitting diodes (LEDs) are a promising candidate for the future solid state lighting. In this contribution, we study direct correlations of structural and optical properties of the core-shell LEDs using highly spatially-resolved cathodoluminescence spectroscopy (CL) in combination with scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM). Temperature-dependent resonant photoluminescence (PL) spectroscopy has been performed to understand recombination mechanisms and to estimate the internal quantum efficiency (IQE).

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

PubMed

Tran, Daniel N; Smith, Sandy A B C; Brown, David A; Parker, Andrew J C; Joseph, Joanne E; Armstrong, Nicola; Sewell, William A

2017-03-01

There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Advanced SLMs for microscopy

NASA Astrophysics Data System (ADS)

Linnenberger, A.

2018-02-01

Wavefront shaping devices such as deformable mirrors, liquid crystal spatial light modulators (SLMs), and active lenses are of considerable interest in microscopy for aberration correction, volumetric imaging, and programmable excitation. Liquid crystal SLMs are high resolution phase modulators capable of creating complex phase profiles to reshape, or redirect light within a three-dimensional (3D) volume. Recent advances in Meadowlark Optics (MLO) SLMs reduce losses by increasing fill factor from 83.4% to 96%, and improving resolution from 512 x 512 pixels to 1920 x 1152 pixels while maintaining a liquid crystal response time of 300 Hz at 1064 nm. This paper summarizes new SLM capabilities, and benefits for microscopy.

Pterygodermatites (Mesopectines) quentini (Nematoda, Rictulariidae), a parasite of Praomys rostratus (Rodentia, Muridae) in Mali: scanning electron and light microscopy

PubMed Central

2013-01-01

Pterygodermatites (Mesopectines) quentini n. sp. (Nematoda, Rictulariidae) is described from the murine host Praomys rostratus in the south of the Republic of Mali. It differs from other species of the subgenus by the morphology of the head, which bears four simple cephalic papillae and a nearly axial oral opening, the number of caudal papillae, the number of precloacal cuticular formations, unequal spicules and the ratio of spicule lengths/body length. The use of scanning electron microscopy in combination with conventional light microscopy enabled us to give a detailed description of the morphological characters of this new species. PMID:24025692

Visible Light Assisted Photocatalytic Hydrogen Generation by Ta 2O 5/Bi 2O 3, TaON/Bi 2O 3, and Ta 3N 5/Bi 2O 3 Composites

DOE PAGES

Adhikari, Shiba; Hood, Zachary D.; More, Karren Leslie; ...

2015-06-15

Composites comprised of two semiconducting materials with suitable band gaps and band positions have been reported to be effective at enhancing photocatalytic activity in the visible light region of the electromagnetic spectrum. Here, we report the synthesis, complete structural and physical characterizations, and photocatalytic performance of a series of semiconducting oxide composites. UV light active tantalum oxide (Ta2O5) and visible light active tantalum oxynitride (TaON) and tantalum nitride (Ta 3N 5) were synthesized, and their composites with Bi 2O 3 were prepared in situ using benzyl alcohol as solvent. The composite prepared using equimolar amounts of Bi 2O 3 andmore » Ta 2O 5 leads to the formation of the ternary oxide, bismuth tantalate (BiTaO 4) upon calcination at 1000 °C. The composites and single phase bismuth tantalate formed were characterized by powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), Brunauer–Emmett–Teller (BET) surface area measurement, scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV–Vis diffuse reflectance spectroscopy, and photoluminescence. The photocatalytic activities of the catalysts were evaluated for generation of hydrogen using aqueous methanol solution under visible light irradiation (λ ≥ 420 nm). The results show that as-prepared composite photocatalysts extend the light absorption range and restrict photogenerated charge-carrier recombination, resulting in enhanced photocatalytic activity compared to individual phases. The mechanism for the enhanced photocatalytic activity for the heterostructured composites is elucidated based on observed activity, band positions calculations, and photoluminescence data.« less

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

PubMed

Ryan, Duncan P; Gould, Elizabeth A; Seedorf, Gregory J; Masihzadeh, Omid; Abman, Steven H; Vijayaraghavan, Sukumar; Macklin, Wendy B; Restrepo, Diego; Shepherd, Douglas P

2017-09-20

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

Multispectral plasmon coupling microscopy and its application in bio-imaging

NASA Astrophysics Data System (ADS)

Wang, Hongyun

A broad range of cellular activities, including receptor mediated endocytosis, signaling and receptor clustering, involve multi-body interactions between different cellular functionalities. Many of these interactions are dynamic in nature, making optical tools the method of choice for their investigation. Conventional optical microscopy has a resolution about 300nm, limited by the diffraction of light, which is insufficient to explore processes that occur on nanometer or tens of nanometer length scales. The aim of this thesis is to develop and validate a plasmon coupling microscopy (PCM), which utilizes the distance dependent spectral properties of coupled noble metal nanoparticles (NPs) to resolve distance changes between NP labels on deeply sub-diffraction length scales. This colorimetric approach is augmented with a polarization sensitive analysis of the scattered light of individual dimers to monitor simultaneously distance and orientation changes. The distance dependent polarization anisotropy in discrete dimers is investigated experimentally and theoretically. The performed analysis reveals that the polarization anisotropy is robust even against relatively large refractive index changes. The polarization sensitive PCM is then applied to characterize the lateral spatial organization of mammalian plasma membranes by analyzing the translational and rotational motion as well as the extension of discrete NP dimers during their diffusion on lysed HeLa cell membranes. The membrane is found to be compartmentalized with typical domain sizes on the order of 70nm. The functionality of plasmon coupling based imaging method is expanded further by developing a multispectral imaging modality for a quantitative analysis of the plasmon coupling between many noble metal immunolabels in a large field of view simultaneously. This approach provides information about the spatial organization of the silver nanoparticle labels and thus of targeted EGF receptor densities on the surface of epidermoid carcinoma cells (A431). Finally, multispectral plasmon coupling microscopy is applied to investigate the uptake and subsequent intracellular spatial distribution of silver nanoparticles in murine macrophage cells (J774A.1). The studies reveal that NP uptake is mediated by scavenger receptors and that the intracellular NP association and distribution are heterogeneous among cells in a cellular ensemble. The heterogeneity is demonstrated to be correlated with the maturation status of the macrophages.

Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

PubMed Central

Mertz, Jerome; Kim, Jinhyun

2010-01-01

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast. PMID:20210471

Calibrating excitation light fluxes for quantitative light microscopy in cell biology

PubMed Central

Grünwald, David; Shenoy, Shailesh M; Burke, Sean; Singer, Robert H

2011-01-01

Power output of light bulbs changes over time and the total energy delivered will depend on the optical beam path of the microscope, filter sets and objectives used, thus making comparison between experiments performed on different microscopes complicated. Using a thermocoupled power meter, it is possible to measure the exact amount of light applied to a specimen in fluorescence microscopy, regardless of the light source, as the light power measured can be translated into a power density at the sample. This widely used and simple tool forms the basis of a new degree of calibration precision and comparability of results among experiments and setups. Here we describe an easy-to-follow protocol that allows researchers to precisely estimate excitation intensities in the object plane, using commercially available opto-mechanical components. The total duration of this protocol for one objective and six filter cubes is 75 min including start-up time for the lamp. PMID:18974739

Radial super-resolution in digital holographic microscopy using structured illumination with circular symmetry

NASA Astrophysics Data System (ADS)

Yin, Yujian; Su, Ping; Ma, Jianshe

2018-01-01

A method to improve the radial resolution using special structured light is proposed in the field of digital holographic microscopy (DHM). A specimen is illuminated with circular symmetrical structured light that makes the spectrum have radial movement, so that high frequency components of the specimen are moved into the passband of the receiver to overcome the diffraction limit. In the DHM imaging system, Computer Generated Hologram (CGH) technology is used to generate the required structured light grating. Then the grating is loaded into a spatial light modulator (SLM) to obtain specific structured illumination. After recording the hologram, digital reconstruction, for the microstructure of a binary optical element that needs to observe radial distribution, the radial resolution of the specimen is improved experimentally compare it with the result of one-dimensional sinusoidal structured light imaging. And a method of designing structured light is presented.

High Prevalence of Giardia duodenalis Assemblage B Infection and Association with Underweight in Rwandan Children

PubMed Central

Klotz, Christian; Steininger, Christian; Shyirambere, Cyprien; Lyng, Michel; Musemakweri, Andre; Aebischer, Toni; Martus, Peter; Harms, Gundel; Mockenhaupt, Frank P.

2012-01-01

Background Giardia duodenalis is highly endemic in East Africa but its effects on child health, particularly of submicroscopic infections, i.e., those below the threshold of microscopy, and of genetic subgroups (assemblages), are not well understood. We aimed at addressing these questions and at examining epidemiological characteristics of G. duodenalis in southern highland Rwanda. Methodology/Principal Findings In 583 children <5 years of age from communities and health facilities, intestinal parasites were assessed by triplicate light microscopy and by PCR assays, and G. duodenalis assemblages were genotyped. Cluster effects of villages were taken into account in statistical analysis. The prevalence of G. duodenalis as detected by microscopy was 19.8% but 60.1% including PCR results. Prevalence differed with residence, increased with age, and was reduced by breastfeeding. In 492 community children without, with submicroscopic and with microscopic infection, underweight (weight-for-age z-score <−2 standard deviations) was observed in 19.7%, 22.1%, and 33.1%, respectively, and clinically assessed severe malnutrition in 4.5%, 9.5%, and 16.7%. Multivariate analysis identified microscopically detectable G. duodenalis infection as an independent predictor of underweight and clinically assessed severe malnutrition. Submicroscopic infection showed respective trends. Overall, G. duodenalis was not associated with gastrointestinal symptoms but assemblages A parasites (proportion, 13%) were increased among children with vomiting and abdominal pain. Conclusions/Significance The prevalence of G. duodenalis in high-endemicity areas may be greatly underestimated by light microscopy, particularly when only single stool samples are analysed. Children with submicroscopic infections show limited overt manifestation, but constitute unrecognized reservoirs of transmission. The predominance of assemblage B in Rwanda may be involved in the seemingly unimposing manifestation of G. duodenalis infection. However, the association with impaired child growth points to its actual relevance. Longitudinal studies considering abundant submicroscopic infections are needed to clarify the actual contribution of G. duodenalis to morbidity in areas of high endemicity. PMID:22720102

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy, morphometry and confocal laser scanning microscopy.

PubMed

Souza, Joyce; Garcia, Juberlan; Neves, Renata H; Machado-Silva, José Roberto; Maldonado, Arnaldo

2013-12-01

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy. Copyright © 2013. Published by Elsevier Inc.

Diatomite silica nanoparticles for drug delivery

NASA Astrophysics Data System (ADS)

Ruggiero, Immacolata; Terracciano, Monica; Martucci, Nicola M.; De Stefano, Luca; Migliaccio, Nunzia; Tatè, Rosarita; Rendina, Ivo; Arcari, Paolo; Lamberti, Annalisa; Rea, Ilaria

2014-07-01

Diatomite is a natural fossil material of sedimentary origin, constituted by fragments of diatom siliceous skeletons. In this preliminary work, the properties of diatomite nanoparticles as potential system for the delivery of drugs in cancer cells were exploited. A purification procedure, based on thermal treatments in strong acid solutions, was used to remove inorganic and organic impurities from diatomite and to make them a safe material for medical applications. The micrometric diatomite powder was reduced in nanoparticles by mechanical crushing, sonication, and filtering. Morphological analysis performed by dynamic light scattering and transmission electron microscopy reveals a particles size included between 100 and 300 nm. Diatomite nanoparticles were functionalized by 3-aminopropyltriethoxysilane and labeled by tetramethylrhodamine isothiocyanate. Different concentrations of chemically modified nanoparticles were incubated with cancer cells and confocal microscopy was performed. Imaging analysis showed an efficient cellular uptake and homogeneous distribution of nanoparticles in cytoplasm and nucleus, thus suggesting their potentiality as nanocarriers for drug delivery.

A polyphasic taxonomic approach in isolated strains of Cyanobacteria from thermal springs of Greece.

PubMed

Bravakos, Panos; Kotoulas, Georgios; Skaraki, Katerina; Pantazidou, Adriani; Economou-Amilli, Athena

2016-05-01

Strains of Cyanobacteria isolated from mats of 9 thermal springs of Greece have been studied for their taxonomic evaluation. A polyphasic taxonomic approach was employed which included: morphological observations by light microscopy and scanning electron microscopy, maximum parsimony, maximum likelihood and Bayesian analysis of 16S rDNA sequences, secondary structural comparisons of 16S-23S rRNA Internal Transcribed Spacer sequences, and finally environmental data. The 17 cyanobacterial isolates formed a diverse group that contained filamentous, coccoid and heterocytous strains. These included representatives of the polyphyletic genera of Synechococcus and Phormidium, and the orders Oscillatoriales, Spirulinales, Chroococcales and Nostocales. After analysis, at least 6 new taxa at the genus level provide new evidence in the taxonomy of Cyanobacteria and highlight the abundant diversity of thermal spring environments with many potential endemic species or ecotypes. Copyright © 2016 Elsevier Inc. All rights reserved.

Extracellular space preservation aids the connectomic analysis of neural circuits.

PubMed

Pallotto, Marta; Watkins, Paul V; Fubara, Boma; Singer, Joshua H; Briggman, Kevin L

2015-12-09

Dense connectomic mapping of neuronal circuits is limited by the time and effort required to analyze 3D electron microscopy (EM) datasets. Algorithms designed to automate image segmentation suffer from substantial error rates and require significant manual error correction. Any improvement in segmentation error rates would therefore directly reduce the time required to analyze 3D EM data. We explored preserving extracellular space (ECS) during chemical tissue fixation to improve the ability to segment neurites and to identify synaptic contacts. ECS preserved tissue is easier to segment using machine learning algorithms, leading to significantly reduced error rates. In addition, we observed that electrical synapses are readily identified in ECS preserved tissue. Finally, we determined that antibodies penetrate deep into ECS preserved tissue with only minimal permeabilization, thereby enabling correlated light microscopy (LM) and EM studies. We conclude that preservation of ECS benefits multiple aspects of the connectomic analysis of neural circuits.

Nanostructured zirconium phosphate as ion exchanger: Synthesis, size dependent property and analytical application in radiochemical separation.

PubMed

Chakraborty, Rajesh; Bhattacharaya, Koustava; Chattopadhyay, Pabitra

2014-02-01

Nanostructured zirconium phosphates (ZPs) of different sizes were synthesized using Tritron X-100 (polyethylene glycol-p-isooctylphenyl ether) surfactant. The materials were characterized by FTIR and powdered X-ray diffraction (XRD). The structural and morphological details of the material were established by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM study was followed by energy dispersive spectroscopic analysis (EDS) for elemental analysis of the sample. The particle sizes were determined by dynamic light scattering (DLS) method. Ion exchange capacity of these nanomaterials towards different metal ions was measured and size-dependent ion exchange property of the materials was investigated thoroughly. The nanomaterial of the smallest size (ca. 21.04nm) was employed to separate carrier-free (137m)Ba from (137)Cs in column chromatographic technique using 1.0M HNO3 as eluting agent at pH=5. © 2013 Elsevier Ltd. All rights reserved.

Photofunctionalization and non-thermal plasma activation of titanium surfaces.

PubMed

Henningsen, Anders; Smeets, Ralf; Hartjen, Philip; Heinrich, Oliver; Heuberger, Roman; Heiland, Max; Precht, Clarissa; Cacaci, Claudio

2018-03-01

The aim of this study was to compare UV light and non-thermal plasma (NTP) treatment regarding the improvement of physical material characteristics and cell reaction on titanium surfaces in vitro after short-term functionalization. Moderately rough (Ra 1.8-2.0 μm) sandblasted and acid-etched titanium disks were treated by UV light (0.05 mW/cm 2 at λ = 360 nm and 2 mW/cm 2 at λ = 250 nm) or by NTP (24 W, -0.5 mbar) of argon or oxygen for 12 min each. Surface structure was investigated by scanning electron microscopy, confocal microscopy and X-ray photoelectron spectroscopy (XPS). Hydrophilicity was assessed by dynamic contact angle measurement. Cell attachment, viability, cell proliferation and cytotoxicity were assessed in vitro using murine osteoblast-like cells. UV irradiation or NTP treatment of titanium surfaces did not alter the surface structure. XPS analysis revealed a significantly increased oxidation of the surface and a decrease of carbon after the use of either method. NTP and UV light led to a significant better cell attachment of murine osteoblasts; significantly more osteoblasts grew on the treated surfaces at each time point (p < 0.001). UV light as well as NTP modified the surface of titanium and significantly improved the conditions for murine osteoblast cells in vitro. However, results indicate a slight advantage for NTP of argon and oxygen in a short time interval of surface functionalization compared to UV. UV light and NTP are able to improve surface conditions of dental implants made of titanium.

Hydrodynamic fractionation of finite size gold nanoparticle clusters.

PubMed

Tsai, De-Hao; Cho, Tae Joon; DelRio, Frank W; Taurozzi, Julian; Zachariah, Michael R; Hackley, Vincent A

2011-06-15

We demonstrate a high-resolution in situ experimental method for performing simultaneous size classification and characterization of functional gold nanoparticle clusters (GNCs) based on asymmetric-flow field flow fractionation (AFFF). Field emission scanning electron microscopy, atomic force microscopy, multi-angle light scattering (MALS), and in situ ultraviolet-visible optical spectroscopy provide complementary data and imagery confirming the cluster state (e.g., dimer, trimer, tetramer), packing structure, and purity of fractionated populations. An orthogonal analysis of GNC size distributions is obtained using electrospray-differential mobility analysis (ES-DMA). We find a linear correlation between the normalized MALS intensity (measured during AFFF elution) and the corresponding number concentration (measured by ES-DMA), establishing the capacity for AFFF to quantify the absolute number concentration of GNCs. The results and corresponding methodology summarized here provide the proof of concept for general applications involving the formation, isolation, and in situ analysis of both functional and adventitious nanoparticle clusters of finite size. © 2011 American Chemical Society

Investigating portable fluorescent microscopy (CyScope) as an alternative rapid diagnostic test for malaria in children and women of child-bearing age.

PubMed

Sousa-Figueiredo, José Carlos; Oguttu, David; Adriko, Moses; Besigye, Fred; Nankasi, Andrina; Arinaitwe, Moses; Namukuta, Annet; Betson, Martha; Kabatereine, Narcis B; Stothard, J Russell

2010-08-27

Prompt and correct diagnosis of malaria is crucial for accurate epidemiological assessment and better case management, and while the gold standard of light microscopy is often available, it requires both expertise and time. Portable fluorescent microscopy using the CyScope offers a potentially quicker, easier and more field-applicable alternative. This article reports on the strengths, limitations of this methodology and its diagnostic performance in cross-sectional surveys on young children and women of child-bearing age. 552 adults (99% women of child-bearing age) and 980 children (99% ≤ 5 years of age) from rural and peri-urban regions of Ugandan were examined for malaria using light microscopy (Giemsa-stain), a lateral-flow test (Paracheck-Pf) and the CyScope. Results from the surveys were used to calculate diagnostic performance (sensitivity and specificity) as well as to perform a receiver operating characteristics (ROC) analyses, using light microscopy as the gold-standard. Fluorescent microscopy (qualitative reads) showed reduced specificity (<40%), resulting in higher community prevalence levels than those reported by light microscopy, particularly in adults (+180% in adults and +20% in children). Diagnostic sensitivity was 92.1% in adults and 86.7% in children, with an area under the ROC curve of 0.63. Importantly, optimum performance was achieved for higher parasitaemia (>400 parasites/μL blood): sensitivity of 64.2% and specificity of 86.0%. Overall, the diagnostic performance of the CyScope was found inferior to that of Paracheck-Pf. Fluorescent microscopy using the CyScope is certainly a field-applicable and relatively affordable solution for malaria diagnoses especially in areas where electrical supplies may be lacking. While it is unlikely to miss higher parasitaemia, its application in cross-sectional community-based studies leads to many false positives (i.e. small fluorescent bodies of presently unknown origin mistaken as malaria parasites). Without recourse to other technologies, arbitration of these false positives is presently equivocal, which could ultimately lead to over-treatment; something that should be further explored in future investigations if the CyScope is to be more widely implemented.

Using SRμCT to define water transport capacity in Picea abies

NASA Astrophysics Data System (ADS)

Lautner, Silke; Lenz, Claudia; Hammel, Jörg; Moosmann, Julian; Kühn, Michael; Caselle, Michele; Vogelgesang, Matthias; Kopmann, Andreas; Beckmann, Felix

2017-10-01

Water transport from roots to shoots is a vital necessity in trees in order to sustain their photosynthetic activity and, hence, their physiological activity. The vascular tissue in charge is the woody body of root, stem and branches. In gymnosperm trees, like spruce trees (Picea abies (L.) Karst.), vascular tissue consists of tracheids: elongated, protoplast- free cells with a rigid cell wall that allow for axial water transport via their lumina. In order to analyze the over-all water transport capacity within one growth ring, time-consuming light microscopy analysis of the woody sample still is the conventional approach for calculating tracheid lumen area. In our investigations at the Imaging Beamline (IBL) operated by the Helmholtz-Zentrum Geesthacht (HZG) at PETRA III storage ring of the Deutsches Elektronen-Synchrotron DESY, Hamburg, we applied SRμCT on small wood samples of spruce trees in order to visualize and analyze size and formation of xylem elements and their respective lumina. The selected high-resolution phase-contrast technique makes full use of the novel 20 MPixel CMOS area detector developed within the cooperation of HZG and the Karlsruhe data by light microscopy analysis and, hence, prove, that μCT is a most appropriate method to gain valid information on xylem cell structure and tree water transport capacity.

Scanning electron microscopy and roughness study of dental composite degradation.

PubMed

Soares, Luís Eduardo Silva; Cortez, Louise Ribeiro; Zarur, Raquel de Oliveira; Martin, Airton Abrahão

2012-04-01

Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350® 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist®; group 3, Listerine®; group 4, Colgate Plax®; LED LCU, group 5, saliva; group 6, Pepsi Twist®; group 7, Listerine®; group 8, Colgate Plax®. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist® groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite.

Detection of oxygen species generated by WO{sub 3} modification fullerene/TiO{sub 2} in the degradation of 1,5-diphenyl carbazide

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

Meng, Ze-Da; Department of Advanced Materials Science and Engineering, Hanseo University, Seosan-si, Chungnam-do 356-706; Zhu, Lei

2014-08-15

Highlights: • Reactive oxygen species were detected through oxidation reaction from DPCI to DPCO. • Generated reactive oxygen species and hydroxyl radicals can be analysed by DPCI degradation. • C{sub 60} have good effect in photo/ultrasonic degradation process. • Enhanced adsorption capacity can increase the degradation effect. • Photocatalytic activity attributed to photo-absorption effect by C{sub 60} and cooperative effect of WO{sub 3} and TiO{sub 2}. - Abstract: In the present work, powders with different relative mole ratios of WO{sub 3}-fullerene/TiO{sub 2} were irradiated by visible light and ultrasonic. The composite obtained was characterized by BET surface area measurements, X-raymore » diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), fourier transform infrared (FT-IR), transmission electron microscopy (TEM) and UV–vis analysis. A methylene blue (MB) solution under visible light and ultrasonic irradiation was used to determine the catalytic activity. The generation of reactive oxygen species were detected through the oxidation reaction from 1,5-diphenyl carbazide (DPCI) to 1,5-diphenyl carbazone (DPCO). Finally, we performed experiments to find the optimum relative mole ratio of fullerene for the degradation of MB.« less

Palladium-doped-ZrO2-multiwalled carbon nanotubes nanocomposite: an advanced photocatalyst for water treatment

NASA Astrophysics Data System (ADS)

Anku, William Wilson; Oppong, Samuel Osei-Bonsu; Shukla, Sudheesh Kumar; Agorku, Eric Selorm; Govender, Poomani Penny

2016-06-01

The photocatalytic degradation of organic pollutants from water using palladium-doped-zirconium oxide-multiwalled carbon nanotubes (Pd-ZrO2-MWCNTs) nanocomposites is presented. A series of Pd doped-ZrO2-MWCNTs nanocomposites with varying percentage compositions of Pd were prepared by the homogenous co-precipitation method. The photocatalytic applicability of the materials was investigated by the degradation of acid blue 40 dye in water under simulated solar light. The optical, morphological and structural properties of the nanocomposites were evaluated using X-ray powder diffraction, Fourier transformer infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, BET surface area analysis and (UV-Vis) spectroscopy. The Pd-ZrO2-MWCNTs nanocomposites showed enhanced photocatalytic activity toward the degradation of the acid blue 40 dye under visible light compared with bare ZrO2 and ZrO2-MWCNTs alone. The remarkable photocatalytic activity of Pd-ZrO2-MWCNTs nanocomposites in the visible light makes it an ideal photocatalyst for the removal of organic pollutants in water. The 0.5 % Pd-ZrO2-MWCNT was the most efficient photocatalyst with 98 % degradation after 3 h with corresponding K a and band gap values of 16.8 × 10-3 m-1 and 2.79 eV, respectively.

Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

PubMed

Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian

2015-03-01

Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

2012-03-01

Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

Surface analysis of graphite fiber reinforced polyimide composites

NASA Technical Reports Server (NTRS)

Messick, D. L.; Progar, D. J.; Wightman, J. P.

1983-01-01

Several techniques have been used to establish the effect of different surface pretreatments on graphite-polyimide composites. Composites were prepared from Celion 6000 graphite fibers and the polyimide LARC-160. Pretreatments included mechanical abrasion, chemical etching and light irradiation. Scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used in the analysis. Contact angle of five different liquids of varying surface tensions were measured on the composites. SEM results showed polymer-rich peaks and polymer-poor valleys conforming to the pattern of the release cloth used durng fabrication. Mechanically treated and light irradiated samples showed varying degrees of polymer peak removal, with some degradation down to the graphite fibers. Minimal changes in surface topography were observed on concentrations of surface fluorine even after pretreatment. The light irradiation pretreatment was most effective at reducing surface fluorine concentrations whereas chemical pretreatment was the least effective. Critical surface tensions correlated directly with the surface fluorine to carbon ratios as calculated from XPS.

Microstructure of Dense Thin Sheets of gamma-TiAl Fabricated by Hot Isostatic Pressing of Tape-Cast Monotapes (Preprint)

DTIC Science & Technology

2007-02-01

fabrication of dense thin sheets of gamma titanium aluminide . Polarized light microscopy revealed a fine-grained microstructure but a few isolated...HIPed (near-gamma) microstructure occurred. 15. SUBJECT TERMS gamma titanium aluminide , thin sheet, tape casting, hot isostatic pressing 16...sheets (250–300 μm thick) of gamma titanium aluminide (γ-TiAl). Polarized light microscopy revealed a fine-grained microstructure (average grain

Subcellular pigment distribution is altered under far-red light acclimation in cyanobacteria that contain chlorophyll f.

PubMed

Majumder, Erica L-W; Wolf, Benjamin M; Liu, Haijun; Berg, R Howard; Timlin, Jerilyn A; Chen, Min; Blankenship, Robert E

2017-11-01

Far-Red Light (FRL) acclimation is a process that has been observed in cyanobacteria and algae that can grow solely on light above 700 nm. The acclimation to FRL results in rearrangement and synthesis of new pigments and pigment-protein complexes. In this study, cyanobacteria containing chlorophyll f, Synechococcus sp. PCC 7335 and Halomicronema hongdechloris, were imaged as live cells with confocal microscopy. H. hongdechloris was further studied with hyperspectral confocal fluorescence microscopy (HCFM) and freeze-substituted thin-section transmission electron microscopy (TEM). Under FRL, phycocyanin-containing complexes and chlorophyll-containing complexes were determined to be physically separated and the synthesis of red-form phycobilisome and Chl f was increased. The timing of these responses was observed. The heterogeneity and eco-physiological response of the cells was noted. Additionally, a gliding motility for H. hongdechloris is reported.

Multiphoton imaging with high peak power VECSELs

NASA Astrophysics Data System (ADS)

Mirkhanov, Shamil; Quarterman, Adrian H.; Swift, Samuel; Praveen, Bavishna B.; Smyth, Conor J. C.; Wilcox, Keith G.

2016-03-01

Multiphoton imaging (MMPI) has become one of thee key non-invasive light microscopy techniques. This technique allows deep tissue imaging with high resolution and less photo-damage than conventional confocal microscopy. MPI is type of laser-scanning microscopy that employs localized nonlinear excitation, so that fluorescence is excited only with is scanned focal volume. For many years, Ti: sapphire femtosecond lasers have been the leading light sources for MPI applications. However, recent developments in laser sources and new types of fluorophores indicate that longer wavelength excitation could be a good alternative for these applications. Mode-locked VECSEELs have the potential to be low cost, compact light sources for MPI systems, with the additional advantage of broad wavelength coverage through use of different semiconductor material systems. Here, we use a femtosecond fibber laser to investigate the effect average power and repetition rate has on MPI image quality, to allow us to optimize our mode-locked VVECSELs for MPI.

Advances in Light Microscopy for Neuroscience

PubMed Central

Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

2010-01-01

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

Perspectives in Super-resolved Fluorescence Microscopy: What comes next?

NASA Astrophysics Data System (ADS)

Cremer, Christoph; Birk, Udo

2016-04-01

The Nobel Prize in Chemistry 2014 has been awarded to three scientists involved in the development of STED and PALM super-resolution fluorescence microscopy (SRM) methods. They have proven that it is possible to overcome the hundred year old theoretical limit for the resolution potential of light microscopy (of about 200 nm for visible light), which for decades has precluded a direct glimpse of the molecular machinery of life. None of the present-day super-resolution techniques have invalidated the Abbe limit for light optical detection; however, they have found clever ways around it. In this report, we discuss some of the challenges still to be resolved before arising SRM approaches will be fit to bring about the revolution in Biology and Medicine envisaged. Some of the challenges discussed are the applicability to image live and/or large samples, the further enhancement of resolution, future developments of labels, and multi-spectral approaches.

Phase Sensitive Demodulation in Multiphoton Microscopy

NASA Astrophysics Data System (ADS)

Fisher, Walt G.; Piston, David W.; Wachter, Eric A.

2002-06-01

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

Multispectral digital lensless holographic microscopy: from femtosecond laser to white light LED

NASA Astrophysics Data System (ADS)

Garcia-Sucerquia, J.

2015-04-01

The use of femtosecond laser radiation and super bright white LED in digital lensless holographic microscopy is presented. For the ultrafast laser radiation two different configurations of operation of the microscope are presented and the dissimilar performance of each one analyzed. The microscope operating with a super bright white light LED in combination with optical filters shows very competitive performance as it is compared with more expensive optical sources. The broadband emission of both radiation sources allows the multispectral imaging of biological samples to obtain spectral responses and/or full color images of the microscopic specimens; sections of the head of a Drosophila melanogaster fly are imaged in this contribution. The simple, solid, compact, lightweight, and reliable architecture of digital lensless holographic microscopy operating with broadband light sources to image biological specimens exhibiting micrometer-sized details is evaluated in the present contribution.

Microcircuit failure analysis using the SEM. [Scanning Electron Microscopes

NASA Technical Reports Server (NTRS)

Nicolas, D. P.

1974-01-01

The scanning electron microscope adds a new dimension to the knowledge that can be obtained from a failed microcircuit. When used with conventional techniques, SEM assists and clarifies the analysis, but it does not replace light microscopy. The most advantageous features for microcircuit analysis are long working distances and great depth of field. Manufacturer related failure modes of microcircuits are metallization defects, poor bonding, surface and particle contamination, and design and fabrication faults. User related failure modes are caused by abuse, such as overstress. The Physics of Failure Procedure followed by the Astrionics Laboratory in failure analysis is described, which is designed to obtain maximum information available from each step.

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

PubMed

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

An Investigation of the Incorporation of Virtual Microscopy in the Cytotechnology Educational Curriculum

ERIC Educational Resources Information Center

Mukherjee, Maheswari S.

2012-01-01

Traditionally, cytotechnology (CT) students have been trained by using light microscopy (LM) and glass slides. However, this method of training has some drawbacks. Several other educational programs with similar issues have incorporated virtual microscopy (VM) in their curricula. In VM, the specimens on glass slides are converted into virtual…

Scanning Capacitance Microscopy | Materials Science | NREL

Science.gov Websites

obtained using scanning capacitance microscopy. Top Right: Image of p-type and n-type material, obtained 'fingers' of light-colored n-type material on a yellow and blue background representing p-type material ; measurement data were obtained using scanning capacitance microscopy. Bottom Right: Image of p-type and n-type

Electronic Blending in Virtual Microscopy

ERIC Educational Resources Information Center

Maybury, Terrence S.; Farah, Camile S.

2010-01-01

Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats

PubMed Central

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis. PMID:27078690

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

PubMed Central

HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

2015-01-01

Summary In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells. PMID:25786567

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis.

Use of a white light supercontinuum laser for confocal interference-reflection microscopy

PubMed Central

Chiu, L-D; Su, L; Reichelt, S; Amos, WB

2012-01-01

Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

Characterisation of a major phytoplankton bloom in the River Thames (UK) using flow cytometry and high performance liquid chromatography.

PubMed

Moorhouse, H L; Read, D S; McGowan, S; Wagner, M; Roberts, C; Armstrong, L K; Nicholls, D J E; Wickham, H D; Hutchins, M G; Bowes, M J

2018-05-15

Recent river studies have observed rapid phytoplankton dynamics, driven by diurnal cycling and short-term responses to storm events, highlighting the need to adopt new high-frequency characterisation methods to understand these complex ecological systems. This study utilised two such analytical methods; pigment analysis by high performance liquid chromatography (HPLC) and cell counting by flow cytometry (FCM), alongside traditional chlorophyll spectrophotometry and light microscopy screening, to characterise the major phytoplankton bloom of 2015 in the River Thames, UK. All analytical techniques observed a rapid increase in chlorophyll a concentration and cell abundances from March to early June, caused primarily by a diatom bloom. Light microscopy identified a shift from pennate to centric diatoms during this period. The initial diatom bloom coincided with increased HPLC peridinin concentrations, indicating the presence of dinoflagellates which were likely to be consuming the diatom population. The diatom bloom declined rapidly in early June, coinciding with a storm event. There were low chlorophyll a concentrations (by both HPLC and spectrophotometric methods) throughout July and August, implying low biomass and phytoplankton activity. However, FCM revealed high abundances of pico-chlorophytes and cyanobacteria through July and August, showing that phytoplankton communities remain active and abundant throughout the summer period. In combination, these techniques are able to simultaneously characterise a wider range of phytoplankton groups, with greater certainty, and provide improved understanding of phytoplankton functioning (e.g. production of UV inhibiting pigments by cyanobacteria in response to high light levels) and ecological status (through examination of pigment degradation products). Combined HPLC and FCM analyses offer rapid and cost-effective characterisation of phytoplankton communities at appropriate timescales. This will allow a more-targeted use of light microscopy to capture phytoplankton peaks or to investigate periods of rapid community succession. This will lead to greater system understanding of phytoplankton succession in response to biogeochemical drivers. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

40 CFR 61.146 - Standard for spraying.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Microscopy, except as provided in paragraph (c) of this section. (b) For spray-on application of materials..., subpart E, 40 CFR part 763, section 1, Polarized Light Microscopy, on equipment and machinery, except as...

Nano titania aided clustering and adhesion of beneficial bacteria to plant roots to enhance crop growth and stress management.

PubMed

Palmqvist, N G M; Bejai, S; Meijer, J; Seisenbaeva, G A; Kessler, V G

2015-05-13

A novel use of Titania nanoparticles as agents in the nano interface interaction between a beneficial plant growth promoting bacterium (Bacillus amyloliquefaciens UCMB5113) and oilseed rape plants (Brassica napus) for protection against the fungal pathogen Alternaria brassicae is presented. Two different TiO2 nanoparticle material were produced by the Sol-Gel approach, one using the patented Captigel method and the other one applying TiBALDH precursor. The particles were characterized by transmission electron microscopy, thermogravimetric analysis, X-ray diffraction, dynamic light scattering and nano particle tracking analysis. Scanning electron microscopy showed that the bacterium was living in clusters on the roots and the combined energy-dispersive X-ray spectroscopy analysis revealed that titanium was present in these cluster formations. Confocal laser scanning microscopy further demonstrated an increased bacterial colonization of Arabidopsis thaliana roots and a semi-quantitative microscopic assay confirmed an increased bacterial adhesion to the roots. An increased amount of adhered bacteria was further confirmed by quantitative fluorescence measurements. The degree of infection by the fungus was measured and quantified by real-time-qPCR. Results showed that Titania nanoparticles increased adhesion of beneficial bacteria on to the roots of oilseed rape and protected the plants against infection.

Nano titania aided clustering and adhesion of beneficial bacteria to plant roots to enhance crop growth and stress management

NASA Astrophysics Data System (ADS)

Palmqvist, N. G. M.; Bejai, S.; Meijer, J.; Seisenbaeva, G. A.; Kessler, V. G.

2015-05-01

A novel use of Titania nanoparticles as agents in the nano interface interaction between a beneficial plant growth promoting bacterium (Bacillus amyloliquefaciens UCMB5113) and oilseed rape plants (Brassica napus) for protection against the fungal pathogen Alternaria brassicae is presented. Two different TiO2 nanoparticle material were produced by the Sol-Gel approach, one using the patented Captigel method and the other one applying TiBALDH precursor. The particles were characterized by transmission electron microscopy, thermogravimetric analysis, X-ray diffraction, dynamic light scattering and nano particle tracking analysis. Scanning electron microscopy showed that the bacterium was living in clusters on the roots and the combined energy-dispersive X-ray spectroscopy analysis revealed that titanium was present in these cluster formations. Confocal laser scanning microscopy further demonstrated an increased bacterial colonization of Arabidopsis thaliana roots and a semi-quantitative microscopic assay confirmed an increased bacterial adhesion to the roots. An increased amount of adhered bacteria was further confirmed by quantitative fluorescence measurements. The degree of infection by the fungus was measured and quantified by real-time-qPCR. Results showed that Titania nanoparticles increased adhesion of beneficial bacteria on to the roots of oilseed rape and protected the plants against infection.

Super-Resolution Imaging Strategies for Cell Biologists Using a Spinning Disk Microscope

PubMed Central

Hosny, Neveen A.; Song, Mingying; Connelly, John T.; Ameer-Beg, Simon; Knight, Martin M.; Wheeler, Ann P.

2013-01-01

In this study we use a spinning disk confocal microscope (SD) to generate super-resolution images of multiple cellular features from any plane in the cell. We obtain super-resolution images by using stochastic intensity fluctuations of biological probes, combining Photoactivation Light-Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM) methodologies. We compared different image analysis algorithms for processing super-resolution data to identify the most suitable for analysis of particular cell structures. SOFI was chosen for X and Y and was able to achieve a resolution of ca. 80 nm; however higher resolution was possible >30 nm, dependant on the super-resolution image analysis algorithm used. Our method uses low laser power and fluorescent probes which are available either commercially or through the scientific community, and therefore it is gentle enough for biological imaging. Through comparative studies with structured illumination microscopy (SIM) and widefield epifluorescence imaging we identified that our methodology was advantageous for imaging cellular structures which are not immediately at the cell-substrate interface, which include the nuclear architecture and mitochondria. We have shown that it was possible to obtain two coloured images, which highlights the potential this technique has for high-content screening, imaging of multiple epitopes and live cell imaging. PMID:24130668

Nano titania aided clustering and adhesion of beneficial bacteria to plant roots to enhance crop growth and stress management

PubMed Central

Palmqvist, N. G. M.; Bejai, S.; Meijer, J.; Seisenbaeva, G. A.; Kessler, V. G.

2015-01-01

A novel use of Titania nanoparticles as agents in the nano interface interaction between a beneficial plant growth promoting bacterium (Bacillus amyloliquefaciens UCMB5113) and oilseed rape plants (Brassica napus) for protection against the fungal pathogen Alternaria brassicae is presented. Two different TiO2 nanoparticle material were produced by the Sol-Gel approach, one using the patented Captigel method and the other one applying TiBALDH precursor. The particles were characterized by transmission electron microscopy, thermogravimetric analysis, X-ray diffraction, dynamic light scattering and nano particle tracking analysis. Scanning electron microscopy showed that the bacterium was living in clusters on the roots and the combined energy-dispersive X-ray spectroscopy analysis revealed that titanium was present in these cluster formations. Confocal laser scanning microscopy further demonstrated an increased bacterial colonization of Arabidopsis thaliana roots and a semi-quantitative microscopic assay confirmed an increased bacterial adhesion to the roots. An increased amount of adhered bacteria was further confirmed by quantitative fluorescence measurements. The degree of infection by the fungus was measured and quantified by real-time-qPCR. Results showed that Titania nanoparticles increased adhesion of beneficial bacteria on to the roots of oilseed rape and protected the plants against infection. PMID:25970693

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.

Visualization of HIV T Cell Virological Synapses and Virus-Containing Compartments by Three-Dimensional Correlative Light and Electron Microscopy

PubMed Central

Wang, Lili; Eng, Edward T.; Law, Kenneth; Gordon, Ronald E.; Rice, William J.

2016-01-01

ABSTRACT Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses. PMID:27847357

Optimization of the excitation light sheet in selective plane illumination microscopy

PubMed Central

Gao, Liang

2015-01-01

Selective plane illumination microscopy (SPIM) allows rapid 3D live fluorescence imaging on biological specimens with high 3D spatial resolution, good optical sectioning capability and minimal photobleaching and phototoxic effect. SPIM gains its advantage by confining the excitation light near the detection focal plane, and its performance is determined by the ability to create a thin, large and uniform excitation light sheet. Several methods have been developed to create such an excitation light sheet for SPIM. However, each method has its own strengths and weaknesses, and tradeoffs must be made among different aspects in SPIM imaging. In this work, we present a strategy to select the excitation light sheet among the latest SPIM techniques, and to optimize its geometry based on spatial resolution, field of view, optical sectioning capability, and the sample to be imaged. Besides the light sheets discussed in this work, the proposed strategy is also applicable to estimate the SPIM performance using other excitation light sheets. PMID:25798312

Vasotropic light-chain amyloidosis and ischaemic cholangiopathy.

PubMed

Johnston, Emma L; Wilkinson, Mark; Knisely, A S

2015-06-25

A 75-year-old woman was incidentally found to have deranged liver function tests (LFTs). She was well, apart from 2 years of dyspnoea. Investigations had revealed atrial fibrillation and a right pleural effusion, without identified aetiology. On examination, the only finding was a palpable liver edge. Initial blood and ultrasound screening suggested no cause. The patient underwent liver biopsy. Microscopy showed κ-immunoglobulin light chains deposited exclusively in portal tracts, within blood vessel and bile duct walls. This pattern, although unusual, raised the possibility of κ-light chain disease. Serum electrophoresis was normal, as were serum immunoglobulin values. Serum concentrations of κ-light chains were elevated and microscopy of aspirated bone marrow found light-chain deposits with 10% plasmacytosis. Serum amyloid P (SAP) scintigraphy demonstrated splenic uptake. Myeloma, κ-light chain, with light-chain amyloidosis was diagnosed. The patient has responded well to cyclophosphamide, bortazomib and dexamethasone chemotherapy, and her LFTs are now nearly normal. 2015 BMJ Publishing Group Ltd.

Fractal propagation method enables realistic optical microscopy simulations in biological tissues

PubMed Central

Glaser, Adam K.; Chen, Ye; Liu, Jonathan T.C.

2017-01-01

Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method’s flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy. PMID:28983499

Optical spectroscopy and microscopy of radiation-induced light-emitting point defects in lithium fluoride crystals and films

NASA Astrophysics Data System (ADS)

Montereali, R. M.; Bonfigli, F.; Menchini, F.; Vincenti, M. A.

2012-08-01

Broad-band light-emitting radiation-induced F2 and F3+ electronic point defects, which are stable and laser-active at room temperature in lithium fluoride crystals and films, are used in dosimeters, tuneable color-center lasers, broad-band miniaturized light sources and novel radiation imaging detectors. A brief review of their photoemission properties is presented, and their behavior at liquid nitrogen temperatures is discussed. Some experimental data from optical spectroscopy and fluorescence microscopy of these radiation-induced point defects in LiF crystals and thin films are used to obtain information about the coloration curves, the efficiency of point defect formation, the effects of photo-bleaching processes, etc. Control of the local formation, stabilization, and transformation of radiation-induced light-emitting defect centers is crucial for the development of optically active micro-components and nanostructures. Some of the advantages of low temperature measurements for novel confocal laser scanning fluorescence microscopy techniques, widely used for spatial mapping of these point defects through the optical reading of their visible photoluminescence, are highlighted.

Correlative SEM SERS for quantitative analysis of dimer nanoparticles.

PubMed

Timmermans, F J; Lenferink, A T M; van Wolferen, H A G M; Otto, C

2016-11-14

A Raman microscope integrated with a scanning electron microscope was used to investigate plasmonic structures by correlative SEM-SERS analysis. The integrated Raman-SEM microscope combines high-resolution electron microscopy information with SERS signal enhancement from selected nanostructures with adsorbed Raman reporter molecules. Correlative analysis is performed for dimers of two gold nanospheres. Dimers were selected on the basis of SEM images from multi aggregate samples. The effect of the orientation of the dimer with respect to the polarization state of the laser light and the effect of the particle gap size on the Raman signal intensity is observed. Additionally, calculations are performed to simulate the electric near field enhancement. These simulations are based on the morphologies observed by electron microscopy. In this way the experiments are compared with the enhancement factor calculated with near field simulations and are subsequently used to quantify the SERS enhancement factor. Large differences between experimentally observed and calculated enhancement factors are regularly detected, a phenomenon caused by nanoscale differences between the real and 'simplified' simulated structures. Quantitative SERS experiments reveal the structure induced enhancement factor, ranging from ∼200 to ∼20 000, averaged over the full nanostructure surface. The results demonstrate correlative Raman-SEM microscopy for the quantitative analysis of plasmonic particles and structures, thus enabling a new analytical method in the field of SERS and plasmonics.

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

NASA Astrophysics Data System (ADS)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

USGS Publications Warehouse

Walker, G.K.; Black, M.G.; Edwards, C.A.

1996-01-01

Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

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.

Mapping whole-brain activity with cellular resolution by light-sheet microscopy and high-throughput image analysis (Conference Presentation)

NASA Astrophysics Data System (ADS)

Silvestri, Ludovico; Rudinskiy, Nikita; Paciscopi, Marco; Müllenbroich, Marie Caroline; Costantini, Irene; Sacconi, Leonardo; Frasconi, Paolo; Hyman, Bradley T.; Pavone, Francesco S.

2016-03-01

Mapping neuronal activity patterns across the whole brain with cellular resolution is a challenging task for state-of-the-art imaging methods. Indeed, despite a number of technological efforts, quantitative cellular-resolution activation maps of the whole brain have not yet been obtained. Many techniques are limited by coarse resolution or by a narrow field of view. High-throughput imaging methods, such as light sheet microscopy, can be used to image large specimens with high resolution and in reasonable times. However, the bottleneck is then moved from image acquisition to image analysis, since many TeraBytes of data have to be processed to extract meaningful information. Here, we present a full experimental pipeline to quantify neuronal activity in the entire mouse brain with cellular resolution, based on a combination of genetics, optics and computer science. We used a transgenic mouse strain (Arc-dVenus mouse) in which neurons which have been active in the last hours before brain fixation are fluorescently labelled. Samples were cleared with CLARITY and imaged with a custom-made confocal light sheet microscope. To perform an automatic localization of fluorescent cells on the large images produced, we used a novel computational approach called semantic deconvolution. The combined approach presented here allows quantifying the amount of Arc-expressing neurons throughout the whole mouse brain. When applied to cohorts of mice subject to different stimuli and/or environmental conditions, this method helps finding correlations in activity between different neuronal populations, opening the possibility to infer a sort of brain-wide 'functional connectivity' with cellular resolution.

Microwave-assisted hydrothermal synthesis of marigold-like ZnIn2S4 microspheres and their visible light photocatalytic activity

NASA Astrophysics Data System (ADS)

Chen, Zhixin; Li, Danzhen; Xiao, Guangcan; He, Yunhui; Xu, Yi-Jun

2012-02-01

Marigold-like ZnIn2S4 microspheres were synthesized by a microwave-assisted hydrothermal method with the temperature ranging from 80 to 195 °C. X-ray diffraction, X-ray photoelectron spectroscopy, nitrogen sorption analysis, UV-visible spectroscopy, scanning electron microscopy and transmission electron microscopy were used to characterize the products. It was found that the crystallographic structure and optical property of the products synthesized at different temperatures were almost the same. The degradation of methyl orange (MO) under the visible light irradiation has been used as a probe reaction to investigate the photocatalytic activity of as-prepared ZnIn2S4, which shows that the ZnIn2S4 sample synthesized at 195 °C shows the best photocatalytic activity for MO degradation. In addition, the photocatalytic activities of all the samples prepared by the microwave-assisted hydrothermal method are better than those prepared by a normal hydrothermal method, which could be attributed to the formation of more defect sites during the microwave-assisted hydrothermal treatment.

Preparation high photocatalytic activity of CdS/halloysite nanotubes (HNTs) nanocomposites with hydrothermal method

NASA Astrophysics Data System (ADS)

Xing, Weinan; Ni, Liang; Huo, Pengwei; Lu, Ziyang; Liu, Xinlin; Luo, Yingying; Yan, Yongsheng

2012-10-01

A novel nanocatalyst CdS/halloysite nanotubes (HNTs) was synthesized by hydrothermal method with direct growth of CdS nanoparticles on the surface of HNTs. The as-prepared photocatalysts had been characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), UV-vis diffuse reflectance spectra (UV-vis DRS), Fourier transform infrared (FT-IR) and the thermo gravimetric analysis (TGA). The photocatalytic activity of the sample was evaluated by the degradation of tetracycline (TC) under visible light irradiation. Benefit from the excellent properties of CdS and HNTs, the photocatalyst exhibited good photocatalytic activity and stability. In order to find out the optimum synthesis condition to obtain the best photocatalytic activity, a series of experiments were performed with different CdS loading capacity, different sources of sulfide and different hydrothermal temperatures, etc. The best photodegradation rate could reach 93% in 60 min under visible light irradiation. Therefore, the combination of CdS nanoparticles with HNTs endowed this material with a potential use in environmental treatments in industries.

Ultraviolet light and laser irradiation enhances the antibacterial activity of glucosamine-functionalized gold nanoparticles

PubMed Central

Govindaraju, Saravanan; Ramasamy, Mohankandhasamy; Baskaran, Rengarajan; Ahn, Sang Jung; Yun, Kyusik

2015-01-01

Here we report a novel method for the synthesis of glucosamine-functionalized gold nanoparticles (GlcN-AuNPs) using biocompatible and biodegradable glucosamine for antibacterial activity. GlcN-AuNPs were prepared using different concentrations of glucosamine. The synthesized AuNPs were characterized for surface plasmon resonance, surface morphology, fluorescence spectroscopy, and antibacterial activity. The minimum inhibitory concentrations (MICs) of the AuNPs, GlcN-AuNPs, and GlcN-AuNPs when irradiated by ultraviolet light and laser were investigated and compared with the MIC of standard kanamycin using Escherichia coli by the microdilution method. Laser-irradiated GlcN-AuNPs exhibited significant bactericidal activity against E. coli. Flow cytometry and fluorescence microscopic analysis supported the cell death mechanism in the presence of GlcN-AuNP-treated bacteria. Further, morphological changes in E. coli after laser treatment were investigated using atomic force microscopy and transmission electron microscopy. The overall results of this study suggest that the prepared nanoparticles have potential as a potent antibacterial agent for the treatment of a wide range of disease-causing bacteria. PMID:26345521

Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

PubMed Central

Yang, Zhe; Mei, Li; Xia, Fei; Luo, Qingming; Fu, Ling; Gong, Hui

2015-01-01

In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit’s width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons’ activities and help reveal the potential functional connections in the whole zebrafish’s brain. PMID:26137381

High-speed polarized light microscopy for in situ, dynamic measurement of birefringence properties

NASA Astrophysics Data System (ADS)

Wu, Xianyu; Pankow, Mark; Shadow Huang, Hsiao-Ying; Peters, Kara

2018-01-01

A high-speed, quantitative polarized light microscopy (QPLM) instrument has been developed to monitor the optical slow axis spatial realignment during controlled medium to high strain rate experiments at acquisition rates up to 10 kHz. This high-speed QPLM instrument is implemented within a modified drop tower and demonstrated using polycarbonate specimens. By utilizing a rotating quarter wave plate and a high-speed camera, the minimum acquisition time to generate an alignment map of a birefringent specimen is 6.1 ms. A sequential analysis method allows the QPLM instrument to generate QPLM data at the high-speed camera imaging frequency 10 kHz. The obtained QPLM data is processed using a vector correlation technique to detect anomalous optical axis realignment and retardation changes throughout the loading event. The detected anomalous optical axis realignment is shown to be associated with crack initiation, propagation, and specimen failure in a dynamically loaded polycarbonate specimen. The work provides a foundation for detecting damage in biological tissues through local collagen fiber realignment and fracture during dynamic loading.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.

PubMed

György, Bence; Módos, Károly; Pállinger, Eva; Pálóczi, Krisztina; Pásztói, Mária; Misják, Petra; Deli, Mária A; Sipos, Aron; Szalai, Anikó; Voszka, István; Polgár, Anna; Tóth, Kálmán; Csete, Mária; Nagy, György; Gay, Steffen; Falus, András; Kittel, Agnes; Buzás, Edit I

2011-01-27

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Preparation of Ag/AgCl/BiMg{sub 2}VO{sub 6} composite and its visible-light photocatalytic activity

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

Guo, Rui; Zhang, Gaoke, E-mail: gkzhang@whut.edu.cn; Liu, Jiu

2013-05-15

Graphical abstract: - Abstract: A novel composite photocatalyst Ag/AgCl/BiMg{sub 2}VO{sub 6} was synthesized by depositing Ag/AgCl nanoparticles on BiMg{sub 2}VO{sub 6} substrate via a precipitation–photoreduction method and characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDXA), X-ray photoelectron spectroscopy (XPS) and UV–vis diffuse reflectance spectrophotometer (UV–vis DRS). The photocatalyst showed high and stable photocatalytic activity for photocatalytic degradation of acid red G under visible-light irradiation (λ > 420 nm). In addition, the active ·O{sub 2}{sup −} and h{sup +}, as main reactive species, played the major roles during the reaction process.more » The high photocatalytic activity of the composite may be related to the efficient electron–hole pairs separation at the photocatalyst interfaces, as well as the surface plasmon resonance of Ag nanoparticles formed on AgCl particles in the degradation reaction.« less

Fe2O3/ZnO/ZnFe2O4 composites for the efficient photocatalytic degradation of organic dyes under visible light

NASA Astrophysics Data System (ADS)

Li, Xiaojuan; Jin, Bo; Huang, Jingwen; Zhang, Qingchun; Peng, Rufang; Chu, Shijin

2018-06-01

In this study, novel ternary Fe2O3/ZnO/ZnFe2O4 (ZFO) composites were successfully prepared through a simple hydrothermal reaction with subsequent thermal treatment. The as-prepared products were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Brunauer-Emmett-Teller (BET) analysis, Barrett-Joyner-Halenda (BJH) measurement, and UV-vis diffuse reflectance spectroscopy (UV-vis DRS). The photocatalytic degradation of rhodamine B (Rh B) under visible light irradiation indicated that the ZFO composites calcined at 500 °C has the best photocatalytic activity (the photocatalytic degradation efficiency can reach up to 95.7% within 60 min) and can maintain a stable photocatalytic degradation efficiency for at least three cycles. In addition, the photocatalytic activity of ZFO composites toward dye decomposition follows the order cationic Rh B > anionic methyl orange. Finally, using different scavengers, superoxide and hydroxyl radicals were identified as the primary active species during the degradation reaction of Rh B.

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.

Intracellular inclusion bodies in 14 patients with B cell lymphoproliferative disorders.

PubMed Central

Peters, O; Thielemans, C; Steenssens, L; De Waele, M; Hijmans, W; Van Camp, B

1984-01-01

Two types of intracytoplasmic inclusion were detected by immunofluorescence microscopy in 12 patients with chronic lymphocytic leukaemia and two patients with a leukaemic phase of well differentiated lymphocytic lymphoma. Further analysis with light- and electron microscopy, showed that most inclusion bodies were rod-like crystalline structures. However, in three patients they consisted of amorphous vesicular precipitates. Immunological studies revealed the presence of immunoglobulins of the same class and type at the cell surface as well as in the inclusion bodies. The monoclonal immunoglobulins were all of lambda type except in two cases. The origin of immunoglobulin inclusion bodies in B cell malignancies is discussed in relation to published data and our own observation in one patient followed during treatment. Images PMID:6323543

One-step, low-temperature fabrication of CdS quantum dots by watermelon rind: a green approach

PubMed Central

Lakshmipathy, Rajasekhar; Sarada, Nallani Chakravarthula; Chidambaram, K; Pasha, Sk Khadeer

2015-01-01

We investigated the one-step synthesis of CdS nanoparticles via green synthesis that used aqueous extract of watermelon rind as a capping and stabilizing agent. Preliminary phytochemical analysis depicted the presence of carbohydrates which can act as capping and stabilizing agents. Synthesized CdS nanoparticles were characterized using UV-visible, Fourier transform infrared spectroscopy, X-ray diffraction, EDX, dynamic light scattering, transmission electron microscopy, and atomic force microscopy techniques. The CdS nanoparticles were found to be size- and shape-controlled and were stable even after 3 months of synthesis. The results suggest that watermelon rind, an agro-waste, can be used for synthesis of CdS nanoparticles without any addition of stabilizing and capping agents. PMID:26491319

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

Deep ultraviolet scanning near-field optical microscopy for the structural analysis of organic and biological materials

NASA Astrophysics Data System (ADS)

Aoki, Hiroyuki; Hamamatsu, Toyohiro; Ito, Shinzaburo

2004-01-01

Scanning near-field optical microscopy (SNOM) using a deep ultraviolet (DUV) light source was developed for in situ imaging of a variety of chemical species without staining. Numerous kinds of chemical species have a carbon-carbon double bond or aromatic group in their chemical structure, which can be excited at the wavelength below 300 nm. In this study, the wavelength range available for SNOM imaging was extended to the DUV region. DUV-SNOM allowed the direct imaging of polymer thin films with high detection sensitivity and spatial resolution of several tens of nanometers. In addition to the polymer materials, we demonstrated the near-field imaging of a cell without using a fluorescence label.

Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

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

Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu

2011-07-11

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RCmore » within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.« less

Challenges in the size analysis of a silica nanoparticle mixture as candidate certified reference material

NASA Astrophysics Data System (ADS)

Kestens, Vikram; Roebben, Gert; Herrmann, Jan; Jämting, Åsa; Coleman, Victoria; Minelli, Caterina; Clifford, Charles; De Temmerman, Pieter-Jan; Mast, Jan; Junjie, Liu; Babick, Frank; Cölfen, Helmut; Emons, Hendrik

2016-06-01

A new certified reference material for quality control of nanoparticle size analysis methods has been developed and produced by the Institute for Reference Materials and Measurements of the European Commission's Joint Research Centre. The material, ERM-FD102, consists of an aqueous suspension of a mixture of silica nanoparticle populations of distinct particle size and origin. The characterisation relied on an interlaboratory comparison study in which 30 laboratories of demonstrated competence participated with a variety of techniques for particle size analysis. After scrutinising the received datasets, certified and indicative values for different method-defined equivalent diameters that are specific for dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), particle tracking analysis (PTA) and asymmetrical-flow field-flow fractionation (AF4) were assigned. The value assignment was a particular challenge because metrological concepts were not always interpreted uniformly across all participating laboratories. This paper presents the main elements and results of the ERM-FD102 characterisation study and discusses in particular the key issues of measurand definition and the estimation of measurement uncertainty.

Nanocrystals of [Cu3(btc)2] (HKUST-1): a combined time-resolved light scattering and scanning electron microscopy study.

PubMed

Zacher, Denise; Liu, Jianing; Huber, Klaus; Fischer, Roland A

2009-03-07

The formation of [Cu(3)(btc)(2)] (HKUST-1; btc = 1,3,5-benzenetricarboxylate) nanocrystals from a super-saturated mother solution at room temperature was monitored by time-resolved light scattering (TLS); the system is characterized by a rapid growth up to a size limit of 200 nm within a few minutes, and the size and shape of the crystallites were also determined by scanning electron microscopy (SEM).

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes[C][W

PubMed Central

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I.

2013-01-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation. PMID:23898030

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

PubMed

Janik, Ewa; Bednarska, Joanna; Zubik, Monika; Puzio, Michal; Luchowski, Rafal; Grudzinski, Wojciech; Mazur, Radoslaw; Garstka, Maciej; Maksymiec, Waldemar; Kulik, Andrzej; Dietler, Giovanni; Gruszecki, Wieslaw I

2013-06-01

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

The use of light-emitting diode fluorescence to diagnose mycobacterial lymphadenitis in fine-needle aspirates from children

PubMed Central

van Wyk, A. C.; Marais, B. J.; Warren, R. M.; van Wyk, S. S.; Wright, C. A.

2011-01-01

SUMMARY BACKGROUND Fine-needle aspiration biopsy (FNAB) is a simple, safe and effective method for investigating suspected mycobacterial lymphadenitis in children. Fluorescence microscopy can provide rapid mycobacterial confirmation. Light-emitting diodes (LEDs) provide a cheap and robust excitation light source, making fluorescence microscopy feasible in resource-limited settings. OBJECTIVE To compare the diagnostic performance of LED fluorescence microscopy on Papanicolaou (PAP) stained smears with the conventional mercury vapour lamp (MVL). METHODS FNAB smears routinely collected from palpable lymph nodes in children with suspected mycobacterial disease were PAP-stained and evaluated by two independent microscopists using different excitatory light sources (MVL and LED). Mycobacterial culture results provided the reference standard. A manually rechargeable battery-powered LED power source was evaluated in a random subset. RESULTS We evaluated 182 FNAB smears from 121 children (median age 31 months, interquartile range 10–67). Mycobacterial cultures were positive in 84 of 121 (69%) children. The mean sensitivity with LED (mains-powered), LED (rechargeable battery-powered) and MVL was respectively 48.2%, 50.0% and 51.8% (specificity 78.4%, 86.7% and 78.4%). Inter-observer variation was similar for LED and MVL (κ = 0.5). CONCLUSION LED fluorescence microscopy provides a reliable alternative to conventional methods and has many favourable attributes that would facilitate improved, decentralised diagnostic services. PMID:21276297

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

PubMed

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

Super-resolution photoacoustic microscopy using a localized near-field of a plasmonic nanoaperture: a three-dimensional simulation study

NASA Astrophysics Data System (ADS)

Park, Byullee; Lee, Hongki; Upputuri, Paul Kumar; Pramanik, Manojit; Kim, Donghyun; Kim, Chulhong

2018-02-01

Super-resolution microscopy has been increasingly important to delineate nanoscale biological structures or nanoparticles. With these increasing demands, several imaging modalities, including super-resolution fluorescence microscope (SRFM) and electron microscope (EM), have been developed and commercialized. These modalities achieve nanoscale resolution, however, SRFM cannot image without fluorescence, and sample preparation of EM is not suitable for biological specimens. To overcome those disadvantages, we have numerically studied the possibility of superresolution photoacoustic microscopy (SR-PAM) based on near-field localization of light. Photoacoustic (PA) signal is generally acquired based on optical absorption contrast; thus it requires no agents or pre-processing for the samples. The lateral resolution of the conventional photoacoustic microscopy is limited to 200 nm by diffraction limit, therefore reducing the lateral resolution is a major research impetus. Our approach to breaking resolution limit is to use laser pulses of extremely small spot size as a light source. In this research, we simulated the PA signal by constructing the three dimensional SR-PAM system environment using the k-Wave toolbox. As the light source, we simulated ultrashort light pulses using geometrical nanoaperture with near-field localization of surface plasmons. Through the PA simulation, we have successfully distinguish cuboids spaced 3 nm apart. In the near future, we will develop the SR-PAM and it will contribute to biomedical and material sciences.

Microscopic video observation of capillary vessel systems using diffuse back lighting

NASA Astrophysics Data System (ADS)

Sakai, Minako; Arai, Hiroki; Iwai, Toshiaki

2017-04-01

We have been developing a simple and practical video microscopy system based on absorption spectra of biological substance to perform spectroscopic observation of living tissues. The diffuse backlighting effect is actively used in the developed system, which is generated by multiple light scattering in the tissue. It is demonstrated that the light specularly reflected from the skin surface can be completely suppressed in the microscopic observation and the biological activity of the capillary vessel systems distributed under the skin can be successfully observed. As a result, we can confirm the effectiveness of the video microscopy system using diffuse backlighting and the applicability of our developed system.

Thermal Images of Seeds Obtained at Different Depths by Photoacoustic Microscopy (PAM)

NASA Astrophysics Data System (ADS)

Domínguez-Pacheco, A.; Hernández-Aguilar, C.; Cruz-Orea, A.

2015-06-01

The objective of the present study was to obtain thermal images of a broccoli seed ( Brassica oleracea) by photoacoustic microscopy, at different modulation frequencies of the incident light beam ((0.5, 1, 5, and 20) Hz). The thermal images obtained in the amplitude of the photoacoustic signal vary with each applied frequency. In the lowest light frequency modulation, there is greater thermal wave penetration in the sample. Likewise, the photoacoustic signal is modified according to the structural characteristics of the sample and the modulation frequency of the incident light. Different structural components could be seen by photothermal techniques, as shown in the present study.

High-resolution corneal topography and tomography of fish eye using wide-field white light interference microscopy

NASA Astrophysics Data System (ADS)

Srivastava, Vishal; Nandy, Sreyankar; Singh Mehta, Dalip

2013-04-01

Topography and tomography of fish cornea is reconstructed using high resolution white light interference microscopy. White light interferograms at different depths were recorded by moving the object axially. For each depth position, five phase shifted interferograms were recorded and analyzed. From the reconstructed phase maps, the corneal topography and hence the refractive index was determined and from amplitude images the cross-sectional image of fish cornea was reconstructed. In the present method, we utilize a nearly common-path interference microscope and wide field illumination and hence do not require any mechanical B-scan. Therefore, the phase stability of the recorded data is improved.

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

Quantification of Nanoscale Density Fluctuations in Biological Cells/Tissues: Inverse Participation Ratio (IPR) Analysis of Transmission Electron Microscopy Images and Implications for Early-Stage Cancer Detection

NASA Astrophysics Data System (ADS)

Pradhan, Prabhakar; Damania, Dhwanil; Joshi, Hrushikesh; Taflove, Allen; Roy, Hemant; Dravid, Vinayak; Backman, Vadim

2010-03-01

We report a study of the nanoscale mass density fluctuations of biological cells and tissues by quantifying their nanoscale light-localization properties. Transmission electron microscope (TEM) images of human cells and tissues are used to construct corresponding effective disordered optical lattices. Light-localization properties are studied by statistical analysis of the inverse participation ratio (IPR) of the eigenfunctions of these optical lattices at the nanoscales. Our results indicate elevation of the nanoscale disorder strength (e.g., refractive index fluctuations) in early carcinogenesis. Importantly, our results demonstrate that the increase in the nanoscale disorder represents the earliest structural alteration in cells undergoing carcinogenesis known to-date. Potential applications of the technique for early stage cancer detection will be discussed.

Wave optics theory and 3-D deconvolution for the light field microscope

PubMed Central

Broxton, Michael; Grosenick, Logan; Yang, Samuel; Cohen, Noy; Andalman, Aaron; Deisseroth, Karl; Levoy, Marc

2013-01-01

Light field microscopy is a new technique for high-speed volumetric imaging of weakly scattering or fluorescent specimens. It employs an array of microlenses to trade off spatial resolution against angular resolution, thereby allowing a 4-D light field to be captured using a single photographic exposure without the need for scanning. The recorded light field can then be used to computationally reconstruct a full volume. In this paper, we present an optical model for light field microscopy based on wave optics, instead of previously reported ray optics models. We also present a 3-D deconvolution method for light field microscopy that is able to reconstruct volumes at higher spatial resolution, and with better optical sectioning, than previously reported. To accomplish this, we take advantage of the dense spatio-angular sampling provided by a microlens array at axial positions away from the native object plane. This dense sampling permits us to decode aliasing present in the light field to reconstruct high-frequency information. We formulate our method as an inverse problem for reconstructing the 3-D volume, which we solve using a GPU-accelerated iterative algorithm. Theoretical limits on the depth-dependent lateral resolution of the reconstructed volumes are derived. We show that these limits are in good agreement with experimental results on a standard USAF 1951 resolution target. Finally, we present 3-D reconstructions of pollen grains that demonstrate the improvements in fidelity made possible by our method. PMID:24150383

Hybrid of two-photon microscopy and optical multimodality imaging for multi-scale imaging of small animals

NASA Astrophysics Data System (ADS)

Li, Tianmeng; Hui, Hui; Ma, He; Yang, Xin; Tian, Jie

2018-02-01

Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

Degradation product analysis from the photocatalytic oxidation/reduction of 2,4-dichlorophenol in the presence of mesoporous silica encapsulated TiO2 particles and TiO2 dispersions (presentation)

EPA Science Inventory

Thin films of Degussa P-25 TiO2 encapsulated in an SBA-15 mesoporous silica matrix were prepared. The TiO2/SBA-15 thin film structure was verified using transmission electron microscopy (TEM) and small angle X-ray diffraction (XRD). During irradiation with 350 nm light, the TiO...

Analysis of plastid number, size, and distribution in Arabidopsis plants by light and fluorescence microscopy.

PubMed

Pyke, Kevin

2011-01-01

Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis.

Necrotizing Infundibular Crystalline Folliculitis: A Case Report of an Exceptional Lesion of Unknown Etiology

PubMed Central

Saxer-Sekulic, Nikolina; Vion-Gauthey, Brigitte; Kaya, Gürkan

2014-01-01

Necrotizing infundibular crystalline folliculitis is a rare follicular lesion of which the etiology is not well understood. Here we describe the case of a 71-year-old male patient presenting with multiple hyperkeratotic lesions localized on the forehead. Histopathological analysis of one of the lesions revealed a follicular invagination containing cellular debris and keratin lamellae containing filamentous mucinous material and numerous crystals birefringent in polarized light microscopy. PMID:27047916

3D X-ray ultra-microscopy of bone tissue.

PubMed

Langer, M; Peyrin, F

2016-02-01

We review the current X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. We further review the different ultra-structural features that have so far been resolved: the lacuno-canalicular network, collagen orientation, nano-scale mineralization and their use as basis for mechanical simulations. X-ray computed tomography at the micro-metric scale is increasingly considered as the reference technique in imaging of bone micro-structure. The trend has been to push towards increasingly higher resolution. Due to the difficulty of realizing optics in the hard X-ray regime, the magnification has mainly been due to the use of visible light optics and indirect detection of the X-rays, which limits the attainable resolution with respect to the wavelength of the visible light used in detection. Recent developments in X-ray optics and instrumentation have allowed to implement several types of methods that achieve imaging that is limited in resolution by the X-ray wavelength, thus enabling computed tomography at the nano-scale. We review here the X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. Further, we review the different ultra-structural features that have so far been resolved and the applications that have been reported: imaging of the lacuno-canalicular network, direct analysis of collagen orientation, analysis of mineralization on the nano-scale and use of 3D images at the nano-scale to drive mechanical simulations. Finally, we discuss the issue of going beyond qualitative description to quantification of ultra-structural features.

Evaluation of Left Ventricular Diastolic Dysfunction with Early Systolic Dysfunction Using Two-Dimensional Speckle Tracking Echocardiography in Canine Heart Failure Model.

PubMed

Wu, Wei-Chun; Ma, Hong; Xie, Rong-Ai; Gao, Li-Jian; Tang, Yue; Wang, Hao

2016-04-01

This study evaluated the role of two-dimensional speckle tracking echocardiography (2DSTE) for predicting left ventricular (LV) diastolic dysfunction in pacing-induced canine heart failure. Pacing systems were implanted in 8 adult mongrel dogs, and continuous rapid right ventricular pacing (RVP, 240 beats/min) was maintained for 2 weeks. The obtained measurements from 2DSTE included global strain rate during early diastole (SRe) and during late diastole (SRa) in the longitudinal (L-SRe, L-SRa), circumferential (C-SRe, C-SRa), and radial directions (R-SRe, R-SRa). Changes in heart morphology were observed by light microscopy and transmission electron microscopy at 2 weeks. The onset of LV diastolic dysfunction with early systolic dysfunction occurred 3 days after RVP initiation. Most of the strain rate imaging indices were altered at 1 or 3 days after RVP onset and continued to worsen until heart failure developed. Light and transmission electron microscopy showed myocardial vacuolar degeneration and mitochondrial swelling in the left ventricular at 2 weeks after RVP onset. Pearson's correlation analysis revealed that parameters of conventional echocardiography and 2DSTE showed moderate correlation with LV pressure parameters, including E/Esep' (r = 0.58, P < 0.01), L-SRe (r = -0.58, P < 0.01), E/L-SRe (r = 0.65, P < 0.01), and R-SRe (r = 0.53, P < 0.01). ROC curves analysis showed that these indices of conventional echocardiography and strain rate imaging could effectively predict LV diastolic dysfunction (area under the curve: E/Esep' 0.78; L-SRe 0.84; E/L-SRe 0.80; R-SRe 0.80). 2DSTE was a sensitive and accurate technique that could be used for predicting LV diastolic dysfunction in canine heart failure model. © 2015, Wiley Periodicals, Inc.

Fast Two-Dimensional Bubble Analysis of Biopolymer Filamentous Networks Pore Size from Confocal Microscopy Thin Data Stacks

PubMed Central

Molteni, Matteo; Magatti, Davide; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

2013-01-01

The average pore size ξ0 of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ0 is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ0 consistent with results from elastic light scattering data. PMID:23473499

Limitations of using Raman microscopy for the analysis of high-content-carbon-filled ethylene propylene diene monomer rubber.

PubMed

Ghanbari-Siahkali, Afshin; Almdal, Kristoffer; Kingshott, Peter

2003-12-01

The effects of laser irradiation on changes to the surface chemistry and structure of a commercially available ethylene propylene diene monomer (EPDM) rubber sample after Raman microscopy analysis was investigated. The Raman measurements were carried out with different levels of laser power on the sample, ranging from 4.55 mW to 0.09 mW. The surface of the EPDM was analyzed before and after laser exposure using X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The techniques have surface probe depths of approximately < or = 10 nm and 1 microm, respectively. Both sets of analysis show that ingredients of the blended EPDM rubber "bloom" to the surface as a result of local heating that takes place due to the absorption of laser by carbon black during the Raman analysis. Scanning electron microscopy (SEM) analysis was also performed on the Raman analyzed areas to visually illustrate the effects created due to laser light exposure (i.e., burning marks). The change in surface chemistry also occurs in regions a few millimeters from the exposed sites, indicating that the effect is quite long range. However, this phenomenon has no major influence, as far as XPS or ATR-FTIR results disclose, on the backbone structure of the rubber sample. The results indicate that precautions should be taken when analyzing complex blended polymer samples using Raman spectroscopy.

A comparative analysis of microscopic alterations in modern and ancient undecalcified and decalcified dry bones.

PubMed

Caruso, Valentina; Cummaudo, Marco; Maderna, Emanuela; Cappella, Annalisa; Caudullo, Giorgio; Scarpulla, Valentina; Cattaneo, Cristina

2018-02-01

The present study aims to evaluate the preservation of the microstructure of skeletal remains collected from four different known burial sites (archaeological and contemporary). Histological analysis on undecalcified and decalcified thin sections was performed in order to assess which of the two techniques is more affected by taphonomic insults. A histological analysis was performed on both undecalcified and decalcified thin sections of 40 long bones and the degree of diagenetic change was evaluated using transmitted and polarized light microscopy according to the Oxford Histological Index (OHI). In order to test the optical behavior of bone tissue, thin sections were observed by polarized light microscopy and the intensity of birefringence was evaluated. The more ancient samples are generally characterized by a low OHI (0-1) with extensive microscopic focal destruction; recent samples exhibited a better preservation of bone micromorphology. When comparing undecalcified to decalcified thin sections, the latter showed an amelioration in the conservation of microscopic structure. As regards the birefringence, it was very low in all the undecalcified thin sections, whereas decalcification process seems to improve its visibility. The preservation of the bone microscopic structure appears to be influenced not only by age, but also by the burial context. Undecalcified bones appear to be more affected by taphonomical alterations, probably because of the thickness of the thin sections; on the contrary, decalcified thin sections proved to be able to tackle this issue allowing a better reading of the bone tissue. © 2017 Wiley Periodicals, Inc.

Developing methods based on light sheet fluorescence microscopy for biophysical investigations of larval zebrafish

NASA Astrophysics Data System (ADS)

Taormina, Michael J.

Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo.

A promising red-emitting phosphor for white-light-emitting diodes prepared by a modified solid-state reaction

NASA Astrophysics Data System (ADS)

Ren, Fuqiang; Chen, Donghua

2010-02-01

Using urea, boric acid and polyethylene glycol (PEG) as auxiliary reagents, the novel red-emitting phosphors Ca 19Zn 2 (PO 4) 14:Eu 3+ have been successfully synthesized by a modified solid-state reaction. Thermogravimetric (TG) analysis, X-ray diffraction (XRD), transmission electron microscopy (TEM) and photoluminescence (PL) spectra were used to characterize the resulting phosphors. The dependence of the photoluminescence properties of Ca 19Zn 2 (PO 4) 14:Eu 3+ phosphors upon urea, boric acid and PEG concentration and the quadric-sintered temperature were investigated. Luminescent measurements showed that the phosphors can be efficiently excited by ultraviolet (UV) to visible region, emitting a red light with a peak wavelength of 616 nm. The material has potential application as a fluorescent material for ultraviolet light-emitting diodes (UV-LEDs).

Visualizing light with electrons

NASA Astrophysics Data System (ADS)

Fitzgerald, J. P. S.; Word, R. C.; Koenenkamp, R.

2014-03-01

In multiphoton photoemission electron microscopy (nP-PEEM) electrons are emitted from surfaces at a rate proportional to the surface electromagnetic field amplitude. We use 2P-PEEM to give nanometer scale visualizations of light of diffracted and waveguide fields around various microstructures. We use Fourier analysis to determine the phase and amplitude of surface fields in relation to incident light from the interference patterns. To provide quick and intuitive simulations of surface fields, we employ two dimensional Fresnel-Kirchhoff integration, a technique based on freely propagating waves and Huygens' principle. We find generally good agreement between simulations and experiment. Additionally diffracted wave simulations exhibit greater phase accuracy, indicating that these waves are well represented by a two dimensional approximation. The authors gratefully acknowledge funding of this research by the US-DOE Basic Science Office under Contract DE-FG02-10ER46406.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

PubMed Central

Bertrand, Vincent; Lenne, Pierre-François

2014-01-01

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time. PMID:24836407

Biomechanical and Histopathologic Effects of Pulsed-Light Accelerated Epithelium-On/-Off Corneal Collagen Cross-Linking.

PubMed

Zhang, Xiaoyu; Sun, Ling; Shen, Yang; Tian, Mi; Zhao, Jing; Zhao, Yu; Li, Meiyan; Zhou, Xingtao

2017-07-01

This study aimed to compare the biomechanical and histopathologic effects of transepithelial and accelerated epithelium-off pulsed-light accelerated corneal collagen cross-linking (CXL). A total of 24 New Zealand rabbits were analyzed after sham operation (control) or transepithelial or epithelium-off operation (45 mW/cm for both). The transepithelial group was treated with pulsed-light ultraviolet A for 5 minutes 20 seconds, and the epithelium-off group was treated for 90 seconds. Biomechanical testing, including ultimate stress, Young modulus, and the physiological modulus, was analyzed. Histological changes were evaluated by light microscopy and transmission electron microscopy. The stress-strain curve was nonlinear in both accelerated transepithelial and epithelium-off CXL groups. The stress and elastic moduli were all significantly higher in both experimental groups compared with the control group (P < 0.05), whereas there were no significant differences between the 2 treatment groups (P > 0.05). Six months after the operation, hematoxylin and eosin staining and transmission electron microscopy showed that the subcutaneous collagen fibers were arranged in a regular pattern, and the fiber density was higher in the experimental groups. Both transepithelial and accelerated epithelium-off CXL produced biomechanical and histopathologic improvements, which were not significantly different between the 2 pulsed-light accelerated CXL treatments.

AccessScope project: Accessible light microscope for users with upper limb mobility or visual impairments.

PubMed

Mansoor, Awais; Ahmed, Wamiq M; Samarapungavan, Ala; Cirillo, John; Schwarte, David; Robinson, J Paul; Duerstock, Bradley S

2010-01-01

A web-based application was developed to remotely view slide specimens and control all functions of a research-level light microscopy workstation, called AccessScope. Students and scientists with upper limb mobility and visual impairments are often unable to use a light microscope by themselves and must depend on others in its operation. Users with upper limb mobility impairments and low vision were recruited to assist in the design process of the AccessScope personal computer (PC) user interface. Participants with these disabilities were evaluated in their ability to use AccessScope to perform microscopical tasks. AccessScope usage was compared with inspecting prescanned slide images by grading participants' identification and understanding of histological features and knowledge of microscope operation. With AccessScope subjects were able to independently perform common light microscopy functions through an Internet browser by employing different PC pointing devices or accessibility software according to individual abilities. Subjects answered more histology and microscope usage questions correctly after first participating in an AccessScope test session. AccessScope allowed users with upper limb or visual impairments to successfully perform light microscopy without assistance. This unprecedented capability is crucial for students and scientists with disabilities to perform laboratory coursework or microscope-based research and pursue science, technology, engineering, and mathematics fields.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

Spectral characterization and white light generation by yttrium silicate nanopowders undoped and doped with Ytterbium(III) at different concentrations when excited by a laser diode at 975 nm

NASA Astrophysics Data System (ADS)

Cinkaya, Hatun; Eryurek, Gonul; Bilir, Gokhan; Collins, John; Di Bartolo, Baldassare

2017-01-01

We have studied nanophosphors of yttrium silicate (YSO) undoped and doped with different concentration of ytterbium (Yb3+) synthesized by using the sol-gel method. Structural and luminescence properties of the nanophosphors were studied experimentally by using different analytical techniques. For the structural analysis, we performed X-ray diffraction (XRD), Transmission Electron Microscopy (TEM) and Energy Dispersive X-ray Spectrometry (EDS) measurements. Upconversion (UC) and the white light (WL) emission properties were investigated by using the near infrared cw laser excitation of 975 nm. The spectral properties have been found to depend on several physical parameters.

Nonlinear side effects of fs pulses inside corneal tissue during photodisruption

NASA Astrophysics Data System (ADS)

Heisterkamp, A.; Ripken, T.; Mamom, T.; Drommer, W.; Welling, H.; Ertmer, W.; Lubatschowski, H.

In order to evaluate the potential for refractive surgery, fs laser pulses of 150-fs pulse duration were used to process corneal tissue of dead and living animal eyes. By focusing the laser radiation down to spot sizes of several microns, very precise cuts could be achieved inside the treated cornea, accompanied with minimum collateral damage to the tissue by thermal or mechanical effects. During histo-pathological analysis by light and transmission electron microscopy considerable side effects of fs photodisruption were found. Due to the high intensities at the focal region several nonlinear effects occurred. Self-focusing, photodissociation, UV-light production were observed, leading to streak formation inside the cornea.

Phase noise optimization in temporal phase-shifting digital holography with partial coherence light sources and its application in quantitative cell imaging.

PubMed

Remmersmann, Christian; Stürwald, Stephan; Kemper, Björn; Langehanenberg, Patrik; von Bally, Gert

2009-03-10

In temporal phase-shifting-based digital holographic microscopy, high-resolution phase contrast imaging requires optimized conditions for hologram recording and phase retrieval. To optimize the phase resolution, for the example of a variable three-step algorithm, a theoretical analysis on statistical errors, digitalization errors, uncorrelated errors, and errors due to a misaligned temporal phase shift is carried out. In a second step the theoretically predicted results are compared to the measured phase noise obtained from comparative experimental investigations with several coherent and partially coherent light sources. Finally, the applicability for noise reduction is demonstrated by quantitative phase contrast imaging of pancreas tumor cells.

Supra-organization and optical anisotropies of the extracellular matrix in the amniotic membrane and limbal stroma before and after explant culture

PubMed Central

Valdetaro, Gisele P.; Aldrovani, Marcela; Padua, Ivan R. M.; Cristovam, Priscila C.; Gomes, José A. P.; Laus, José L.

2016-01-01

In this research we evaluated the supramolecular organizations and the optical anisotropical properties of the de-epithelialized human amniotic membrane and rabbit limbal stroma, before and after explant culture. Birefringence, monochromatic light spectral absorption and linear dichroism of the main extracellular matrix biopolymers, that is, the fibrillar collagens and proteoglycans, were investigated by polarized light microscopy combined with image analysis. Our results demonstrated that the culture procedure–induced stimuli altered the supra-organizational characteristics (in terms of collagens/proteoglycans spatial orientation and ordered-aggregational state) of the amniotic and limbal extracellular matrix, which led to changes in optical anisotropical properties. PMID:28018719

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research.

PubMed

Moretti, Elena; Sutera, Gaetano; Collodel, Giulia

2016-06-01

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility's therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology. transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Induced structural defects in Ti-doped ZnO and its two-photon-excitation

NASA Astrophysics Data System (ADS)

Martínez Julca, Milton A.; Rivera, Ivonnemary; Santillan Mercado, Jaime; Sierra, Heidy; Perales-Pérez, Oscar

2016-03-01

ZnO is a well-known luminescent material that reacts with light to generate free radicals enabling its use in cancer treatment by Photodynamic Therapy (PDT). Unfortunately, up to know, the photo-excitation of ZnO-based materials' requires excitation with ultraviolet light, which limits their biomedical applications. In this regard, this work investigates the effect of Ti species incorporation into the lattice of ZnO nanoparticles (NPs) with the aim of improving the corresponding optical properties and enabling the two-photoexcitation with 690nm-light (near infrared light). A modified polyol-based route was used to synthesize pure and Ti-doped (9% at.) ZnO NPs. X-ray diffraction confirmed the formation of ZnO-wurtzite whereas Scanning Electron Microscopy confirmed the formation of monodispersed 100-nm NPs. Raman Spectroscopy measurements evidenced the presence of zinc interstitials (Zni) and oxygen vacancies (VO) in the host oxide strcuture. Asynthesized NPs were excited using the technique of two-photon fluorescence microscopy (TPFM). The photoluminescence (PL) spectra generated from the analysis of TPFM images revealed a high emission peak presence in the green region (555 nm) that was assigned to VO. Also, a weak but noticeable band at 420 nm was detected, which is attributed to electron transition from the shallow donor level of Zni to the valence band. These PL transitions will favor triplet states formation necessary to yield cytotoxic reactive oxygen species. Furthermore, the presence of the PL peaks confirmed the Ti-ZnO NPs capacity to be excited by 690-nm light, thus, opening new possibilities for this NPs to be used in lightinduced bio-medical applications.