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Sample records for two-photon scanning microscopy

  1. Comparison of calcium imaging in dorsal root ganglion neurons by using laser scanning confocal and two-photon microscopy

    Science.gov (United States)

    Huang, Yimei; Yang, Hongqin; Chen, Jiangxu; Shen, Xiuqiu; Zheng, Liqin; Wang, Yuhua; Xie, Shusen

    2012-03-01

    As one of the most important second messengers, calcium in nerve cells plays a critical role in neuronal processes, including excitability, neurotransmitter release, synaptic plasticity. Modulation of the calcium concentration is an important means of regulating diverse neuronal functions. To evaluate the role of calcium, quantitative measurement of cytosolic free calcium concentrations is necessary. There are several optical techniques that are available for measurement of calcium in live cells. Laser scanning confocal microscopy and two-photon microscopy are two prevalent techniques for their advantage in spatial resolution. In this paper, calcium in dorsal root ganglion neurons was imaged by laser scanning confocal microscopy and two-photon microscopy with Fluo-3, a calcium specific fluorescence probe. Both of spatial resolution and photobleaching, two common limitations of optical image modality, were compared between laser scanning confocal microscopy and two-photon microscopy, respectively. Three dimension images showed that laser scanning confocal microscopy and two-photon microscopy had not only similar lateral resolution but also parallel vertical resolution. However, Laser scanning confocal microscopy had an advantage over the two-photon microcopy in photobleaching. These results indicated that laser scanning confocal microscopy was more suitable than two-photon microscopy to be applied in imaging calcium in dorsal root ganglion neurons with Fluo-3.

  2. All-optical histology using two photon laser scanning microscopy and ablation with ultrashort pulses

    Science.gov (United States)

    Tsai, Philbert S.

    This dissertation discusses the use of ultrashort laser pulses to image and manipulate tissue for the purpose of three-dimensional histological reconstruction of extended brain structures. Two photon laser scanning microscopy (TPLSM) and ultrashort pulsed laser ablation are used to provide in situ three-dimensional imaging through thick preparations of fixed tissue. Surface regions of fixed tissue are first imaged using TPLSM. The imaged regions are then removed by ablation with amplified, ultrashort laser pulses, thereby exposing a previously underlying tissue region for imaging. This process of imaging and ablation proceeds iteratively until the desired tissue volume has been processed. First, the principles, design, and construction of a two photon laser scanning microscope are discussed, followed by a discussion of the physical mechanisms of tissue ablation with ultrashort laser pulses. The compatibility of tissue ablation using ultrashort pulses with subsequent histological analysis, particularly with fluorescent microscopy, is evaluated. Tissue ablation with ultrashort laser pulses is found to produce ablated tissue surfaces that are smooth to within a micrometer. Intrinsic fluorescence as well as immunoreactivity are found to be resilient to the ablation process. The all-optical histological technique is demonstrated on brain tissue from rats and mice, including tissue from embryonic mouse as early at E15. The ablation process is shown to preserve both macroscopic and microscopic structures within tissue. To facilitate the all-optical histological analysis of neuronal vasculature and its relative distribution to surrounding neuronal tissue, a fluorescent gel perfusion technique is developed that provides a temperature-stabilized fluorescent label of the neuronal vasculature. The use of immunohistochemistry to label specific cell populations throughout an 800 micrometer-thick tissue section is demonstrated. Additionally, the immersion of fixed tissue in high

  3. Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors.

    Science.gov (United States)

    Salomé, R; Kremer, Y; Dieudonné, S; Léger, J-F; Krichevsky, O; Wyart, C; Chatenay, D; Bourdieu, L

    2006-06-30

    Two-photon scanning microscopy (TPSM) is a powerful tool for imaging deep inside living tissues with sub-cellular resolution. The temporal resolution of TPSM is however strongly limited by the galvanometric mirrors used to steer the laser beam. Fast physiological events can therefore only be followed by scanning repeatedly a single line within the field of view. Because acousto-optic deflectors (AODs) are non-mechanical devices, they allow access at any point within the field of view on a microsecond time scale and are therefore excellent candidates to improve the temporal resolution of TPSM. However, the use of AOD-based scanners with femtosecond pulses raises several technical difficulties. In this paper, we describe an all-digital TPSM setup based on two crossed AODs. It includes in particular an acousto-optic modulator (AOM) placed at 45 degrees with respect to the AODs to pre-compensate for the large spatial distortions of femtosecond pulses occurring in the AODs, in order to optimize the spatial resolution and the fluorescence excitation. Our setup allows recording from freely selectable point-of-interest at high speed (1kHz). By maximizing the time spent on points of interest, random-access TPSM (RA-TPSM) constitutes a promising method for multiunit recordings with millisecond resolution in biological tissues.

  4. Changes in cortical microvasculature during misery perfusion measured by two-photon laser scanning microscopy.

    Science.gov (United States)

    Tajima, Yosuke; Takuwa, Hiroyuki; Kokuryo, Daisuke; Kawaguchi, Hiroshi; Seki, Chie; Masamoto, Kazuto; Ikoma, Yoko; Taniguchi, Junko; Aoki, Ichio; Tomita, Yutaka; Suzuki, Norihiro; Kanno, Iwao; Saeki, Naokatsu; Ito, Hiroshi

    2014-08-01

    This study aimed to examine the cortical microvessel diameter response to hypercapnia in misery perfusion using two-photon laser scanning microscopy (TPLSM). We evaluated whether the vascular response to hypercapnia could represent the cerebrovascular reserve. Cerebral blood flow (CBF) during normocapnia and hypercapnia was measured by laser-Doppler flowmetry through cranial windows in awake C57/BL6 mice before and at 1, 7, 14, and 28 days after unilateral common carotid artery occlusion (UCCAO). Diameters of the cortical microvessels during normocapnia and hypercapnia were also measured by TPLSM. Cerebral blood flow and the vascular response to hypercapnia were decreased after UCCAO. Before UCCAO, vasodilation during hypercapnia was found primarily in arterioles (22.9%±3.5%). At 14 days after UCCAO, arterioles, capillaries, and venules were autoregulatorily dilated by 79.5%±19.7%, 57.2%±32.3%, and 32.0%±10.8%, respectively. At the same time, the diameter response to hypercapnia in arterioles was significantly decreased to 1.9%±1.5%. A significant negative correlation was observed between autoregulatory vasodilation and the diameter response to hypercapnia in arterioles. Our findings indicate that arterioles play main roles in both autoregulatory vasodilation and hypercapnic vasodilation, and that the vascular response to hypercapnia can be used to estimate the cerebrovascular reserve.

  5. Advances in two photon scanning and scanless microscopy technologies for functional neural circuit imaging.

    Science.gov (United States)

    Schultz, Simon R; Copeland, Caroline S; Foust, Amanda J; Quicke, Peter; Schuck, Renaud

    2017-01-01

    Recent years have seen substantial developments in technology for imaging neural circuits, raising the prospect of large scale imaging studies of neural populations involved in information processing, with the potential to lead to step changes in our understanding of brain function and dysfunction. In this article we will review some key recent advances: improved fluorophores for single cell resolution functional neuroimaging using a two photon microscope; improved approaches to the problem of scanning active circuits; and the prospect of scanless microscopes which overcome some of the bandwidth limitations of current imaging techniques. These advances in technology for experimental neuroscience have in themselves led to technical challenges, such as the need for the development of novel signal processing and data analysis tools in order to make the most of the new experimental tools. We review recent work in some active topics, such as region of interest segmentation algorithms capable of demixing overlapping signals, and new highly accurate algorithms for calcium transient detection. These advances motivate the development of new data analysis tools capable of dealing with spatial or spatiotemporal patterns of neural activity, that scale well with pattern size.

  6. Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

    Science.gov (United States)

    Thériault, Gabrielle; Cottet, Martin; Castonguay, Annie; McCarthy, Nathalie; De Koninck, Yves

    2014-01-01

    Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general. PMID:24904284

  7. NIR-to-NIR Two-Photon Scanning Laser Microscopy Imaging of Single Nanoparticles Doped by Yb(III) Complexes.

    Science.gov (United States)

    Bourdolle, Adrien; D'Aléo, Anthony; Philippot, Cécile; Baldeck, Patrice L; Guyot, Yannick; Dubois, Fabien; Ibanez, Alain; Andraud, Chantal; Brasselet, Sophie; Maury, Olivier

    2016-01-04

    The photophysical and nonlinear optical properties of water-soluble chromophore-functionalised tris-dipicolinate complexes [LnL3](3-) (Ln=Yb and Nd) are thoroughly studied, revealing that only the Yb(III) luminescence can be sensitized by a two-photon excitation process. The stability of the complex in water is strongly enhanced by embedding in dispersible organosilicate nanoparticles (NPs). Finally, the spectroscopic properties of [NBu4]3 [YbL3] are studied in solution and in the solid state. The high brightness of the NPs allows imaging them as single objects using a modified two-photon microscopy setup in a NIR-to-NIR configuration.

  8. Mean cell size and collagen orientation from 2D Fourier analysis on confocal laser scanning microscopy and two-photon fluorescence microscopy on human skin in vivo

    Science.gov (United States)

    Lucassen, Gerald W.; Bakker, Bernard L.; Neerken, Sieglinde; Hendriks, Rob F. M.

    2003-07-01

    We present results from 2D Fourier analysis on 3D stacks of images obtained by confocal laser scanning reflectance microscopy (CLSM) and two-photon fluorescence microscopy (2PM) on human skin in vivo. CLSM images were obtained with a modified commercial system (Vivascope1000, Lucid Inc, excitation wavelength 830 nm) equipped with a piezo-focusing element (350 μm range) for depth positioning of the objective lens. 2PM was performed with a specially designed set-up with excitation wavelength 730 nm. Mean cell size in the epidermal layer and structural orientation in the dermal layer have been determined as a function of depth by 2D Fourier analysis. Fourier analysis on microscopic images enables automatic non-invasive quantitative structural analysis (mean cell size and orientation) of living human skin.

  9. Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

    Directory of Open Access Journals (Sweden)

    Rumelo Amor

    Full Text Available We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca(2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca(2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

  10. Widefield two-photon excitation without scanning: live cell microscopy with high time resolution and low photo-bleaching

    CERN Document Server

    Amor, Rumelo; Robb, Gillian; Wilson, Louise; Rahman, Nor Zaihana Abdul; Dempster, John; Amos, William Bradshaw; Bushell, Trevor J; McConnell, Gail

    2015-01-01

    We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca$^{2+}$ events in primary rat neurone cultures loaded with the fluorescent Ca$^{2+}$ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

  11. A High Performance, Cost-Effective, Open-Source Microscope for Scanning Two-Photon Microscopy that Is Modular and Readily Adaptable

    Science.gov (United States)

    Rosenegger, David G.; Tran, Cam Ha T.; LeDue, Jeffery; Zhou, Ning; Gordon, Grant R.

    2014-01-01

    Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems. PMID:25333934

  12. A high performance, cost-effective, open-source microscope for scanning two-photon microscopy that is modular and readily adaptable.

    Directory of Open Access Journals (Sweden)

    David G Rosenegger

    Full Text Available Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.

  13. Evaluation of transdermal delivery of nanoemulsions in ex vivo porcine skin using two-photon microscopy and confocal laser-scanning microscopy

    Science.gov (United States)

    Choi, Sanghoon; Kim, Jin Woong; Lee, Yong Joong; Delmas, Thomas; Kim, Changhwan; Park, Soyeun; Lee, Ho

    2014-10-01

    This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.

  14. Reduction of the pulse duration of the ultrafast laser pulses of the Two-Photon Laser Scanning Microscopy (2PLSM

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    Reshak Ali

    2008-07-01

    Full Text Available Abstract Background We provide an update of our two-photon laser scanning microscope by compressing or reducing the broadening of the pulse width of ultrafast laser pulses for dispersion precompensation, to enable the pulses to penetrate deeply inside the sample. Findings The broadening comes as the pulses pass through the optical elements. We enhanced and modified the quality and the sharpness of images by enhancing the resolution using special polarizer namely Glan Laser polarizer GL10. This polarizer consists of two prisms separated by air space. This air separation between the two prisms uses to delay the red wavelength when the light leaves the first prism to the air then to second prism. We note a considerable enhancing with using the GL polarizer, and we can see the details of the leaf structure in early stages when we trying to get focus through z-stacks of images in comparison to exactly the same measurements without using GL polarizer. Hence, with this modification we able to reduce the time of exposure the sample to the laser radiation thereby we will reduce the probability of photobleaching and phototoxicity. When the pulse width reduced, the average power of the laser pulses maintained at a constant level. Significant enhancement is found between the two kinds of images of the Two-Photon Excitation Fluorescence (TPEF. Conclusion In summary reduction the laser pulse width allowed to collect more diffraction orders which will used to form the images. The more diffraction orders the higher resolution images.

  15. Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confo-cal laser scanning microscopy

    Institute of Scientific and Technical Information of China (English)

    张春阳; 李艳平; 马辉; 李素文; 薛绍白; 陈瓞延

    2001-01-01

    Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

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

    Directory of Open Access Journals (Sweden)

    Jong Kang Park

    2017-03-01

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

  17. Two-photon excitation laser scanning microscopy of rabbit nasal septal cartilage following Nd:YAG-laser-mediated stress relaxation

    Science.gov (United States)

    Kim, Charlton C.; Wallace, Vincent P.; Coleno, Mariah L.; Dao, Xavier; Tromberg, Bruce J.; Wong, Brian J.

    2000-04-01

    Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within rabbit nasal septal cartilage tissue over a 12-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation. During laser irradiation surface temperature, stress relaxation, and diffuse reflectance, were measured dynamically. Each specimen received one or two sequential laser exposures. The cartilage reached a peak surface temperature of about 61 degrees C during irradiation. Cartilage denatured in 50 percent EtOH was used as a positive control. TPM was performed to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns, immediately following laser exposure, and also following 12 days in culture. Few differences in the pattern or intensity of fluorescence was observed between controls and irradiated specimens imaged immediately following exposure, regardless of the number of laser pulses. However, following twelve days in tissue culture, the irradiated specimens increase, whereas the native tissue diminishes, in intensity and distribution of fluorescence in the cytoplasm. In contrast, the positive control shows only extracellular matrices and empty lacuna, feature consistent with cell membrane lysis.

  18. Two-photon microscopy for chemical neuroscience.

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    Ellis-Davies, Graham C R

    2011-04-20

    Microscopes using non-linear excitation of chromophores with pulsed near-IR light can generate highly localized foci of molecules in the electronic singlet state that are concentrated in volumes of less than one femtoliter. The three-dimensional confinement of excitation arises from the simultaneous absorption of two IR photons of approximately half the energy required for linear excitation. Two-photon microscopy is especially useful for two types of interrogation of neural processes. First, uncaging of signaling molecules such as glutamate, as stimulation is so refined it can be used to mimic normal unitary synaptic levels. In addition, uncaging allows complete control of the timing and position of stimulation, so the two-photon light beam provides the chemical neuroscientist with an "optical conductor's baton" which can command synaptic activity at will. A second powerful feature of two-photon microscopy is that when used for fluorescence imaging it enables the visualization of cellular structure and function in living animals at depths far beyond that possible with normal confocal microscopes. In this review I provide a survey of the many important applications of two-photon microscopy in these two fields of neuroscience, and suggest some areas for future technical development.

  19. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

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    Kurt F Ahrens

    2012-03-01

    Full Text Available A fluorescent voltage sensor protein Flare was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular visual crescent in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo.

  20. Two-photon scanning microscopy of in vivo sensory responses of cortical neurons genetically encoded with a fluorescent voltage sensor in rat

    Science.gov (United States)

    Ahrens, Kurt F.; Heider, Barbara; Lee, Hanson; Isacoff, Ehud Y.; Siegel, Ralph M.

    2012-01-01

    A fluorescent voltage sensor protein “Flare” was created from a Kv1.4 potassium channel with YFP situated to report voltage-induced conformational changes in vivo. The RNA virus Sindbis introduced Flare into neurons in the binocular region of visual cortex in rat. Injection sites were selected based on intrinsic optical imaging. Expression of Flare occurred in the cell bodies and dendritic processes. Neurons imaged in vivo using two-photon scanning microscopy typically revealed the soma best, discernable against the background labeling of the neuropil. Somatic fluorescence changes were correlated with flashed visual stimuli; however, averaging was essential to observe these changes. This study demonstrates that the genetic modification of single neurons to express a fluorescent voltage sensor can be used to assess neuronal activity in vivo. PMID:22461770

  1. Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

    Science.gov (United States)

    Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

    2017-01-01

    Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. In Vivo Time-Course Imaging of Tumor Angiogenesis in Colorectal Liver Metastases in the Same Living Mice Using Two-Photon Laser Scanning Microscopy

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    Koji Tanaka

    2012-01-01

    Full Text Available In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM. We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green. In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.

  3. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances

    Science.gov (United States)

    Diaspro, Alberto

    2001-11-01

    Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references Companion FTP site with full-color photographs The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.

  4. Two-photon excitation laser scanning microscopy of porcine nasal septal cartilage following Nd:YAG laser-mediated stress relaxation

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    Kim, Charlton C.; Wallace, Vincent P.; Rasouli, Alexandre; Coleno, Mariah L.; Dao, Xavier; Tromberg, Bruce J.; Wong, Brian J.

    2000-05-01

    Laser irradiation of hyaline cartilage result in stable shape changes due to temperature dependent stress relaxation. In this study, we determined the structural changes in chondrocytes within porcine nasal septal cartilage tissue over a 4-day period using a two-photon laser scanning microscope (TPM) following Nd:YAG laser irradiation (lambda equals 1.32 micrometer) using parameters that result in mechanical stress relaxation (6.0 W, 5.4 mm spot diameter). TPM excitation (780 nm) result in induction of fluorescence from endogenous agents such as NADH, NADPH, and flavoproteins in the 400 - 500 nm spectral region. During laser irradiation diffuse reflectance (from a probe HeNe laser, (lambda) equals 632.8 nm), surface temperature, and stress relaxation were measured dynamically. Each specimen received one, two, or three sequential laser exposures (average irradiation times of 5, 6, and 8 seconds). The cartilage reached a peak surface temperature of about 70 degrees Celsius during irradiation. Cartilage denatured in 50% EtOH (20 minutes) was used as a positive control. TPM was performed using a mode-locked 780 nm Titanium:Sapphire (Ti:Al203) beam with a, 63X, 1.2 N.A. water immersion objective (working distance of 200 mm) to detect the fluorescence emission from the chondrocytes. Images of chondrocytes were obtained at depths up to 150 microns (lateral resolution equals 35 micrometer X 35 micrometer). Images were obtained immediately following laser exposure, and also after 4 days in culture. In both cases, the irradiated and non-irradiated specimens do not show any discernible difference in general shape or auto fluorescence. In contrast, positive controls (immersed in 50% ethanol), show markedly increased fluorescence relative to both the native and irradiated specimens, in the cytoplasmic region.

  5. Two-photon flow cytometer with laser scanning Bessel beams

    Science.gov (United States)

    Wang, Yongdong; Ding, Yu; Ray, Supriyo; Paez, Aurelio; Xiao, Chuan; Li, Chunqiang

    2016-03-01

    Flow cytometry is an important technique in biomedical discovery for cell counting, cell sorting and biomarker detection. In vivo flow cytometers, based on one-photon or two-photon excited fluorescence, have been developed for more than a decade. One drawback of laser beam scanning two-photon flow cytometer is that the two-photon excitation volume is fairly small due to the short Rayleigh range of a focused Gaussian beam. Hence, the sampling volume is much smaller than one-photon flow cytometry, which makes it challenging to count or detect rare circulating cells in vivo. Bessel beams have narrow intensity profiles with an effective spot size (FWHM) as small as several wavelengths, making them comparable to Gaussian beams. More significantly, the theoretical depth of field (propagation distance without diffraction) can be infinite, making it an ideal solution as a light source for scanning beam flow cytometry. The trade-off of using Bessel beams rather than a Gaussian beam is the fact that Bessel beams have small concentric side rings that contribute to background noise. Two-photon excitation can reduce this noise, as the excitation efficiency is proportional to intensity squared. Therefore, we developed a two-photon flow cytometer using scanned Bessel beams to form a light sheet that intersects the micro fluidic channel.

  6. Two-photon absorbing porphyrins for oxygen microscopy (Conference Presentation)

    Science.gov (United States)

    Esipova, Tatiana V.; Vinogradov, Sergei A.

    2016-03-01

    The ability to quantify oxygen in vivo in 3D with high spatial and temporal resolution is invaluable for many areas of the biomedical science, including ophthalmology, neuroscience, cancer and stem biology. An optical method based on oxygen-dependent quenching of phosphorescence is being developed, that allows quantitative minimally invasive real-time imaging of partial pressure of oxygen (pO2) in tissue. In the past, dendritically protected phosphorescent oxygen probes with controllable quenching parameters and defined bio-distributions have been developed. More recently our probe strategy has extended to encompass two-photon excitable oxygen probes, which brought about first demonstrations of two-photon phosphorescence lifetime microscopy (2PLM) of oxygen in vivo, providing new valuable information for neuroscience and stem cell biology. However, current two-photon oxygen probes suffer from a number of limitations, such as low brightness and high cost of synthesis, which dramatically reduce imaging performance and limit usability of the method. Here we present an approach to new bright phosphorescent chromophores with internally enhanced two-photon absorption cross-sections, which pave a way to novel proves for 2PLM. In addition to substantial increase in performance, the new probes can be synthesized by much more efficient methods, thereby greatly reducing the cost of the synthesis and making the technique accessible to a broader range of researchers across different fields.

  7. Two-photon microscopy using fiber-based nanosecond excitation.

    Science.gov (United States)

    Karpf, Sebastian; Eibl, Matthias; Sauer, Benjamin; Reinholz, Fred; Hüttmann, Gereon; Huber, Robert

    2016-07-01

    Two-photon excitation fluorescence (TPEF) microscopy is a powerful technique for sensitive tissue imaging at depths of up to 1000 micrometers. However, due to the shallow penetration, for in vivo imaging of internal organs in patients beam delivery by an endoscope is crucial. Until today, this is hindered by linear and non-linear pulse broadening of the femtosecond pulses in the optical fibers of the endoscopes. Here we present an endoscope-ready, fiber-based TPEF microscope, using nanosecond pulses at low repetition rates instead of femtosecond pulses. These nanosecond pulses lack most of the problems connected with femtosecond pulses but are equally suited for TPEF imaging. We derive and demonstrate that at given cw-power the TPEF signal only depends on the duty cycle of the laser source. Due to the higher pulse energy at the same peak power we can also demonstrate single shot two-photon fluorescence lifetime measurements.

  8. Insights into esophagus tissue architecture using two-photon confocal microscopy

    Science.gov (United States)

    Liu, Nenrong; Wang, Yue; Feng, Shangyuan; Chen, Rong

    2013-08-01

    In this paper, microstructures of human esophageal mucosa were evaluated using the two-photon laser scanning confocal microscopy (TPLSCM), based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). The distribution of epithelial cells, muscle fibers of muscularis mucosae has been distinctly obtained. Furthermore, esophageal submucosa characteristics with cancer cells invading into were detected. The variation of collagen, elastin and cancer cells is very relevant to the pathology in esophagus, especially early esophageal cancer. Our experimental results indicate that the MPM technique has the much more advantages for label-free imaging, and has the potential application in vivo in the clinical diagnosis and monitoring of early esophageal cancer.

  9. Tailored probes for atomic force microscopy fabricated by two-photon polymerization

    Science.gov (United States)

    Göring, Gerald; Dietrich, Philipp-Immanuel; Blaicher, Matthias; Sharma, Swati; Korvink, Jan G.; Schimmel, Thomas; Koos, Christian; Hölscher, Hendrik

    2016-08-01

    3D direct laser writing based on two-photon polymerization is considered as a tool to fabricate tailored probes for atomic force microscopy. Tips with radii of 25 nm and arbitrary shape are attached to conventionally shaped micro-machined cantilevers. Long-term scanning measurements reveal low wear rates and demonstrate the reliability of such tips. Furthermore, we show that the resonance spectrum of the probe can be tuned for multi-frequency applications by adding rebar structures to the cantilever.

  10. Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination.

    Science.gov (United States)

    Chu, Kengyeh K; Lim, Daryl; Mertz, Jerome

    2007-10-01

    We describe a technique to enhance both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. The technique is based on maintaining a fixed detection power by fast feedback control of the illumination power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. We analyze and demonstrate the benefits of adaptive illumination in two-photon fluorescence microscopy.

  11. In vivo reactive neural plasticity investigation by means of correlative two photon: electron microscopy

    Science.gov (United States)

    Allegra Mascaro, A. L.; Cesare, P.; Sacconi, L.; Grasselli, G.; Mandolesi, G.; Maco, B.; Knott, G.; Huang, L.; De Paola, V.; Strata, P.; Pavone, F. S.

    2013-02-01

    In the adult nervous system, different populations of neurons correspond to different regenerative behavior. Although previous works showed that olivocerebellar fibers are capable of axonal regeneration in a suitable environment as a response to injury1, we have hitherto no details about the real dynamics of fiber regeneration. We set up a model of singularly axotomized climbing fibers (CF) to investigate their reparative properties in the adult central nervous system (CNS) in vivo. Time lapse two-photon imaging has been combined to laser nanosurgery2, 3 to define a temporal pattern of the degenerative event and to follow the structural rearrangement after injury. To characterize the damage and to elucidate the possible formation of new synaptic contacts on the sprouted branches of the lesioned CF, we combined two-photon in vivo imaging with block face scanning electron microscopy (FIB-SEM). Here we describe the approach followed to characterize the reactive plasticity after injury.

  12. Imaging theory and resolution improvement of two-photon confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    唐志列; 杨初平; 裴红津; 梁瑞生; 刘颂豪

    2002-01-01

    The nonlinear effect of two-photon excitation on the imaging property of two-photonconfocal microscopy has been analyzed by the two-photon fluorescence intensity transfer functionderived in this paper. The two-photon fluorescence intensity transfer function in a confocal micros-copy is given. Furthermore the three-dimensional point spread function (3D-PSF) and thethree-dimensional optical transfer function (3D-OTF) of two-photon confocal microscopy are de-rived based on the nonlinear effect of two-photon excitation. The imaging property of two-photonconfocal microscopy is discussed in detail based on 3D-OTF. Finally the spatial resolution limit oftwo-photon confocal microscopy is discussed according to the uncertainty principle.

  13. Integrated single- and two-photon light sheet microscopy using accelerating beams

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2017-01-01

    We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode, a...

  14. Two-photon microscopy with diffractive optical elements and spatial light modulators

    Directory of Open Access Journals (Sweden)

    Brendon O Watson

    2010-09-01

    Full Text Available Two-photon microscopy is often performed at slow frame rates, due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE generates a fixed number of beamlets that are scanned in parallel, resulting in a corresponding increase in speed, or in signal-to-noise ratio, in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM, to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image, such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions, including light path corrections or as adaptive optical devices.

  15. A new approach to dual-color two-photon microscopy with fluorescent proteins

    Directory of Open Access Journals (Sweden)

    Rebane Aleks

    2010-02-01

    Full Text Available Abstract Background Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA efficiency. Results Here we present a new method of dual-color two-photon microscopy that uses the simultaneous excitation of the lowest-energy electronic transition of a blue fluorescent protein and a higher-energy electronic transition of a red fluorescent protein. Conclusion Our method does not require large differences in Stokes shifts and can be extended to a variety of FP pairs with larger 2PA efficiency and more optimal imaging properties.

  16. A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy.

    Science.gov (United States)

    Day, Richard N; Tao, Wen; Dunn, Kenneth W

    2016-11-01

    Genetically encoded fluorescent protein (FP)-based biosensor probes are useful tools for monitoring cellular events in living cells and tissues. Because these probes were developed for one-photon excitation approaches, their broad two-photon excitation (2PE) and poorly understood photobleaching characteristics have made their implementation in studies using two-photon laser-scanning microscopy (TPLSM) challenging. Here we describe a protocol that simplifies the use of Förster resonance energy transfer (FRET)-based biosensors in TPLSM. First, the TPLSM system is evaluated and optimized using FRET standards expressed in living cells, which enables the determination of spectral bleed-through (SBT) and the confirmation of FRET measurements from the known standards. Next, we describe how to apply the approach experimentally using a modified version of the A kinase activity reporter (AKAR) protein kinase A (PKA) biosensor as an example-first in cells in culture and then in hepatocytes in the liver of living mice. The microscopic imaging can be accomplished in a day in laboratories that routinely use TPLSM.

  17. Correction-free remotely scanned two-photon in vivo mouse retinal imaging

    Institute of Scientific and Technical Information of China (English)

    Adi Schejter Bar-Noam; Nairouz Farah; Shy Shoham

    2016-01-01

    Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications.The in vivotwo-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue.However,to date,this has only been possible using relatively complex adaptive-optics systems.Here,the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality,optically sectioned fundus images.By remotely scanning the focus using an electronically tunable lens,high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired,thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging.Moreover,the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator,GCaMP6s.These results and the simplicity of the new add-on optics are an important step toward several structural,functional,and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.

  18. Two-Photon and Second Harmonic Microscopy in Clinical and Translational Cancer Research

    Science.gov (United States)

    PERRY, SETH W.; BURKE, RYAN M.; BROWN, EDWARD B.

    2012-01-01

    Application of two-photon microscopy (TPM) to translational and clinical cancer research has burgeoned over the last several years, as several avenues of pre-clinical research have come to fruition. In this review, we focus on two forms of TPM—two-photon excitation fluorescence microscopy, and second harmonic generation microscopy—as they have been used for investigating cancer pathology in ex vivo and in vivo human tissue. We begin with discussion of two-photon theory and instrumentation particularly as applicable to cancer research, followed by an overview of some of the relevant cancer research literature in areas that include two-photon imaging of human tissue biopsies, human skin in vivo, and the rapidly developing technology of two-photon microendoscopy. We believe these and other evolving two-photon methodologies will continue to help translate cancer research from the bench to the bedside, and ultimately bring minimally invasive methods for cancer diagnosis and treatment to therapeutic reality. PMID:22258888

  19. QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

    Directory of Open Access Journals (Sweden)

    Merete Krog Raarup

    2011-05-01

    Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

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

    CERN Document Server

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

    2015-01-01

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

  1. Two-photon microscopy with double-circle trajectories for in vivo cerebral blood flow measurements

    Science.gov (United States)

    Landolt, Andrin; Obrist, Dominik; Wyss, Matthias; Barrett, Matthew; Langer, Dominik; Jolivet, Renaud; Soltysinski, Tomasz; Roesgen, Thomas; Weber, Bruno

    2013-05-01

    Scanning microscopes normally use trajectories which produce full-frame images of an object at a low frame rate. Time-resolved measurements are possible if scans along a single line are repeated at a high rate. In conjunction with fluorescence labeling techniques, in vivo recording of blood flow in single capillaries is possible. The present work investigates scanning with double-circle trajectories to measure blood flow simultaneously in several vessels of a capillary network. With the trajectory centered near a bifurcation, a double circle crosses each vessel twice, creating a sensing gate for passing dark red blood cells in fluorescently labeled plasma. From the stack of scans repeated at 1,300 Hz, the time-resolved velocity is retrieved using an image correlation approach. Single bifurcation events can be identified from a few fluorescently labeled red blood cells. The applicability of the method for in vivo measurements is illustrated on the basis of two-photon laser scanning microscopy of the cerebral capillary network of mice. Its performance is assessed with synthetic data generated from a two-phase model for the perfusion in a capillary network. The calculation of velocities is found to be sufficiently robust for a wide range of conditions. The achievable limits depend significantly on the experimental conditions and are estimated to be in the 1 μm/s (velocity) and 0.1 s (time resolution) ranges, respectively. Some manual fine-tuning is required for optimal performance in terms of accuracy and time resolution. Further work may lead to improved reliability with which bifurcation events are identified in the algorithm and to include red blood cell flux and hematocrit measurements. With the capability for time-resolved measurements in all vessels of a bifurcation, double-circle scanning trajectories allow a detailed study of the dynamics in vascular networks.

  2. Femtosecond laser subsurface scleral treatment in cadaver human sclera and evaluation using two-photon and confocal microscopy

    Science.gov (United States)

    Sun, Hui; Fan, Zhongwei; Yan, Ying; Lian, Fuqiang; Kurtz, Ron; Juhasz, Tibor

    2016-03-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial-thickness drainage channels can be created with femtosecond laser in the translucent sclera for the potential treatment of glaucoma. We demonstrate the creation of partial-thickness subsurface drainage channels with the femtosecond laser in the cadaver human eyeballs and describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. A femtosecond laser operating at a wavelength of 1700 nm was scanned along a rectangular raster pattern to create the partial thickness subsurface drainage channels in the sclera of cadaver human eyes. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in cadaver human eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such partial thickness subsurface scleral channels. Our studies suggest that the confocal and two-photon microscopy can be used to investigate femtosecond-laser created partial-thickness drainage channels in the sclera of cadaver human eyes.

  3. Two-photon luminescence microscopy of field enhancement at gold nanoparticles

    DEFF Research Database (Denmark)

    Beermann, Jonas; Bozhevolnyi, Sergey I.

    2005-01-01

    Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both polarizat......Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both...

  4. Visualization of laser tattoo removal treatment effects in a mouse model by two-photon microscopy

    Science.gov (United States)

    Jang, Won Hyuk; Yoon, Yeoreum; Kim, Wonjoong; Kwon, Soonjae; Lee, Seunghun; Song, Duke; Choi, Jong Woon; Kim, Ki Hean

    2017-01-01

    Laser tattoo removal is an effective method of eliminating tattoo particles in the skin. However, laser treatment cannot always remove the unwanted tattoo completely, and there are risks of either temporary or permanent side effects. Studies using preclinical animal models could provide detailed information on the effects of laser treatment in the skin, and might help to minimize side effects in clinical practices. In this study, two-photon microscopy (TPM) was used to visualize the laser treatment effects on tattoo particles in both phantom specimens and in vivo mouse models. Fluorescent tattoo ink was used for particle visualization by TPM, and nanosecond (ns) and picosecond (ps) lasers at 532 nm were used for treatment. In phantom specimens, TPM characterized the fragmentation of individual tattoo particles by tracking them before and after the laser treatment. These changes were confirmed by field emission scanning electron microscopy (FE-SEM). TPM was used to measure the treatment efficiency of the two lasers at different laser fluences. In the mouse model, TPM visualized clusters of tattoo particles in the skin and detected their fragmentation after the laser treatment. Longitudinal TPM imaging observed the migration of cells containing tattoo particles after the laser treatment. These results show that TPM may be useful for the assessment of laser tattoo removal treatment in preclinical studies. PMID:28856046

  5. Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2015-03-01

    High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas.

  6. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N.; Wientjes, Emilie; Amerongen, van Herbert

    2016-01-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was ac

  7. Polarization-resolved two-photon luminescence microscopy of V-groove arrays

    DEFF Research Database (Denmark)

    Beermann, J.; Novikov, S. M.; Holmgaard, T.

    2012-01-01

    Using two-photon luminescence (TPL) microscopy and local reflection spectroscopy we investigate electromagnetic field enhancement effects from a mu m-sized composition of 450-nm-deep V-grooves milled by focused ion beam in a thick gold film and assembled to feature, within the same structure...

  8. Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves

    NARCIS (Netherlands)

    Broess, K.; Borst, J.W.; Amerongen, van H.

    2009-01-01

    This study investigates to which extent two-photon excitation (TPE) fluorescence lifetime imaging microscopy can be applied to study picosecond fluorescence kinetics of individual chloroplasts in leaves. Using femtosecond 860 nm excitation pulses, fluorescence lifetimes can be measured in leaves of

  9. Polarization-resolved two-photon luminescence microscopy of V-groove arrays

    DEFF Research Database (Denmark)

    Beermann, J.; Novikov, S. M.; Holmgaard, T.;

    2012-01-01

    Using two-photon luminescence (TPL) microscopy and local reflection spectroscopy we investigate electromagnetic field enhancement effects from a mu m-sized composition of 450-nm-deep V-grooves milled by focused ion beam in a thick gold film and assembled to feature, within the same structure...... obtained to evaluation of local field enhancements using TPL microscopy, especially when investigating extended structures exhibiting different radiation channels, are discussed. (C)2011 Optical Society of America...

  10. Imaging marine virus CroV and its host Cafeteria roenbergensis with two-photon microscopy

    Science.gov (United States)

    Cao, Bin; Chakraborty, Sayan; Sun, Wenqing; Aghvami, Seyedmohammadali; Fischer, Matthias G.; Qian, Wei; Xiao, Chuan; Li, Chunqiang

    2014-02-01

    We use two-photon microscopy to monitor the infection process of marine zooplankton, Cafeteria roenbergensis (C.roenbergensis), by Cafeteria roenbergensis virus (CroV), a giant DNA virus named after its host. Here, we image C.roenbergensis in culture by two-photon excited NADH autofluorescence at video-rate (30 frame/s), and the movement of C.roenbergensis is recorded in live videos. Moreover, CroV is stained with DNA dye SYBR gold and recorded simultaneously with this two-photon microscope. We observed the initial infection moment with this method. The result demonstrates the potential use of two-photon microscopy to investigate the fast dynamic interaction between C.roenbergensis with virus CroV. After catching this initial moment, we will freeze the sample in liquid nitrogen for cryo-electron microscopy (EM) study to resolve the virus-host interaction at molecular level. The long-term goal is to study similar fast moving pathogen-host interaction process which could lead to important medical applications.

  11. Imaging zebrafish embryos by two-photon excitation time-lapse microscopy.

    Science.gov (United States)

    Carvalho, Lara; Heisenberg, Carl-Philipp

    2009-01-01

    The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. In particular, 2PE microscopy has been shown to be useful for imaging deep cell layers within the embryo and following tissue morphogenesis over long periods. This chapter describes how to use 2PE microscopy to study morphogenetic movements during early zebrafish embryonic development, providing a general blueprint for its use in zebrafish.

  12. Evaluation of human sclera after femtosecond laser ablation using two photon and confocal microscopy

    Science.gov (United States)

    Sun, Hui; Kurtz, Ronald; Juhasz, Tibor

    2012-08-01

    Glaucoma is the second-leading cause of blindness worldwide and is often associated with elevated intraocular pressure (IOP). Partial thickness intrascleral channels can be created with a femtosecond laser operating at a wavelength of 1700 nm. Such channels have the potential to increase outflow facility and reduce elevated IOP. Analysis of the dimensions and location of these channels is important in understanding their effects. We describe the application of two-photon microscopy and confocal microscopy for noninvasive imaging of the femtosecond laser created partial-thickness scleral channels in human cadaver eyes. High-resolution images, hundreds of microns deep in the sclera, were obtained to allow determination of the shape and dimension of such channels. This demonstrates that concept of integrating femtosecond laser surgery, and two-photon and confocal imaging has the future potential for image-guided high-precision surgery in transparent and translucent tissue.

  13. Functional screening of intracardiac cell transplants using two-photon fluorescence microscopy.

    Science.gov (United States)

    Tao, Wen; Soonpaa, Mark H; Field, Loren J; Chen, Peng-Sheng; Firulli, Anthony B; Shou, Weinian; Rubart, Michael

    2012-08-01

    Although the adult mammalian myocardium exhibits a limited ability to undergo regenerative growth, its intrinsic renewal rate is insufficient to compensate for myocyte loss during cardiac disease. Transplantation of donor cardiomyocytes or cardiomyogenic stem cells is considered a promising strategy for reconstitution of cardiac mass, provided the engrafted cells functionally integrate with host myocardium and actively contribute to its contractile force. The authors previously developed a two-photon fluorescence microscopy-based assay that allows in situ screening of donor cell function after intracardiac delivery of the cells. This report reviews the techniques of two-photon fluorescence microscopy and summarizes its application for quantifying the extent to which a variety of donor cell types stably and functionally couple with the recipient myocardium.

  14. Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy (Conference Presentation)

    Science.gov (United States)

    Kwok, Sheldon J. J.; Kuznetsov, Ivan A.; Kim, Moonseok; Choi, Myunghwan; Scarcelli, Giuliano; Yun, Seok-Hyun

    2017-02-01

    Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively. Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.

  15. Phosphorescent probes for two-photon microscopy of oxygen (Conference Presentation)

    Science.gov (United States)

    Vinogradov, Sergei A.; Esipova, Tatiana V.

    2016-03-01

    The ability to quantify oxygen in vivo in 3D with high spatial and temporal resolution is much needed in many areas of biological research. Our laboratory has been developing the phosphorescence quenching technique for biological oximetry - an optical method that possesses intrinsic microscopic capability. In the past we have developed dendritically protected oxygen probes for quantitative imaging of oxygen in tissue. More recently we expanded our design on special two-photon enhanced phosphorescent probes. These molecules brought about first demonstrations of the two-photon phosphorescence lifetime microscopy (2PLM) of oxygen in vivo, providing new information for neouroscience and stem cell biology. However, current two-photon oxygen probes suffer from a number of limitations, such as sub-optimal brightness and high cost of synthesis, which dramatically reduce imaging performance and limit usability of the method. In this paper we discuss principles of 2PLM and address the interplay between the probe chemistry, photophysics and spatial and temporal imaging resolution. We then present a new approach to brightly phosphorescent chromophores with internally enhanced two-photon absorption cross-sections, which pave a way to a new generation of 2PLM probes.

  16. [Intensity loss of two-photon excitation fluorescence microscopy images of mouse oocyte chromosomes].

    Science.gov (United States)

    Zhao, Feng-Ying; Wu, Hong-Xin; Chen, Die-Yan; Ma, Wan-Yun

    2014-07-01

    As an optical microscope with high resolution, two-photon excitation (TPE) fluorescence microscope is widely used in noninvasive 3D optical imaging of biological samples. Compared with confocal laser scanning microscope, TPE fluorescence microscope provides a deeper detecting depth. In spite of that, the image quality of sample always declines as the detecting depth increases when a noninvasive 3D optical imaging of thicker samples is performed. Mouse oocytes with a large diameter, which play an important role in clinical and biological fields, have obvious absorption and scattering effects. In the present paper, we performed compensation for two-photon fluorescence images of mouse oocyte chromosomes. Using volume as a parameter, the attenuation degree of these chromosomes was also studied. The result of our data suggested that there exists a severe axial intensity loss in two-photon microscopic images of mouse oocytes due to the absorption and scattering effects. It is necessary to make compensation for these images of mouse oocyte chromosomes obtained from two-photon microscopic system. It will be specially needed in studying the quantitative three-dimensional information of mouse oocytes.

  17. Two-photon microscopy for imaging germinal centers and T follicular helper cells.

    Science.gov (United States)

    Clatworthy, Menna R

    2015-01-01

    One of the principle features of immune cells is their dynamic nature. Lymphocytes circulate in the blood between secondary lymphoid organs and tissues in an effort to maximize the likelihood of a rapid and appropriate immune response to invading pathogens and tissue damage. Conventional experimental techniques such as histology and flow cytometry have greatly increased our understanding of immune cells, but in the last decade, two-photon microscopy has revolutionized our ability to interrogate the dynamic behavior of immune cells, a facet so critical to their function. Two-photon microscopy relies on the excitation of fluorophores by simultaneous application of two photons of longer wavelength light. This allows a greater depth of imaging with minimal photodamage. Thus, living tissues can be imaged, including immune cells in lymph nodes. This technique has been used to interrogate the events occurring in a germinal center response and the interactions between cells in the germinal center, including T follicular helper cells (Tfh), germinal center B cells, and follicular dendritic cells (FDC). Herein, a method is described by which the interactions between Tfh and B cells within a germinal center in a popliteal lymph node can be imaged in a live mouse.

  18. Dendritic spine classification using shape and appearance features based on two-photon microscopy.

    Science.gov (United States)

    Ghani, Muhammad Usman; Mesadi, Fitsum; Kanık, Sümeyra Demir; Argunşah, Ali Özgür; Hobbiss, Anna Felicity; Israely, Inbal; Ünay, Devrim; Taşdizen, Tolga; Çetin, Müjdat

    2017-03-01

    Neuronal morphology and function are highly coupled. In particular, dendritic spine morphology is strongly governed by the incoming neuronal activity. The first step towards understanding the structure-function relationships is to classify spine shapes into the main spine types suggested in the literature. Due to the lack of reliable automated analysis tools, classification is mostly performed manually, which is a time-intensive task and prone to subjectivity. We propose an automated method to classify dendritic spines using shape and appearance features based on challenging two-photon laser scanning microscopy (2PLSM) data. Disjunctive Normal Shape Models (DNSM) is a recently proposed parametric shape representation. We perform segmentation of spine images by applying DNSM and use the resulting representation as shape features. Furthermore, we use Histogram of oriented gradients (HOG) to extract appearance features. In this context, we propose a kernel density estimation (KDE) based framework for dendritic spine classification, which uses these shape and appearance features. Our shape and appearance features based approach combined with Neural Network (NN) correctly classifies 87.06% of spines on a dataset of 456 spines. Our proposed method outperforms standard morphological feature based approaches. Our KDE based framework also enables neuroscientists to analyze the separability of spine shape classes in the likelihood ratio space, which leads to further insights about nature of the spine shape analysis problem. Results validate that performance of our proposed approach is comparable to a human expert. It also enable neuroscientists to study shape statistics in the likelihood ratio space. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Wide-field two-photon microscopy: features and advantages for biomedical applications

    Science.gov (United States)

    Wachsmann-Hogiu, S.; Hwang, J. Y.; Lindsley, E.; Farkas, D. L.

    2007-02-01

    We describe a simple fluorescence microscope based on wide-field two-photon excitation. While still taking advantage of some inherent properties of non-linear (two-photon) microscopy, such as increased penetration depth through tissue and reduced phototoxicity, this approach provides video frame rate imaging, can be easily coupled to fluorescence spectral and lifetime detection modules, and makes efficient use of the high average power currently available from ultrashort pulsed lasers. For a standard histopathology specimen, we were able to identify different structures based on spectral and fluorescence lifetime detection and analysis. We examined the use of 200fs and 2ps pulses from Spectra Physics MaiTai and Tsunami lasers, respectively, with average power ranging from 50mW to 500mW.

  20. Measurement of degenerate two-photon absorption spectra of a series of developed two-photon initiators using a dispersive white light continuum Z-scan

    Science.gov (United States)

    Ajami, Aliasghar; Husinsky, Wolfgang; Tromayer, Maximilian; Gruber, Peter; Liska, Robert; Ovsianikov, Aleksandr

    2017-08-01

    To achieve efficient micro- and nanostructuring based on two-photon polymerization (2PP), the development and evaluation of specialized two-photon initiators (2PIs) are essential. Hence, a reliable method to determine the two-photon absorption (2PA) spectra of the synthesized 2PIs used for 2PP structuring is crucial. A technique by which absolute visible-to-near-infrared 2PA spectra of degenerate nature can be determined via performing a single dispersive white-light continuum (WLC) Z-scan has been realized. Using a dispersed white light beam containing 8 fs pulses at wavelengths ranging from 650 nm to 950 nm, the nonlinear transmittance as a function of the sample position can be measured for all spectral components by performing a single scan along the laser beam propagation direction. In this work, the 2PA spectrum of three different 2PIs was determined using this technique. 2PP structuring was also accomplished using the developed 2PIs at different wavelengths. Tuning the wavelength of the laser to match the peak of the 2PA spectra of the developed 2PIs resulted in lower intensity thresholds and facilitated higher structuring speeds. As an example, using M2CMK 2PI for 2PP, the scanning speed can be increased up to 5 folds when the laser wavelength is tuned to 760 nm (i.e., 2PA maximum) instead of the conventionally used 800 nm.

  1. Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

    Directory of Open Access Journals (Sweden)

    Philipp Steven

    Full Text Available BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM. Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible

  2. Two-Photon Microscopy Allows Imaging and Characterization of Cochlear Microvasculature In Vivo

    Directory of Open Access Journals (Sweden)

    Friedrich Ihler

    2015-01-01

    Full Text Available Impairment of cochlear blood flow has been discussed as factor in the pathophysiology of various inner ear disorders. However, the microscopic study of cochlear microcirculation is limited due to small scale and anatomical constraints. Here, two-photon fluorescence microscopy is applied to visualize cochlear microvessels. Guinea pigs were injected with Fluorescein isothiocyanate- or Texas red-dextrane as plasma marker. Intravital microscopy was performed in four animals and explanted cochleae from four animals were studied. The vascular architecture of the cochlea was visualized up to a depth of 90.0±22.7 μm. Imaging yielded a mean contrast-to-noise ratio (CNR of 3.3±1.7. Mean diameter in vivo was 16.5±6.0 μm for arterioles and 8.0±2.4 μm for capillaries. In explanted cochleae, the diameter of radiating arterioles and capillaries was measured with 12.2±1.6 μm and 6.6±1.0 μm, respectively. The difference between capillaries and arterioles was statistically significant in both experimental setups (P<0.001 and P=0.022, two-way ANOVA. Measured vessel diameters in vivo and ex vivo were in agreement with published data. We conclude that two-photon fluorescence microscopy allows the investigation of cochlear microvessels and is potentially a valuable tool for inner ear research.

  3. Two-photon luminescence microscopy of field enhancement at gold nanoparticles

    DEFF Research Database (Denmark)

    Beermann, Jonas; Bozhevolnyi, Sergey I.

    2005-01-01

    Using a reflection scanning optical microscope detecting two-photon luminescence (TPL) we have imaged square gold bumps positioned in a periodic array either on a smooth gold film or directly on a glass substrate. The second-harmonic (SH) and TPL response from these structures show both...... polarization and wavelength dependence. The gold bumps on gold film showed extremely high sensitivity to the incident field, with the strongest TPL response from the gold bumps being enhanced nearly 103 times compared to the TPL response from the smooth gold surface. For gold bumps directly on glass...

  4. Fiber-optic raster scanning two-photon endomicroscope using a tubular piezoelectric actuator

    Science.gov (United States)

    Do, Dukho; Yoo, Hongki; Gweon, Dae-Gab

    2014-06-01

    A nonresonant, fiber-optic raster scanning endomicroscope was developed using a quarter-tubular piezoelectric (PZT) actuator. A fiber lever mechanism was utilized to enhance the small actuation range of the tubular PZT actuator and to increase its field-of-view. Finite element method simulation of the endoscopic probe was conducted for various conditions to maximize its scanning range. After fabricating the probe using a double clad fiber, we obtained two-photon fluorescence images using raster beam scanning of the fiber. The outer diameter of the probe was 3.5 mm and its rigid distal length was 30 mm including a high numerical aperture gradient index lens. These features are sufficient for input into the instrumental channel of a commercial colonoscope or gastroscope to obtain high resolution images in vivo.

  5. Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

    Science.gov (United States)

    Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

    2016-01-01

    The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling. PMID:27576922

  6. Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo

    Science.gov (United States)

    Song, Wei; Xu, Qiang; Zhang, Yang; Zhan, Yang; Zheng, Wei; Song, Liang

    2016-08-01

    The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.

  7. Identification of calcifications in intracranial neoplasms using two photon excitation fluorescence microscopy

    Science.gov (United States)

    Lin, Peihua; Wang, Xingfu; Wu, Zanyi; Fang, Na; Li, Lianhuang; Kang, Dezhi; Chen, Jianxin

    2016-10-01

    Calcifications within brain tumors may be an indicator of a relatively long survival because a long time is required for the formation of calcium deposits, and may present a novel biomarker associated with response and improved outcome of therapy. In this paper, we describe the use of two-photon excitation fluorescent (TPEF) microscopy combined second harmonic generation (SHG) microscopy for high-resolution imaging that can be applied in identification of intratumoral calcifications. Our results demonstrate that the calcification has stronger TPEF signal than the area around it and the emission spectra shows the difference between the two areas clearly. The TPEF image of calcified region corresponds well with the corresponding H&E stained image. In this work, we present that the label-free imaging technique is able to distinguish the calcified mass lesions in intracranial neoplasms reliably.

  8. Fully integrated reflection-mode photoacoustic/two-photon microscopy in vivo (Conference Presentation)

    Science.gov (United States)

    Song, Liang; Song, Wei; Zhang, Yang; Zheng, Wei

    2016-03-01

    Using a water-immersion optical objective in conjunction with a miniature 40-MHz ultrasonic transducer, we developed reflection-mode photoacoustic microscopy with a transverse resolution as high as 320 nm. Here, we further integrated two-photon microscopy capability into the system to enable multimodality in vivo biomedical imaging at submicron resolution. As a result, the system is capable of tri-modality label-free imaging of microvasculature, collagen, and cell morphology, based on the contrast of optical absorption, second-harmonic generation, and autofluorescence, respectively. In addition, we demonstrated simultaneous microscopic imaging of neuron and microvasculature in the brain cortex of a living mouse, which may offer new opportunities for studying the mechanisms of neurovascular coupling.

  9. Photothermal imaging scanning microscopy

    Science.gov (United States)

    Chinn, Diane; Stolz, Christopher J.; Wu, Zhouling; Huber, Robert; Weinzapfel, Carolyn

    2006-07-11

    Photothermal Imaging Scanning Microscopy produces a rapid, thermal-based, non-destructive characterization apparatus. Also, a photothermal characterization method of surface and subsurface features includes micron and nanoscale spatial resolution of meter-sized optical materials.

  10. Brain refractive index measured in vivo with high-NA defocus-corrected full-field OCT and consequences for two-photon microscopy.

    Science.gov (United States)

    Binding, Jonas; Ben Arous, Juliette; Léger, Jean-François; Gigan, Sylvain; Boccara, Claude; Bourdieu, Laurent

    2011-03-14

    Two-photon laser scanning microscopy (2PLSM) is an important tool for in vivo tissue imaging with sub-cellular resolution, but the penetration depth of current systems is potentially limited by sample-induced optical aberrations. To quantify these, we measured the refractive index n' in the somatosensory cortex of 7 rats in vivo using defocus optimization in full-field optical coherence tomography (ff-OCT). We found n' to be independent of imaging depth or rat age. From these measurements, we calculated that two-photon imaging beyond 200 µm into the cortex is limited by spherical aberration, indicating that adaptive optics will improve imaging depth.

  11. Automated filtering of intrinsic movement artifacts during two-photon intravital microscopy.

    Directory of Open Access Journals (Sweden)

    Denis Soulet

    Full Text Available In vivo imaging using two-photon microscopy is an essential tool to explore the dynamic of physiological events deep within biological tissues for short or extended periods of time. The new capabilities offered by this technology (e.g. high tissue penetrance, low toxicity have opened a whole new era of investigations in modern biomedical research. However, the potential of using this promising technique in tissues of living animals is greatly limited by the intrinsic irregular movements that are caused by cardiac and respiratory cycles and muscular and vascular tone. Here, we show real-time imaging of the brain, spinal cord, sciatic nerve and myenteric plexus of living mice using a new automated program, named Intravital_Microscopy_Toolbox, that removes frames corrupted with motion artifacts from time-lapse videos. Our approach involves generating a dissimilarity score against precalculated reference frames in a specific reference channel, thus allowing the gating of distorted, out-of-focus or translated frames. Since the algorithm detects the uneven peaks of image distortion caused by irregular animal movements, the macro allows a fast and efficient filtering of the image sequence. In addition, extra features have been implemented in the macro, such as XY registration, channel subtraction, extended field of view with maximum intensity projection, noise reduction with average intensity projections, and automated timestamp and scale bar overlay. Thus, the Intravital_Microscopy_Toolbox macro for ImageJ provides convenient tools for biologists who are performing in vivo two-photon imaging in tissues prone to motion artifacts.

  12. Evaluation of cryo-preserved skin tissues using two-photon microscopy

    Science.gov (United States)

    Riemann, Iris; Beier, Axel; Schwarz, Martin; Dörr, Daniel; Stracke, Frank; Zimmermann, Heiko

    2010-02-01

    If no fresh skin samples can be obtained or used, it is important for research and industries to have models and stored tissue samples as close to the native state as possible at disposal. One way to preserve tissues for a longer timeframe is to use deep freezing cryo-techniques. Unfortunately much damage can be induced during the cooling and the thawing processes like disruption of cells and extra-cellular matrices due to the formation of ice crystals. This could lead to a disturbance of the united cell structure up to the point of a loss of cell viability. Two-photon microscopy is able to gather information about cells and tissue components via excitation of the autofluorescence deep inside the sample with a high resolution in both, frozen and thawed states. It is possible to monitor the samples before and after and, important, observe events during the freezing process like the formation of ice crystals. To determine the state of skin tissues after slow rate freezing and the quick process of vitrification, the samples were examined with two-photon microscopy. To establish an optimized freezing-protocol for skin tissues, morphological changes, changes in autofluorescence of endogenous fluorophores (NADH, keratin, flavins, elastin) or changes in second harmonic generation of collagen fibres could provide information about the quality of the used freezing parameters and protective additives and lead to an optimized freezing-protocol with a new set of parameters to obtain mostly intact tissue samples. Multiphoton microscopy has been established as a useful tool for optical in situ quality control of frozen tissues.

  13. Polarization-Sensitive Two-Photon Microscopy Study of the Organization of Liquid-Crystalline DNA

    Science.gov (United States)

    Mojzisova, Halina; Olesiak, Joanna; Zielinski, Marcin; Matczyszyn, Katarzyna; Chauvat, Dominique; Zyss, Joseph

    2009-01-01

    Abstract Highly concentrated DNA solutions exhibit self-ordering properties such as the generation of liquid-crystalline phases. Such organized domains may play an important role in the global chromatin topology but can also be used as a simple model for the study of more complex 3D DNA structures. In this work, using polarized two-photon fluorescence microscopy, we report on the orientation of DNA molecules in liquid-crystalline phases. For this purpose, we analyze the signal emitted by fluorophores that are noncovalently bound to DNA strands. In nonlinear processes, excitation occurs exclusively in the focal volume, which offers advantages such as the reduction of photobleaching of out-of-focus molecules and intrinsic 3D sectioning capability. Propidium iodide and Hoechst, two fluorophores with different DNA binding modes, have been considered. Polarimetric measurements show that the dyes follow the alignment with respect to the DNA strands and allow the determination of the angles between the emission dipoles and the longitudinal axis of the DNA double strand. These results provide a useful starting point toward the application of two-photon polarimetry techniques to determine the local orientation of condensed DNA in physiological conditions. PMID:19843467

  14. Two-photon luminescence contrast by tip-sample coupling in femtosecond near-field optical microscopy

    Science.gov (United States)

    Horneber, Anke; Wackenhut, Frank; Braun, Kai; Wang, Xiao; Wang, Jiyong; Zhang, Dai; Meixner, Alfred J.

    2017-01-01

    We investigate the role of tip-sample interaction in nonlinear optical scanning near-field microscopy. The experiment was performed by tightly focusing femtosecond laser pulses onto a sharp gold tip that was positioned in close proximity to the surface of a sample with gold nanostructures on a Si-substrate by shear force feedback. The nonlinear optical signal consists of two-photon photoluminescence and second harmonic signal from the gold tip and the gold nanostructures. These signals can be used to characterize different coupling parameters such as geometry, material and width of the tip-sample gap and enable to reveal the mechanism responsible for the image contrast. Under the excitation with 776-nm and 110-fs laser pulses nonlinear imaging is almost background free and yields super resolution showing features with dimensions significantly below the diffraction limit with a signal intensity following quadratic excitation power law.

  15. Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.

    Science.gov (United States)

    Winter, Peter W; York, Andrew G; Nogare, Damian Dalle; Ingaramo, Maria; Christensen, Ryan; Chitnis, Ajay; Patterson, George H; Shroff, Hari

    2014-09-20

    Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 μm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

  16. A novel Kalman filter based video image processing scheme for two-photon fluorescence microscopy

    Science.gov (United States)

    Sun, Wenqing; Huang, Xia; Li, Chunqiang; Xiao, Chuan; Qian, Wei

    2016-03-01

    Two-photon fluorescence microscopy (TPFM) is a perfect optical imaging equipment to monitor the interaction between fast moving viruses and hosts. However, due to strong unavoidable background noises from the culture, videos obtained by this technique are too noisy to elaborate this fast infection process without video image processing. In this study, we developed a novel scheme to eliminate background noises, recover background bacteria images and improve video qualities. In our scheme, we modified and implemented the following methods for both host and virus videos: correlation method, round identification method, tree-structured nonlinear filters, Kalman filters, and cell tracking method. After these procedures, most of noises were eliminated and host images were recovered with their moving directions and speed highlighted in the videos. From the analysis of the processed videos, 93% bacteria and 98% viruses were correctly detected in each frame on average.

  17. Nanoshells for in vivo imaging using two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gao Liang; Nammalvar, Vengadesan [Department of Bioengineering, Rice University, Houston, TX 77005 (United States); Vadakkan, Tegy J, E-mail: lg3@rice.edu, E-mail: venkyn@rice.edu [Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 (United States)

    2011-09-07

    Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.

  18. Nanoshells for in vivo imaging using two-photon excitation microscopy.

    Science.gov (United States)

    Gao, Liang; Vadakkan, Tegy J; Nammalvar, Vengadesan

    2011-09-07

    Gold nanoshells have been intensively investigated and applied to various biomedical fields because of their flexible optical tunability and biological compatibility. They hold great potential to serve as luminescent contrast agents excitable with near-infrared (NIR) lasers. In this paper, we describe the development of nanoshells with a peak of plasmon resonance at 800 nm and their subsequent use for in vivo blood vessel imaging using two-photon excitation microscopy at an excitation wavelength of 750 nm. We were able to image single nanoshell particles in blood vessels and generate optical contrast for blood vessel structure using luminescent signals. These results confirm the feasibility of engineering nanoshells with controlled optical properties for single-particle-based in vivo imaging.

  19. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs.

    Science.gov (United States)

    Cahalan, Michael D; Parker, Ian

    2008-01-01

    The immune system is the most diffuse cellular system in the body. Accordingly, long-range migration of cells and short-range communication by local chemical signaling and by cell-cell contacts are vital to the control of an immune response. Cellular homing and migration within lymphoid organs, antigen recognition, and cell signaling and activation are clearly vital during an immune response, but these events had not been directly observed in vivo until recently. Introduced to the field of immunology in 2002, two-photon microscopy is the method of choice for visualizing living cells deep within native tissue environments, and it is now revealing an elegant cellular choreography that underlies the adaptive immune response to antigen challenge. We review cellular dynamics and molecular factors that contribute to basal motility of lymphocytes in the lymph node and cellular interactions leading to antigen capture and recognition, T cell activation, B cell activation, cytolytic effector function, and antibody production.

  20. Electric field allowed molecular transitions for one and two photon excitation microscopy.

    Science.gov (United States)

    Mondal, Partha Pratim; Diaspro, Alberto

    2008-07-01

    We propose an excitation technique for observing single and two photon excitation in those molecules for which such transitions are forbidden by the selection rules. This is possible by the application of an external electric field that perturbs the molecular orbitals, thereby resulting in a significant shift of energy levels. Such a shift of energy levels may bring those levels in resonance with the radiation field which is normally forbidden by selection rules. Further, parity of the these states may significantly improve the emission process. The external electric field results in the mixing of excited (short lifetime) and metastable states (long lifetime), thus reducing the lifetime of metastable (or near metastable) states. This may provide an effective channel for allowing transition from the metastable states. An application of electric field may result in the excitation of poorly excitable biomolecules. This excitation technique may find applications in single- and multi-photon fluorescence microscopy, bioimaging and optical devices.

  1. Rotary-scanning optical resolution photoacoustic microscopy

    Science.gov (United States)

    Qi, Weizhi; Xi, Lei

    2016-10-01

    Optical resolution photoacoustic microscopy (ORPAM) is currently one of the fastest evolving photoacoustic imaging modalities. It has a comparable spatial resolution to pure optical microscopic techniques such as epifluorescence microscopy, confocal microscopy, and two-photon microscopy, but also owns a deeper penetration depth. In this paper, we report a rotary-scanning (RS)-ORPAM that utilizes a galvanometer scanner integrated with objective to achieve rotary laser scanning. A 15 MHz cylindrically focused ultrasonic transducer is mounted onto a motorized rotation stage to follow optical scanning traces synchronously. To minimize the loss of signal to noise ratio, the acoustic focus is precisely adjusted to reach confocal with optical focus. Black tapes and carbon fibers are firstly imaged to evaluate the performance of the system, and then in vivo imaging of vasculature networks inside the ears and brains of mice is demonstrated using this system.

  2. Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy

    Science.gov (United States)

    Bukara, Katarina; Jovanić, Svetlana; Drvenica, Ivana T.; Stančić, Ana; Ilić, Vesna; Rabasović, Mihailo D.; Pantelić, Dejan; Jelenković, Branislav; Bugarski, Branko; Krmpot, Aleksandar J.

    2017-02-01

    The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells' shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

  3. Scanning ultrafast electron microscopy.

    Science.gov (United States)

    Yang, Ding-Shyue; Mohammed, Omar F; Zewail, Ahmed H

    2010-08-24

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

  4. Scanning ultrafast electron microscopy

    Science.gov (United States)

    Yang, Ding-Shyue; Mohammed, Omar F.; Zewail, Ahmed H.

    2010-01-01

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability. PMID:20696933

  5. Visualizing heterogeneity of photosynthetic properties of plant leaves with two-photon fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Iermak, Ievgeniia; Vink, Jochem; Bader, Arjen N; Wientjes, Emilie; van Amerongen, Herbert

    2016-09-01

    Two-photon fluorescence lifetime imaging microscopy (FLIM) was used to analyse the distribution and properties of Photosystem I (PSI) and Photosystem II (PSII) in palisade and spongy chloroplasts of leaves from the C3 plant Arabidopsis thaliana and the C4 plant Miscanthus x giganteus. This was achieved by separating the time-resolved fluorescence of PSI and PSII in the leaf. It is found that the PSII antenna size is larger on the abaxial side of A. thaliana leaves, presumably because chloroplasts in the spongy mesophyll are "shaded" by the palisade cells. The number of chlorophylls in PSI on the adaxial side of the A. thaliana leaf is slightly higher. The C4 plant M. x giganteus contains both mesophyll and bundle sheath cells, which have a different PSI/PSII ratio. It is shown that the time-resolved fluorescence of bundle sheath and mesophyll cells can be analysed separately. The relative number of chlorophylls, which belong to PSI (as compared to PSII) in the bundle sheath cells is at least 2.5 times higher than in mesophyll cells. FLIM is thus demonstrated to be a useful technique to study the PSI/PSII ratio and PSII antenna size in well-defined regions of plant leaves without having to isolate pigment-protein complexes.

  6. Two-photon microscopy measurement of CMRO2 using periarteriolar PO2 gradients(Conference Presentation)

    Science.gov (United States)

    Sakadžić, Sava; Yaseen, Mohammad A.; Jaswal, Rajeshwer S.; Roussakis, Emmanuel; Dale, Anders M.; Buxton, Richard B.; Vinogradov, Sergei A.; Boas, David A.; Devor, Anna

    2017-02-01

    The cerebral metabolic rate of oxygen (CMRO2) is an essential parameter for evaluating brain function and pathophysiology. Measurements of CMRO2 with high spatio-temporal resolution are critically important for understanding how the brain copes with metabolic and blood perfusion changes associated with various clinical conditions, such as stroke, periinfarct depolarizations, and various microvasculopathies (e.g., Alzheimer's disease, chronic hypertension). CMRO2 measurements are also important for understanding the physiological underpinnings of functional Magnetic Resonance Imaging signals. However, the currently available approaches for quantifying CMRO2 rely on complex multimodal imaging and mathematical modeling. Here, we introduce a novel method that allows estimation of CMRO2 based on a single measurement modality - two-photon phosphorescence lifetime microscopy (2PLM) imaging of the partial pressure of oxygen (PO2) in cortical tissue. CMRO2 is estimated by fitting the changes of tissue PO2 around cortical penetrating arterioles with the Krogh cylinder model of oxygen diffusion. We measured the baseline CMRO2 in anesthetized rats, and modulated tissue PO2 levels by manipulating the depth of anesthesia. This method has a spatial resolution of approximately 200 μm and it may provide CMRO2 measurements in individual cortical layers or within confined cortical regions such as in ischemic penumbra and the foci of functional activation.

  7. Maximum imaging depth of two-photon autofluorescence microscopy in epithelial tissues.

    Science.gov (United States)

    Durr, Nicholas J; Weisspfennig, Christian T; Holfeld, Benjamin A; Ben-Yakar, Adela

    2011-02-01

    Endogenous fluorescence provides morphological, spectral, and lifetime contrast that can indicate disease states in tissues. Previous studies have demonstrated that two-photon autofluorescence microscopy (2PAM) can be used for noninvasive, three-dimensional imaging of epithelial tissues down to approximately 150 μm beneath the skin surface. We report ex-vivo 2PAM images of epithelial tissue from a human tongue biopsy down to 370 μm below the surface. At greater than 320 μm deep, the fluorescence generated outside the focal volume degrades the image contrast to below one. We demonstrate that these imaging depths can be reached with 160 mW of laser power (2-nJ per pulse) from a conventional 80-MHz repetition rate ultrafast laser oscillator. To better understand the maximum imaging depths that we can achieve in epithelial tissues, we studied image contrast as a function of depth in tissue phantoms with a range of relevant optical properties. The phantom data agree well with the estimated contrast decays from time-resolved Monte Carlo simulations and show maximum imaging depths similar to that found in human biopsy results. This work demonstrates that the low staining inhomogeneity (∼ 20) and large scattering coefficient (∼ 10 mm(-1)) associated with conventional 2PAM limit the maximum imaging depth to 3 to 5 mean free scattering lengths deep in epithelial tissue.

  8. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bickford, Lissett; Sun Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah [Department of Bioengineering, Rice University, Houston, TX 77005 (United States)], E-mail: drezek@rice.edu

    2008-08-06

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions.

  9. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    Science.gov (United States)

    Bickford, Lissett; Sun, Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah

    2008-08-01

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions.

  10. Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues

    Science.gov (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y.

    2010-02-01

    Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

  11. Comparison of reflectance confocal microscopy and two-photon second harmonic generation microscopy in fungal keratitis rabbit model ex vivo

    Science.gov (United States)

    Lee, Jun Ho; Lee, Seunghun; Yoon, Calvin J.; Park, Jin Hyoung; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-01-01

    Fungal keratitis is an infection of the cornea by fungal pathogens. Diagnosis methods based on optical microscopy could be beneficial over the conventional microbiology method by allowing rapid and non-invasive examination. Reflectance confocal microscopy (RCM) and two-photon second harmonic generation microscopy (TPSHGM) have been applied to pre-clinical or clinical studies of fungal keratitis. In this report, RCM and TPSHGM were characterized and compared in the imaging of a fungal keratitis rabbit model ex vivo. Fungal infection was induced by using two strains of fungi: aspergillus fumigatus and candida albicans. The infected corneas were imaged in fresh condition by both modalities sequentially and their images were analyzed. Both RCM and TPSHGM could detect both fungal strains within the cornea based on morphology: aspergillus fumigatus had distinctive filamentous structures, and candida albicans had round structures superficially and elongated structures in the corneal stroma. These imaging results were confirmed by histology. Comparison between RCM and TPSHGM showed several characteristics. Although RCM and TPSHGM images had good correlation each other, their images were slightly different due to difference in contrast mechanism. RCM had relatively low image contrast with the infected turbid corneas due to high background signal. TPSHGM visualized cells and collagen in the cornea clearly compared to RCM, but used higher laser power to compensate low autofluorescence. Since these two modalities provide complementary information, combination of RCM and TPSHGM would be useful for fungal keratitis detection by compensating their weaknesses each other. PMID:26977371

  12. Two-photon microscopy for non-invasive, quantitative monitoring of stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    William L Rice

    Full Text Available BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF and second harmonic generation (SHG as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(PH, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(PH and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool

  13. Steady state anisotropy two-photon microscopy resolves multiple, spectrally similar fluorophores, enabling in vivo multilabel imaging.

    Science.gov (United States)

    Dubach, J Matthew; Vinegoni, Claudio; Weissleder, Ralph

    2014-08-01

    The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy, the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-05-01

    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.

  16. Arduino Due based tool to facilitate in vivo two-photon excitation microscopy.

    Science.gov (United States)

    Artoni, Pietro; Landi, Silvia; Sato, Sebastian Sulis; Luin, Stefano; Ratto, Gian Michele

    2016-04-01

    Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra. This tool is portable, fast, affordable and precise. It allowed studying the impact of scattering and of blood absorption on two-photon excitation light. In this way, we determined the wavelength-dependent deformation of excitation spectra occurring in deep tissues in vivo.

  17. Ultrafast scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Botkin, D.A. [California Univ., Berkeley, CA (United States). Dept. of Physics]|[Lawrence Berkeley Lab., CA (United States)

    1995-09-01

    I have developed an ultrafast scanning tunneling microscope (USTM) based on uniting stroboscopic methods of ultrafast optics and scanned probe microscopy to obtain nanometer spatial resolution and sub-picosecond temporal resolution. USTM increases the achievable time resolution of a STM by more than 6 orders of magnitude; this should enable exploration of mesoscopic and nanometer size systems on time scales corresponding to the period or decay of fundamental excitations. USTM consists of a photoconductive switch with subpicosecond response time in series with the tip of a STM. An optical pulse from a modelocked laser activates the switch to create a gate for the tunneling current, while a second laser pulse on the sample initiates a dynamic process which affects the tunneling current. By sending a large sequence of identical pulse pairs and measuring the average tunnel current as a function of the relative time delay between the pulses in each pair, one can map the time evolution of the surface process. USTM was used to measure the broadband response of the STM`s atomic size tunnel barrier in frequencies from tens to hundreds of GHz. The USTM signal amplitude decays linearly with the tunnel junction conductance, so the spatial resolution of the time-resolved signal is comparable to that of a conventional STM. Geometrical capacitance of the junction does not appear to play an important role in the measurement, but a capacitive effect intimately related to tunneling contributes to the measured signals and may limit the ultimate resolution of the USTM.

  18. Effects of Ox-LDL on Macrophages NAD(P)H Autofluorescence Changes by Two-photon Microscopy

    CERN Document Server

    Lin, Ching-Ting; Lee, Szu-Yuan; Lu, Long-Sheng; Wu, Chau-Chung; Dong, Chen-Yuan; Lin, Chii-Wann

    2007-01-01

    Ox-LDL uptakes by macrophage play a critical role in the happening of atherosclerosis. Because of its low damage on observed cells and better signal-to- background ratio, two-photon excitation fluorescence microscopy is used to observe NAD(P)H autofluorescence of macrophage under difference cultured conditions- bare cover glass, coated with fibronectin or poly-D-lysine. The results show that the optimal condition is fibronectin coated surface, on which, macrophages profile can be clearly identified on NAD(P)H autofluorescence images collected by two-photon microscopy. Moreover, different morphology and intensities of autofluorescence under different conditions were observed as well. In the future, effects of ox-LDL on macrophages will be investigated by purposed system to research etiology of atherosclerosis.

  19. Simple approach to three-color two-photon microscopy by a fiber-optic wavelength convertor.

    Science.gov (United States)

    Li, Kuen-Che; Huang, Lynn L H; Liang, Jhih-Hao; Chan, Ming-Che

    2016-11-01

    A simple approach to multi-color two-photon microscopy of the red, green, and blue fluorescent indicators was reported based on an ultra-compact 1.03-μm femtosecond laser and a nonlinear fiber. Inside the nonlinear fiber, the 1.03-μm laser pulses were simultaneously blue-shifted to 0.6~0.8 μm and red-shifted to 1.2~1.4 μm region by the Cherenkov radiation and fiber Raman gain effects. The wavelength-shifted 0.6~0.8 μm and 1.2~1.4 μm radiations were co-propagated with the residual non-converted 1.03-μm pulses inside the same nonlinear fiber to form a fiber-output three-color femtosecond source. The application of the multi-wavelength sources on multi-color two-photon fluorescence microscopy were also demonstrated. Overall, due to simple system configuration, convenient wavelength conversion, easy wavelength tunability within the entire 0.7~1.35 μm bio-penetration window and less requirement for high power and bulky light sources, the simple approach to multi-color two-photon microscopy could be widely applicable as an easily implemented and excellent research tool for future biomedical and possibly even clinical applications.

  20. Label-free near-infrared reflectance microscopy as a complimentary tool for two-photon fluorescence brain imaging.

    Science.gov (United States)

    Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S

    2015-11-01

    In vivo two-photon imaging combined with targeted fluorescent indicators is currently extensively used for attaining critical insights into brain functionality and structural plasticity. Additional information might be gained from back-scattered photons from the near-infrared (NIR) laser without introducing any exogenous labelling. Here, we describe a complimentary and versatile approach that, by collecting the reflected NIR light, provides structural details on axons and blood vessels in the brain, both in fixed samples and in live animals under a cranial window. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from a Thy1-GFPm mouse, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Interestingly, NIR reflectance microscopy allowed the label-free detection of axonal elongations over the superficial layers of mouse cortex under a cranial window in vivo. Finally, blood flow can be measured in live preparations, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated.

  1. Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy.

    Science.gov (United States)

    Jiang, Xingshan; Zhong, Jiazhao; Liu, Yuchun; Yu, Haibo; Zhuo, Shuangmu; Chen, Jianxin

    2011-01-01

    Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging. Copyright © 2011 Wiley Periodicals, Inc.

  2. Spectral characterization and unmixing of intrinsic contrast in intact normal and diseased gastric tissues using hyperspectral two-photon microscopy.

    Directory of Open Access Journals (Sweden)

    Lauren E Grosberg

    Full Text Available BACKGROUND: Living tissues contain a range of intrinsic fluorophores and sources of second harmonic generation which provide contrast that can be exploited for fresh tissue imaging. Microscopic imaging of fresh tissue samples can circumvent the cost and time associated with conventional histology. Further, intrinsic contrast can provide rich information about a tissue's composition, structure and function, and opens the potential for in-vivo imaging without the need for contrast agents. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used hyperspectral two-photon microscopy to explore the characteristics of both normal and diseased gastrointestinal (GI tissues, relying only on their endogenous fluorescence and second harmonic generation to provide contrast. We obtained hyperspectral data at subcellular resolution by acquiring images over a range of two-photon excitation wavelengths, and found excitation spectral signatures of specific tissue types based on our ability to clearly visualize morphology. We present the two-photon excitation spectral properties of four major tissue types that are present throughout the GI tract: epithelium, lamina propria, collagen, and lymphatic tissue. Using these four excitation signatures as basis spectra, linear unmixing strategies were applied to hyperspectral data sets of both normal and neoplastic tissue acquired in the colon and small intestine. Our results show that hyperspectral unmixing with excitation spectra allows segmentation, showing promise for blind identification of tissue types within a field of view, analogous to specific staining in conventional histology. The intrinsic spectral signatures of these tissue types provide information relating to their biochemical composition. CONCLUSIONS/SIGNIFICANCE: These results suggest hyperspectral two-photon microscopy could provide an alternative to conventional histology either for in-situ imaging, or intraoperative 'instant histology' of fresh tissue

  3. Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

    Directory of Open Access Journals (Sweden)

    Chunqiang Li

    2016-01-01

    Full Text Available Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs, mast cells, vascular smooth muscle cells (SMCs. Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs which participate in the degradation of collagen.

  4. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy.

    Science.gov (United States)

    Bharali, Dhruba J; Lucey, Derrick W; Jayakumar, Harishankar; Pudavar, Haridas E; Prasad, Paras N

    2005-08-17

    A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

  5. Dynamic characterization of hydrophobic and hydrophilic solutes in oleic-acid enhanced transdermal delivery using two-photon fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Te-Yu; Yang, Chiu-Sheng; Chen, Yang-Fang [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Tsai, Tsung-Hua [Department of Dermatology, Far Eastern Memorial Hospital, New Taipei City, Taiwan (China); Dong, Chen-Yuan, E-mail: cydong@phys.ntu.edu.tw [Department of Physics, National Taiwan University, Taipei, Taiwan (China); Center for Quantum Science and Engineering, National Taiwan University, Taipei, Taiwan (China); Center for Optoelectronic Biomedicine, National Taiwan University, Taipei, Taiwan (China)

    2014-10-20

    In this letter, we propose an efficient methodology of investigating dynamic properties of sulforhodamine B and rhodamine B hexyl ester molecules transporting across ex-vivo human stratum corneum with and without oleic acid enhancement. Three-dimensional, time-lapse fluorescence images of the stratum corneum can be obtained using two-photon fluorescence microscopy. Furthermore, temporal quantifications of transport enhancements in diffusion parameters can be achieved with the use of Fick's second law. Dynamic characterization of solutes transporting across the stratum corneum is an effective method for understanding transient phenomena in transdermal delivery of probe molecules, leading to improved delivery strategies of molecular species for therapeutic purposes.

  6. Development and design of up-to-date laser scanning two-photon microscope using in neuroscience

    Science.gov (United States)

    Doronin, Maxim; Popov, Alexander

    2017-02-01

    Today one of the main areas of application of two-photon microscopy is biology. This is due to the fact that this technique allows to obtain 3D images of tissues due to laser focus change, that is possible due to substantially greater penetration depth on the main wavelength into biological tissues. Self-developed microscopy system provides possibility to service it and modify the structure of microscope depending on highly specialized experimental design and scientific goals. This article may be regarded as a quick reference to laboratory staff who are wishing to develop their own microscopy system for self-service and modernization of the system and in order to save the lab budget.

  7. Increased illumination uniformity and reduced photodamage offered by the Lissajous scanning in fiber-optic two-photon endomicroscopy.

    Science.gov (United States)

    Liang, Wenxuan; Murari, Kartikeya; Zhang, Yuying; Chen, Yongping; Li, Ming-Jun; Li, Xingde

    2012-02-01

    We compare the illumination uniformity and the associated effects of the spiral and Lissajous scanning patterns that are commonly used in an endomicroscope. Theoretical analyses and numerical simulations were first performed to quantitatively investigate the area illumination density in the spiral scanning pattern. The results revealed the potential problem of manifest photodamage due to the very high illumination density in the center of the spiral scan. Similar analyses of the Lissajous scanning pattern, which can be conveniently implemented on the same endomicroscope with no hardware modifications, showed a more uniform illumination density with about an 80-fold reduction in the peak illumination density. To underscore the benefit offered by the improved illumination uniformity, we conducted in vitro two-photon fluorescence imaging of cultured cells stained with a LIVE/DEAD viability assay using our home-built, fiber-optic, two-channel endomicroscopy system. Both the spiral and the Lissajous scans were implemented. Our experimental results showed that cells near the spiral scan center experienced obvious photodamage, whereas cells remained alive over the entire region under the Lissajous beam scanning, confirming the predicted advantage offered by the Lissajous scan over this spiral scan in an endomicroscopy setting.

  8. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

    This report is based on a metrological investigation on confocal microscopy technique carried out by Uffe Rolf Arlø Theilade and Paolo Bariani. The purpose of the experimental activity was twofold a metrological instrument characterization and application to assessment of rough PP injection moulded...... replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

  9. Confocal scanning microscopy

    DEFF Research Database (Denmark)

    Bariani, Paolo

    replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

  10. Two-Photon Excitation STED Microscopy with Time-Gated Detection.

    Science.gov (United States)

    Coto Hernández, Iván; Castello, Marco; Lanzanò, Luca; d'Amora, Marta; Bianchini, Paolo; Diaspro, Alberto; Vicidomini, Giuseppe

    2016-01-13

    We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture's complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.

  11. In situ imaging of the mouse cochlea using two-photon microscopy

    Science.gov (United States)

    Yang, Xin; Pu, Ye; Psaltis, Demetri; Stankovic, Konstantina M.

    2013-04-01

    Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea's small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.

  12. Automated image analysis for diameters and branching points of cerebral penetrating arteries and veins captured with two-photon microscopy.

    Science.gov (United States)

    Sugashi, Takuma; Yoshihara, Kouichi; Kawaguchi, Hiroshi; Takuwa, Hiroyuki; Ito, Hiroshi; Kanno, Iwao; Yamada, Yukio; Masamoto, Kazuto

    2014-01-01

    The present study was aimed to characterize 3-dimensional (3D) morphology of the cortical microvasculature (e.g., penetrating artery and emerging vein), using two-photon microscopy and automated analysis for their cross-sectional diameters and branching positions in the mouse cortex. We observed that both artery and vein had variable cross-sectional diameters across cortical depths. The mean diameter was similar for both artery (17 ± 5 μm) and vein (15 ± 5 μm), and there were no detectable differences over depths of 50-400 μm. On the other hand, the number of branches was slightly increased up to 400-μm depth for both the artery and vein. The mean number of branches per 0.1 mm vessel length was 1.7 ± 1.2 and 3.8 ± 1.6 for the artery and vein, respectively. This method allows for quantification of the large volume data of microvascular images captured with two-photon microscopy. This will contribute to the morphometric analysis of the cortical microvasculature in functioning brains.

  13. Cell and brain tissue imaging of the flavonoid fisetin using label-free two-photon microscopy.

    Science.gov (United States)

    Krasieva, Tatiana B; Ehren, Jennifer; O'Sullivan, Thomas; Tromberg, Bruce J; Maher, Pamela

    2015-10-01

    Over the last few years, we have identified an orally active, novel neuroprotective and cognition-enhancing molecule, the flavonoid fisetin. Fisetin not only has direct antioxidant activity but it can also increase the intracellular levels of glutathione, the major intracellular antioxidant. Fisetin can also activate key neurotrophic factor signaling pathways. In addition, it has anti-inflammatory activity against microglia and astrocytes and inhibits the activity of lipoxygenases, thereby reducing the production of pro-inflammatory eicosanoids and their by-products. However, key questions about its targets and brain penetration remain. In this study, we used label-free two-photon microscopy of intrinsic fisetin fluorescence to examine the localization of fisetin in living nerve cells and the brains of living mice. In cells, fisetin but not structurally related flavonols with different numbers of hydroxyl groups, localized to the nucleoli suggesting that key targets of fisetin may reside in this organelle. In the mouse brain, following intraperitoneal injection and oral administration, fisetin rapidly distributed to the blood vessels of the brain followed by a slower dispersion into the brain parenchyma. Thus, these results provide further support for the effects of fisetin on brain function. In addition, they suggest that label-free two-photon microscopy may prove useful for studying the intracellular and tissue distribution of other intrinsically-fluorescent flavonoids. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Scanning Quantum Decoherence Microscopy

    OpenAIRE

    2008-01-01

    The use of qubits as sensitive magnetometers has been studied theoretically and recent demonstrated experimentally. In this paper we propose a generalisation of this concept, where a scanning two-state quantum system is used to probe the subtle effects of decoherence (as well as its surrounding electromagnetic environment). Mapping both the Hamiltonian and decoherence properties of a qubit simultaneously, provides a unique image of the magnetic (or electric) field properties at the nanoscale....

  15. Characterizing the intracellular distribution of metabolites in intact Chlamydia-infected cells by Raman and two-photon microscopy.

    Science.gov (United States)

    Szaszák, Márta; Chang, Jiun Chiun; Leng, Weinan; Rupp, Jan; Ojcius, David M; Kelley, Anne Myers

    2013-06-01

    Chlamydia species are obligate intracellular pathogens that proliferate only within infected cells. Currently, there are no known techniques or systems that can probe the spatial distribution of metabolites of interest within intact Chlamydia-infected cells. Here we investigate the ability of Raman microscopy to probe the chemical composition of different compartments (nucleus, inclusion, and cytoplasm) of Chlamydia trachomatis-infected epithelial cells. The overall intensity of the Raman spectrum is greatest in the inclusions and lowest in the cytoplasm in fixed cells. Difference spectra generated by normalizing to the intensity of the strong 1004 cm(-1) phenylalanine line show distinct differences among the three compartments. Most notably, the concentrations of adenine are greater in both the inclusions and the nucleus than in the cytoplasm, as seen by Raman microscopy. The source of the adenine was explored through a complementary approach, using two-photon microscopy imaging. Autofluorescence measurements of living, infected cells show that the adenine-containing molecules, NAD(P)H and FAD, are present mainly in the cytoplasm, suggesting that these molecules are not the source of the additional adenine signal in the nucleus and inclusions. Experiments of infected cells stained with a DNA-binding dye, Hoechst 33258, reveal that most of the DNA is present in the nucleus and the inclusions, suggesting that DNA/RNA is the main source of the additional Raman adenine signal in the nucleus and inclusions. Thus, Raman and two-photon microscopy are among the few non-invasive methods available to investigate cells infected with Chlamydia and, together, should also be useful for studying infection by other intracellular pathogens that survive within intracellular vacuoles.

  16. [Confocal laser scanning microscopy].

    Science.gov (United States)

    Ulrich, M

    2015-07-01

    Reflectance confocal microscopy (RCM) allows the in vivo evaluation of melanocytic and nonmelanocytic skin tumours with high sensitivity and specificity. RCM represents an optical imaging technique, which enables us to examine the skin at high resolution. Today, RCM represents not only an interesting tool for dermatologic research but has also been introduced as a diagnostic tool in every day clinical practice. As such, RCM is applied for improvement of skin cancer diagnosis adjunct to clinical and dermatoscopic examination. In combination with dermatoscopy RCM has shown an increased specificity with similar sensitivity. In this regard RCM helps to decrease the rate of unnecessary biopsies of benign lesions. Despite its use in dermatooncology RCM may also be used for diagnosis and monitoring of inflammatory diseases. Future developments include technical improvements, teledermatology solutions and the application of ex vivo RCM in Moh's micrographic surgery.

  17. Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser.

    Science.gov (United States)

    Kawakami, Ryosuke; Sawada, Kazuaki; Sato, Aya; Hibi, Terumasa; Kozawa, Yuichi; Sato, Shunichi; Yokoyama, Hiroyuki; Nemoto, Tomomi

    2013-01-01

    In vivo two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power, and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.

  18. Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye.

    Science.gov (United States)

    Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J; Williams, David R; Alexander, Nathan S; Palczewski, Krzysztof

    2014-07-01

    Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

  19. Noninvasive two-photon fluorescence microscopy imaging of mouse retina and RPE through the pupil of the eye

    Science.gov (United States)

    Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J.; Williams, David R.; Alexander, Nathan S.; Palczewski, Krzysztof

    2014-01-01

    Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions. PMID:24952647

  20. The use of two-photon microscopy to study the biological effects of focused ultrasound on the brain

    Science.gov (United States)

    Burgess, Alison; Cho, Eunice E.; Shaffaf, Leila; Nhan, Tam; Poon, Charissa; Hynynen, Kullervo

    2012-03-01

    Focused ultrasound (FUS) has been used to successfully disrupt the blood-brain barrier (BBB), aiding in the delivery of therapeutic agents to the brain and leading to improvements in disease pathology. Although significant progress has been made in the development of FUS technology, there is still a lack of understanding of the biophysical mechanisms of the BBB disruption and the microscopic effects of this disruption on brain cells. In this study, we combine a custom built ultrasound transducer with two-photon microscopy to conduct real time monitoring of BBB disruption in vivo. We have manufactured and tested a single element piezoelectric transducer with frequencies ranging from 1.15 to 1.30 MHz. Sonications were performed using 0.07-0.25 MPa estimated in situ pressure, 10 ms pulses, 1 Hz pulse repetition frequency for a total duration of 120 s in the presence of microbubbles. BBB disruption was observed through a cranial window created in the rat skull after intravenous injection of dextran conjugated- Texas Red (MW: 10,000 - 70,000 Da). Using this experimental setup, we have observed and characterized 3 different leakage patterns following BBB disruption. Our results indicate that varying the acoustic power leading to in situ pressure changes, may allow us to control the mechanism of BBB disruption. Furthermore, we have labelled astrocytes in vivo in order to visualize the effects of FUS on this cell population. Combination of our custom transducers with two-photon microscopy will allow significant advancement in allow significant advancement in the understanding of the mechanisms and cellular effects of FUS-induced BBB disruption.

  1. In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy

    Science.gov (United States)

    Le, Viet-Hoan; Lee, Seunghun; Lee, Seungwon; Wang, Taejun; Hyuk Jang, Won; Yoon, Yeoreum; Kwon, Soonjae; Kim, Hyekang; Lee, Seung-Woo; Hean Kim, Ki

    2017-01-01

    Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies. PMID:28276477

  2. High Resolution Scanning Ion Microscopy

    NARCIS (Netherlands)

    Castaldo, V.

    2011-01-01

    The structure of the thesis is the following. The first chapter is an introduction to scanning microscopy, where the path that led to the Focused Ion Beam (FIB) is described and the main differences between electrons and ion beams are highlighted. Chapter 2 is what is normally referred to (which I d

  3. Volumetric label-free imaging and 3D reconstruction of mammalian cochlea based on two-photon excitation fluorescence microscopy

    Science.gov (United States)

    Zhang, Xianzeng; Geng, Yang; Ye, Qing; Zhan, Zhenlin; Xie, Shusen

    2013-11-01

    The visualization of the delicate structure and spatial relationship of intracochlear sensory cells has relied on the laborious procedures of tissue excision, fixation, sectioning and staining for light and electron microscopy. Confocal microscopy is advantageous for its high resolution and deep penetration depth, yet disadvantageous due to the necessity of exogenous labeling. In this study, we present the volumetric imaging of rat cochlea without exogenous dyes using a near-infrared femtosecond laser as the excitation mechanism and endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. We find that TPEF exhibits strong contrast, allowing cellular and even subcellular resolution imaging of the cochlea, differentiating cell types, visualizing delicate structures and the radial nerve fiber. Our results further demonstrate that 3D reconstruction rendered with z-stacks of optical sections enables better revealment of fine structures and spatial relationships, and easily performed morphometric analysis. The TPEF-based optical biopsy technique provides great potential for new and sensitive diagnostic tools for hearing loss or hearing disorders, especially when combined with fiber-based microendoscopy.

  4. Intravital two-photon microscopy of host-pathogen interactions in a mouse model of Staphylococcus aureus skin abscess formation.

    Science.gov (United States)

    Liese, Jan; Rooijakkers, Suzan H M; van Strijp, Jos A G; Novick, Richard P; Dustin, Michael L

    2013-06-01

    Staphylococcus (S.) aureus is a frequent cause of severe skin infections. The ability to control the infection is largely dependent on the rapid recruitment of neutrophils (PMN). To gain more insight into the dynamics of PMN migration and host-pathogen interactions in vivo, we used intravital two-photon (2-P) microscopy to visualize S. aureus skin infections in the mouse. Reporter S. aureus strains expressing fluorescent proteins were developed, which allowed for detection of the bacteria in vivo. By employing LysM-EGFP mice to visualize PMN, we observed the rapid appearance of PMN in the extravascular space of the dermis and their directed movement towards the focus of infection, which led to the delineation of an abscess within 1 day. Moreover, tracking of transferred labelled bone-marrow neutrophils showed that PMN localization to the site of infection is dependent on the presence of G-protein-coupled receptors on the PMN, whereas Interleukin-1 receptor was required on host cells other than PMN. Furthermore, the S. aureus complement inhibitor Ecb could block PMN accumulation at thesite of infection. Our results establish that 2-P microscopy is a powerful tool to investigate the orchestration of the immune cells, S. aureus location and gene expression in vivo on a single cell level.

  5. Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy.

    Science.gov (United States)

    Norlén, L; Plasencia, I; Bagatolli, L

    2008-12-01

    Skin moisturization is largely a function of stratum corneum barrier capacity, which in turn is a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix [J. Invest. Dermatol.18, 433 (1952); AIChE J. 21, 985 (1975); Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001)]. Three unsolved key questions with respect to this lipid matrix' structural organization [Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001); J. Invest. Dermatol.118, 897 (2002); J. Invest. Dermatol.118, 899 (2002)] are: i) whether the lipid matrix is constituted by a single-gel phase or by co-existing solid (crystalline or gel) domains, ii) whether a separate fluid (liquid crystalline) phase is present and iii) whether the local pH has a direct effect on the lipid matrix' phase behaviour. Using an array of complementary visual-related biophysical techniques (e.g. atomic force microscopy and confocal/two-photon excitation fluorescence microscopy), it was recently shown that reconstituted membranes composed of extracted decontaminated human stratum corneum lipids do not form a fluid phase, but exclusively a single-gel phase that segregates into co-existing microscopic domains below pH 6 [Biophys. J.93, 3142 (2007)]. It was further shown that the role of cholesterol is related to dispersion of ceramide-enriched domains. This effect is counteracted by the presence of free fatty acids, which mix with skin ceramides but not with cholesterol.

  6. Combined nonlinear laser imaging (two-photon excitation fluorescence, second and third-harmonic generation, and fluorescence lifetime imaging microscopies) in ovarian tumors

    Science.gov (United States)

    Adur, J.; Pelegati, V. B.; de Thomaz, A. A.; Bottcher-Luiz, F.; Andrade, L. A. L. A.; Almeida, D. B.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue.

  7. Upconverting nanoparticles: a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging.

    Science.gov (United States)

    Park, Yong Il; Lee, Kang Taek; Suh, Yung Doug; Hyeon, Taeghwan

    2015-03-21

    Lanthanide-doped upconverting nanoparticles (UCNPs) have recently attracted enormous attention in the field of biological imaging owing to their unique optical properties: (1) efficient upconversion photoluminescence, which is intense enough to be detected at the single-particle level with a (nonscanning) wide-field microscope setup equipped with a continuous wave (CW) near-infrared (NIR) laser (980 nm), and (2) resistance to photoblinking and photobleaching. Moreover, the use of NIR excitation minimizes adverse photoinduced effects such as cellular photodamage and the autofluorescence background. Finally, the cytotoxicity of UCNPs is much lower than that of other nanoparticle systems. All these advantages can be exploited simultaneously without any conflicts, which enables the establishment of a novel UCNP-based platform for wide-field two-photon microscopy. UCNPs are also useful for multimodal in vivo imaging because simple variations in the composition of the lattice atoms and dopant ions integrated into the particles can be easily implemented, yielding various distinct biomedical activities relevant to magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). These multiple functions embedded in a single type of UCNPs play a crucial role in precise disease diagnosis. The application of UCNPs is extended to therapeutic fields such as photodynamic and photothermal cancer therapies through advanced surface conjugation schemes.

  8. Effects of cholesterol on plasma membrane lipid order in MCF-7 cells by two-photon microscopy

    Science.gov (United States)

    Zeng, Yixiu; Chen, Jianling; Yang, Hongqin; Wang, Yuhua; Li, Hui; Xie, Shusen

    2014-09-01

    Lipid rafts are cholesterol- and glycosphingolipids- enriched microdomains on plasma membrane surface of mammal cells, involved in a variety of cellular processes. Depleting cholesterol from the plasma membrane by drugs influences the trafficking of lipid raft markers. Optical imaging techniques are powerful tools to study lipid rafts in live cells due to its noninvasive feature. In this study, breast cancer cells MCF-7 were treated with different concentrations of MβCD to deplete cholesterol and an environmentally sensitive fluorescence probe, Laurdan was loaded to image lipid order by two-photon microscopy. The generalized polarization (GP) values were calculated to distinguish the lipid order and disorder phase. GP images and GP distributions of native and cholesterol-depleted MCF-7 cells were obtained. Our results suggest that even at low concentration (0.5 mM) of MβCD, the morphology of the MCF-7 cells changes. Small high GP areas (lipid order phase) decrease more rapidly than low GP areas (lipid disorder phase), indicating that lipid raft structure was altered more severely than nonraft domains. The data demonstrates that cholesterol dramatically affect raft coverage and plasma membrane fluidity in living cells.

  9. In vivo monitoring of intracellular chloroplast movements in intact leaves of C4 plants using two-photon microscopy.

    Science.gov (United States)

    Ryu, Jeongeun; Nam, Hyoseok; Kim, Hae Koo; Joo, Yongjoon; Lee, Sang Joon; Kim, Ki Hean

    2014-10-01

    Dynamic changes in the spatial distribution of chloroplasts are essential for optimizing photosynthetic capacity under changing light conditions. Light-induced movement of chloroplasts has been widely investigated, but most studies were conducted on isolated tissues or protoplasts. In this study, a two-photon microscopy (TPM) system was adapted to monitor the intracellular 3-dimensional (3D) movements of chloroplasts in intact leaves of plants during dark to light transitions. The TPM imaging was based on autofluorescence of chlorophyll generated by a femto-second Ti:Sapphire laser. All chloroplasts did not exhibit the same motion in response to irradiation variation. In the sub-epidermal mesophyll cells, chloroplasts generally moved away from the surface following blue light treatment, however many chloroplasts did not show any movement. Such spatial heterogeneity in chloroplast motility underlines the importance of monitoring intracellular orientation and movement of individual chloroplasts across intact leaves. Our investigation shows that the 3D imaging of chloroplasts using TPM can help to understand the changes in local photosynthetic capacity in intact leaves under changing environmental conditions.

  10. NEAR-IR TWO PHOTON MICROSCOPY IMAGING OF SILICA NANOPARTICLES FUNCTIONALIZED WITH ISOLATED SENSITIZED Yb(III) CENTERS

    Energy Technology Data Exchange (ETDEWEB)

    Lapadula, Giuseppe; Bourdolle, Adrien; Allouche, Florian; Conley, Matthew P.; Maron, Laurent; Lukens, Wayne W.; Guyot, Yannick; Andraud, Chantal; Brasselet, Sophie; Copé; ret, Christophe; Maury, Olivier; Andersen, Richard A.

    2013-01-12

    Bright nano objects emitting in the near infrared with a maximal cross section of 41.4 x 103 GM (Goppert Mayer), were prepared by implanting ca. 180 4,4 diethylaminostyryl 2,2 bipyridine (DEAS) Yb(III) complexes on the surface of 12 nm silica nanoparticles. The surface complexes ([DEAS Ln SiO2], Ln =Y,Yb) were characterized using IR, solid state NMR, UV Vis, EXAFS spectroscopies in combination with the preparation and characterization of similar molecular analogues by analytical techniques (IR, solution NMR, UV Vis, X ray crystallography) as well as DFT calculations. Starting from the partial dehydroxylation of the silica at 700 C on high vacuum having 0.8 OH.nm 2, the grafting of Ln(N(SiMe3)2)3 generate ≤SiO Ln(N(SiMe3)2)2, which upon thermal step and coordination of the DEAS chromophore yields (≤SiO)3Ln(DEAS). Surface and molecular analogues display similar properties, in terms of DEAS binding constants absorption maxima and luminescence properties (intense emission band assigned to a ligand centered CT fluorescence and life time) in the solid state, consistent with the molecular nature of the surface species. The densely functionalized nanoparticles can be dispersed via ultra-sonication in small ca. 15-20 nm aggregates (1 to 6 elementary particles) that were detected using two photon microscopy imaging at 720 nm excitation, making them promising nano objects for bio imaging.

  11. Two-photon laser confocal microscopy of micropermeability of resin-dentin bonds made with water or ethanol wet bonding.

    Science.gov (United States)

    Sauro, Salvatore; Watson, Timothy F; Mannocci, Francesco; Miyake, Katsuya; Huffman, Bradford P; Tay, Franklin R; Pashley, David H

    2009-07-01

    This study evaluated the micropermeability of six etch-and-rinse adhesives bonded to dentin. There were two principal groups: wet bonding with water or wet bonding with absolute ethyl alcohol. After bonding and the creation of composite build-ups, the pulp chambers were filled with 0.1% lucifer yellow. The contents of the pulp chamber were kept under 20 cm H(2)O pressure to simulate pulpal pressure for 3 h. The specimens were vertically sectioned into multiple 0.5-mm thick slabs that were polished and then examined using a two-photon confocal laser scanning microscope (TPCLSM). The results showed that specimens bonded with adhesives using the water wet-bonding condition all showed tracer taken up uniformly by the hybrid layer. This uptake of fluorescent tracer into the hybrid layer was quantified by computer software. The most hydrophobic experimental resins showed the highest fluorescent tracer uptake (ca. 1800 +/- 160 arbitrary fluorescent units/std. surface area). The most hydrophilic experimental resins showed the lowest tracer uptake into water-saturated hybrid layers. When ethanol wet-bonding was used, significantly less fluorescent tracer was seen in hybrid layers. The most hydrophilic experimental resins and Single Bond Plus showed little micropermeability. Clearly, ethanol wet-bonding seals dentin significantly better than water-wet dentin regardless of the adhesive in etch-and-rinse systems.

  12. Non-linear image scanning microscopy (Conference Presentation)

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

    Nowadays, multiphoton microscopy can be considered as a routine method for the observation of living cells, organs, up to whole organisms. Second-harmonics generation (SHG) imaging has evolved to a powerful qualitative and label-free method for studying fibrillar structures, like collagen networks. However, examples of super-resolution non-linear microscopy are rare. So far, such approaches require complex setups and advanced synchronization of scanning elements limiting the image acquisition rates. We describe theory and realization of a super-resolution image scanning microscope [1, 2] using two-photon excited fluorescence as well as second-harmonic generation. It requires only minor modifications compared to a classical two-photon laser-scanning microscope and allows image acquisition at the high frame rates of a resonant galvo-scanner. We achieve excellent sensitivity and high frame-rate in combination with two-times improved lateral resolution. We applied this method to fixed cells, collagen hydrogels, as well as living fly embryos. Further, we proofed the excellent image quality of our setup for deep tissue imaging. 1. Müller C.B. and Enderlein J. (2010) Image scanning microscopy. Phys. Rev. Lett. 104(19), 198101. 2. Sheppard C.J.R. (1988) Super-resolution in confocal imaging. Optik (Stuttg) 80 53-54.

  13. Aperture scanning Fourier ptychographic microscopy

    Science.gov (United States)

    Ou, Xiaoze; Chung, Jaebum; Horstmeyer, Roarke; Yang, Changhuei

    2016-01-01

    Fourier ptychographic microscopy (FPM) is implemented through aperture scanning by an LCOS spatial light modulator at the back focal plane of the objective lens. This FPM configuration enables the capturing of the complex scattered field for a 3D sample both in the transmissive mode and the reflective mode. We further show that by combining with the compressive sensing theory, the reconstructed 2D complex scattered field can be used to recover the 3D sample scattering density. This implementation expands the scope of application for FPM and can be beneficial for areas such as tissue imaging and wafer inspection. PMID:27570705

  14. Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo.

    Directory of Open Access Journals (Sweden)

    Claudia Laperchia

    Full Text Available Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity of dendritic cells (DCs, and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.

  15. The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.

    Science.gov (United States)

    Niesner, Raluca; Andresen, Volker; Neumann, Jens; Spiecker, Heinrich; Gunzer, Matthias

    2007-10-01

    Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.

  16. The translated conceptual survey of physics / stablization of the focal plane in two photon excitation fluorescence microscopy

    Science.gov (United States)

    Wada, Asma

    As a reflection of my career to be an effective college physics teacher, my thesis is in two parts. The first is in education research, the focus of this part is to have a tool to evaluate pedagogies I have learned at the school and plan to apply in my classrooms back home. Consequently, this resulted in the development of the translated conceptual survey of physics ( TCSP). (TCSP) was designed by combining some questions from the Force Conceptual Inventory (FCI), and the Conceptual Survey of Electricity and Magnetism (CSEM) to assess student's understanding of basic concepts of Newtonian mechanics and electricity and magnetism in introductory physics. The idea of developing this questionnaire is to use it in classrooms back home as a part of a long term objective to implement what has been realized in the area of education research to improve the quality of teaching physics there. The survey was initially written in English, validated with interviews with native English speakers, translated into Arabic, and then validated via an interview with a native Arabic speaker. We then administered the survey to two different English-speaking intro physics courses and analyzed the results for consistency. The objective of the second part in my thesis is to expand my knowledge in an area of physics that I have interest in, and getting involved in a scientific research to develop skills I need as a teacher. My research is in optical physics, in particular, I am working on one of the challenges in implementing two photon excitation luorescence (TPEF) microscopy in imaging living systems. (TPEF) microscopy has been shown to be an invaluable tool for investigating biological structure and function in living organisms. The utility of (TPEF) imaging for this application arises from several important factors including it's ability to image deep within tissue, and to do so without harming the organism. Both of these advantages arise from the fact that (TPEF) imaging is done with

  17. Image scanning microscopy with radially polarized light

    Science.gov (United States)

    Xiao, Yun; Zhang, Yunhai; Wei, Tongda; Huang, Wei; Shi, Yaqin

    2017-03-01

    In order to improve the resolution of image scanning microscopy, we present a method based on image scanning microscopy and radially polarized light. According to the theory of image scanning microscopy, we get the effective point spread function of image scanning microscopy with the longitudinal component of radially polarized light and a 1 AU detection area, and obtain imaging results of the analyzed samples using this method. Results show that the resolution can be enhanced by 7% compared with that in image scanning microscopy with circularly polarized light, and is 1.54-fold higher than that in confocal microscopy with a pinhole of 1 AU. Additionally, the peak intensity of ISM is 1.54-fold higher than that of a confocal microscopy with a pinhole of 1 AU. In conclusion, the combination of the image scanning microscopy and the radially polarized light could improve the resolution, and it could realize high-resolution and high SNR imaging at the same time.

  18. A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view.

    Science.gov (United States)

    Kremer, Y; Léger, J-F; Lapole, R; Honnorat, N; Candela, Y; Dieudonné, S; Bourdieu, L

    2008-07-07

    Acousto-optic deflectors (AOD) are promising ultrafast scanners for non-linear microscopy. Their use has been limited until now by their small scanning range and by the spatial and temporal dispersions of the laser beam going through the deflectors. We show that the use of AOD of large aperture (13mm) compared to standard deflectors allows accessing much larger field of view while minimizing spatio-temporal distortions. An acousto-optic modulator (AOM) placed at distance of the AOD is used to compensate spatial and temporal dispersions. Fine tuning of the AOM-AOD setup using a frequency-resolved optical gating (GRENOUILLE) allows elimination of pulse front tilt whereas spatial chirp is minimized thanks to the large aperture AOD.

  19. Two-photon cryomicroscope

    Science.gov (United States)

    Breunig, H. G.; Köhler, C.; König, K.

    2012-03-01

    We report on a new two-photon cryomicroscope which consist of a compact laser-scanning microscope combined with a motorized heating and freezing stage. Samples can be cooled down to -196 °C (77 K) and heated up to 600 °C (873 K) with adjustable heating/freezing rates between 0.01 K / min and 150 K / min. Two-photon imaging is realized by near infrared femtosecond-laser pulse excitation. The abilities of the two-photon cryomicroscope are illustrated in several measurements: imaging of fluorescent microspheres inside a piece of ice illustrates the feasibility of deep-microscopic imaging inside frozen sample. The temperature-dependent structural integrity of collagen is monitored by detection of second harmonic generation signals from porcine cornea. The measurements reveal also the dependence of the collagendenaturation temperature on hydration state of the cornea collagen. Furthermore, the potential of the two-photon cryomicroscope for optimization of freezing and thawing procedures as well as to evaluate the viability of frozen cells and tissue is discussed.

  20. Integrating two-photon microscopy and cryo-electron microscopy for studying the interaction of Cafeteria roenbergensis and CroV

    Science.gov (United States)

    Aghvami, Seyedmohammadali

    Cafeteria roenbergensis (Cro) is a marine zooplankton; its voracious appetite plays a significant role in regulating bacteria populations. The giant virus that lives within Cro, known as Cafeteria roenbergensis virus (CroV), has an important effect on the mortality of Cro populations. Although viral infections are extremely abundant in oceans, the complete procedure of the infection is still unknown. We study the infection process of Cro by CroV to find out whether the initial contact is through phagocytosis or CroV penetrating the host cell membrane directly. Cro is a moving at speed in the range of 10-100 um/s, therefore, there are many difficulties and challenges for traditional imaging techniques to study this viral-host interaction. We apply two-photon fluorescence microscopy to image this infection process. The image is taken at video rate (30 frame/s), which makes us able to catch the moment of interaction. We are able to image host and virus simultaneously where CroV is stained by SYBR gold dye and Cro is excited through NADH autofluorescence. For further structural biology study, we will obtain atomic level resolution information of infection. After catching the initial moment of infection, we will freeze the sample instantly and image it with cryo-electron microscope .

  1. Re-scan confocal microscopy: scanning twice for better resolution

    NARCIS (Netherlands)

    De Luca, G.M.R.; Breedijk, R.M.P.; Brandt, R.A.J.; Zeelenberg, C.H.C.; De Jong, B.E.; Timmermans, W.; Nahidi Azar, L.; Hoebe, R.A.; Stallinga, S.; Manders, E.M.M.

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity wh

  2. Re-scan confocal microscopy : scanning twice for better resolution

    NARCIS (Netherlands)

    De Luca, G.M.R.; Breedijk, R.M.P.; Brandt, R.A.J.; Zeelenberg, C.H.C.; de Jong, B.E.; Timmermans, W.; Azar, L.N.; Hoebe, R.A.; Stallinga, S.; Manders, E.M.M.

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity wh

  3. Label-free NIR reflectance imaging as a complimentary tool for two-photon fluorescence microscopy: multimodal investigation of stroke (Conference Presentation)

    Science.gov (United States)

    Allegra Mascaro, Anna Letizia; Costantini, Irene; Margoni, Emilia; Iannello, Giulio; Bria, Alessandro; Sacconi, Leonardo; Pavone, Francesco S.

    2016-03-01

    Two-photon imaging combined with targeted fluorescent indicators is extensively used for visualizing critical features of brain functionality and structural plasticity. Back-scattered photons from the NIR laser provide complimentary information without introducing any exogenous labelling. Here, we describe a versatile approach that, by collecting the reflected NIR light, provides structural details on the myelinated axons and blood vessels in the brain, both in fixed samples and in live animals. Indeed, by combining NIR reflectance and two-photon imaging of a slice of hippocampus from Thy1-GFPm mice, we show the presence of randomly oriented axons intermingled with sparsely fluorescent neuronal processes. The back-scattered photons guide the contextualization of the fluorescence structure within brain atlas thanks to the recognition of characteristic hippocampal structures. Label-free detection of axonal elongations over the layer 2/3 of mouse cortex under a cranial window was also possible in live brain. Finally, blood flow could be measured in vivo, thus validating label free NIR reflectance as a tool for monitoring hemodynamic fluctuations. The prospective versatility of this label-free technique complimentary to two-photon fluorescence microscopy is demonstrated in a mouse model of photothrombotic stroke in which the axonal degeneration and blood flow remodeling can be investigated simultaneously.

  4. Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

    DEFF Research Database (Denmark)

    Lund, F. W.; Lomholt, M. A.; Solanko, L. M.

    2012-01-01

    Background: Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute...... to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter...... probe has utility for prolonged live-cell imaging of sterol transport. Results: We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements...

  5. Photon Emission and Reabsorption Processes in CH3NH3PbBr3 Single Crystals Revealed by Time-Resolved Two-Photon-Excitation Photoluminescence Microscopy

    Science.gov (United States)

    Yamada, Takumi; Yamada, Yasuhiro; Nakaike, Yumi; Wakamiya, Atsushi; Kanemitsu, Yoshihiko

    2017-01-01

    The dynamical processes of radiative recombination of photocarriers and reabsorption of emitted photons in CH3NH3PbBr3 single crystals are studied using time-resolved two-photon-excitation photoluminescence (PL) microscopy. We find that the PL spectrum and its decay dynamics depend on the excitation-depth profile. As the excitation depth increases, the PL spectrum becomes asymmetric, the peak energy redshifts, and the PL decay time becomes longer. These observations can be well explained by a simple model including photon recycling (photon emission and reabsorption) in thick samples with strong band-to-band transitions and high radiative recombination efficiencies.

  6. Scanning tunneling microscopy II further applications and related scanning techniques

    CERN Document Server

    Güntherodt, Hans-Joachim

    1992-01-01

    Scanning Tunneling Microscopy II, like its predecessor, presents detailed and comprehensive accounts of the basic principles and broad range of applications of STM and related scanning probe techniques. The applications discussed in this volume come predominantly from the fields of electrochemistry and biology. In contrast to those described in Vol. I, these sudies may be performed in air and in liquids. The extensions of the basic technique to map other interactions are described inchapters on scanning force microscopy, magnetic force microscopy, scanning near-field optical microscopy, together with a survey of other related techniques. Also described here is the use of a scanning proximal probe for surface modification. Togehter, the two volumes give a comprehensive account of experimental aspcets of STM. They provide essentialreading and reference material for all students and researchers involvedin this field.

  7. Scanning tunneling microscopy II further applications and related scanning techniques

    CERN Document Server

    Güntherodt, Hans-Joachim

    1995-01-01

    Scanning Tunneling Microscopy II, like its predecessor, presents detailed and comprehensive accounts of the basic principles and broad range of applications of STM and related scanning probe techniques. The applications discussed in this volume come predominantly from the fields of electrochemistry and biology. In contrast to those described in STM I, these studies may be performed in air and in liquids. The extensions of the basic technique to map other interactions are described in chapters on scanning force microscopy, magnetic force microscopy, and scanning near-field optical microscopy, together with a survey of other related techniques. Also described here is the use of a scanning proximal probe for surface modification. Together, the two volumes give a comprehensive account of experimental aspects of STM. They provide essential reading and reference material for all students and researchers involved in this field. In this second edition the text has been updated and new methods are discussed.

  8. Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells

    Directory of Open Access Journals (Sweden)

    Lund Frederik W

    2012-10-01

    Full Text Available Abstract Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE, an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent α ~ 0.63 and an anomalous diffusion constant of Dα = 1.95 x 10-3 μm2/sα. On a longer time scale (t > ~5 s, a transition to superdiffusion consistent with slow directed transport with an average velocity of v ~ 6 x 10-3 μm/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle

  9. Scanning probe microscopy competency development

    Energy Technology Data Exchange (ETDEWEB)

    Hawley, M.E.; Reagor, D.W.; Jia, Quan Xi [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The project collaborators developed an ultra-high vacuum scanning tunneling microscope (UHV-STM) capability, integrated it with existing scanning probe microscopes, and developed new, advanced air-based scanning force techniques (SPMs). Programmatic, basic, and industrially related laboratory research requires the existence of SPMs, as well as expertise capable of providing local nano-scale information. The UHV-STM capability, equipped with load-lock system and several surface science techniques, will allow introduction, examination, and reaction of surfaces prepared under well-controlled vacuum conditions, including the examination of morphology and local bonding associated with the initial stages of film growth under controlled growth conditions. The resulting capabilities will enable the authors to respond to a variety of problems requiring local characterization of conducting and nonconducting surfaces in liquids, air, and UHV.

  10. Re-scan confocal microscopy

    NARCIS (Netherlands)

    De Luca, G.M.R.

    2016-01-01

    One of the instruments that gave insight in the morphology and function of cellular components is the optical microscope. Nowadays, optical microscopy in biomedical applications is commonly combined with fluorescence. One fundamental limit in the possibility to distinguish small structures in the sa

  11. Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

    Science.gov (United States)

    Atmeh, A R; Chong, E Z; Richard, G; Boyde, A; Festy, F; Watson, T F

    2015-02-01

    Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

  12. Introduction to scanning tunneling microscopy

    CERN Document Server

    Chen, C Julian

    2008-01-01

    The scanning tunneling and the atomic force microscope, both capable of imaging individual atoms, were crowned with the Physics Nobel Prize in 1986, and are the cornerstones of nanotechnology today. This is a thoroughly updated version of this 'bible' in the field.

  13. Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording and pharmacology

    Directory of Open Access Journals (Sweden)

    Manuel eLevy

    2012-12-01

    Full Text Available Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1 hyperpolarization by intracellular current injection, (2 pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3 GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5 to 10 nA, which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be

  14. Re-scan confocal microscopy: scanning twice for better resolution.

    Science.gov (United States)

    De Luca, Giulia M R; Breedijk, Ronald M P; Brandt, Rick A J; Zeelenberg, Christiaan H C; de Jong, Babette E; Timmermans, Wendy; Azar, Leila Nahidi; Hoebe, Ron A; Stallinga, Sjoerd; Manders, Erik M M

    2013-01-01

    We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

  15. Scanning probe microscopy on new dental alloys

    Science.gov (United States)

    Reusch, B.; Geis-Gerstorfer, J.; Ziegler, C.

    Surface analytical methods such as scanning force microscopy (SFM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used to determine the surface properties of amalgam substitutes as tooth filling materials. In particular the corrosion and the passivation behavior of new gallium restorative materials were studied. To give relevant practical data, the measurements were performed with and without the alloys being stored in artificial saliva to simulate physiological oral conditions.

  16. Towards high-speed scanning tunneling microscopy

    NARCIS (Netherlands)

    Tabak, Femke Chantal

    2013-01-01

    In this thesis, two routes towards high-speed scanning tunneling microscopy (STM) are described. The first possibility for high-speed scanning that is discussed is the use of MEMS (Micro-Electro Mechanical Systems) devices as high-speed add-ons in STM microscopes. The functionality of these devices

  17. Colloquium: Time-resolved scanning tunneling microscopy

    NARCIS (Netherlands)

    Houselt, van Arie; Zandvliet, Harold J.W.

    2010-01-01

    Scanning tunneling microscopy has revolutionized our ability to image, study, and manipulate solid surfaces on the size scale of atoms. One important limitation of the scanning tunneling microscope (STM) is, however, its poor time resolution. Recording a standard image with a STM typically takes abo

  18. Kinetics and subcellular localization of 5-ALA-induced PpIX in DHL cells via two-photon excitation fluorescence microscopy.

    Science.gov (United States)

    Chen, Rong; Huang, Zufang; Chen, Guannan; Li, Yongzeng; Chen, Xianlian; Chen, Jianxin; Zeng, Haishan

    2008-04-01

    Two-photon excitation fluorescence (TPEF) microscopy was used to measure the 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence in follicular lymphoma DHL cells. Kinetics of 5-ALA-induced PpIX accumulation in DHL cells under various 5-ALA concentrations was studied. We found that during the course of continuous incubation with 5-ALA, the relationship between the DHL cell fluorescence signal and the incubation time showed a biphasic variation. Initially the PpIX signal increased with the incubation time and reached the maximal value at about 3 h, and then it decreased with time during the subsequent incubation period. By labeling the 5-ALA incubated DHL cells with different organelle-specific fluorescence probes: Rhodamine 123 (for mitochondria), DioC6(3) (for endoplasmic reticulum) and LysoTracker Green (for lysosomes) respectively, we found that 5-ALA-induced PpIX was primarily localized in endoplasmic reticulum and mitochondria; its concentration in the lysosome was much lower. The results suggested that 5-ALA could potentially be an effective photosensitizer in photodynamic purging of DHL cells. Two-photon excitation fluorescence microscope is a useful tool for studying 5-ALA-induced PpIX subcellular localization.

  19. Identification of the boundary between normal brain tissue and ischemia region using two-photon excitation fluorescence microscopy

    Science.gov (United States)

    Du, Huiping; Wang, Shu; Wang, Xingfu; Zhu, Xiaoqin; Zhuo, Shuangmu; Chen, Jianxin

    2016-10-01

    Ischemic stroke is one of the common neurological diseases, and it is becoming the leading causes of death and permanent disability around the world. Early and accurate identification of the potentially salvageable boundary region of ischemia brain tissues may enable selection of the most appropriate candidates for early stroke therapies. In this work, TPEF microscopy was used to image the microstructures of normal brain tissues, ischemia regions and the boundary region between normal and ischemia brain tissues. The ischemia brain tissues from Sprague-Dawley (SD) rats were subjected to 6 hours of middle cerebral artery occlusion (MCAO). Our study demonstrates that TPEF microscopy has the ability to not only reveal the morphological changes of the neurons but also identify the boundary between normal brain tissue and ischemia region, which correspond well to the hematoxylin and eosin (H and E) stained images. With the development of miniaturized TPEF microscope imaging devices, TPEF microscopy can be developed into an effectively diagnostic and monitoring tool for cerebral ischemia.

  20. Proximity Scanning Transmission Electron Microscopy/Spectroscopy

    CERN Document Server

    Hwang, Ing-Shouh

    2016-01-01

    Here a new microscopic method is proposed to image and characterize very thin samples like few-layer materials, organic molecules, and nanostructures with nanometer or sub-nanometer resolution using electron beams of energies lower than 20 eV. The microscopic technique achieves high resolution through the proximity (or near-field) effect, as in scanning tunneling microscopy (STM), while it also allows detection of transmitted electrons for imaging and spectroscopy, as in scanning transmission electron microscopy (STEM). This proximity transmission electron microscopy (PSTEM) does not require any lens to focus the electron beam. It also allows detailed characterization of the interaction of low-energy electron with materials. PSTEM can operate in a way very similar to scanning tunneling microscopy, which provides high-resolution imaging of geometric and electronic structures of the sample surface. In addition, it allows imaging and characterization of the interior structures of the sample based on the detected...

  1. 4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM).

    Science.gov (United States)

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

    2016-04-01

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

  2. Analysis of Recombination in CdTe Heterostructures With Time-Resolved Two-Photon Excitation Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kuciauskas, Darius; Wernsing, Keith; Jensen, Soren Alkaersig; Barnes, Teresa M.; Myers, Thomas H.; Bartels, Randy A.

    2016-11-01

    We used time-resolved photoluminescence micro-scopy to analyze charge carrier transport and recombination in CdTe double heterostructures fabricated by molecular beam epitaxy (MBE). This allowed us to determine the charge carrier mobility in this system, which was found to be 500-625 cm2/(V s). Charge carrier lifetimes in the 15-100 ns range are limited by the interface recombination, and the data indicate higher interface recombination velocity near extended defects. This study describes a new method to analyze the spatial distribution of the interface recombination velocity and the interface defects in semiconductor heterostructures.

  3. Effect of detergents on the physico-chemical properties of skin stratum corneum: A two-photon excitation fluorescence microscopy study

    DEFF Research Database (Denmark)

    Bloksgaard, Maria; Brewer, Jonathan R.; Pashkovski, Eugene

    2014-01-01

    performed using two-photon excitation fluorescence microscopy. Fluorescent images of fluorescence reporters sensitive to proton activity and hydration of SC were obtained in excised skin and examined in presence and absence of SCM and SDS detergents. RESULTS: Hydration of the intercellular lipid matrix......OBJECTIVE: Understanding the structural and dynamical features of skin is critical for advancing innovation in personal care and drug discovery. Synthetic detergent mixtures used in commercially available body wash products are thought to be less aggressive towards the skin barrier when compared...... to conventional detergents. The aim of this work is to comparatively characterize the effect of a mild synthetic cleanser mixture (SCM) and sodium dodecyl sulphate (SDS) on the hydration state of the intercellular lipid matrix and on proton activity of excised skin stratum corneum (SC). METHOD: Experiments were...

  4. Spatial heterodyne scanning laser confocal holographic microscopy

    CERN Document Server

    Liu, Changgeng

    2016-01-01

    Scanning laser confocal holographic microscopy using a spatial heterodyne detection method is presented. Spatial heterodyne detection technique employs a Mach-Zehnder interferometer with the reference beam frequency shifted by two acousto-optic modulators (AOM) relative to the object beam frequency. Different from the traditional temporal heterodyne detection technique in which hundreds temporal samples are taken at each scanning point to achieve the complex signal, the spatial heterodyne detection technique generates spatial interference fringes by use of a linear tempo-spatial relation provided by galvanometer scanning in a typical line-scanning confocal microscope or for the slow-scanning on one dimension in a point-scanning confocal microscope, thereby significantly reducing sampling rate and increasing the signal to noise ratio under the same illumination compared to the traditional temporal heterodyne counterpart. The proposed spatial heterodyne detection scheme applies to both line-scanning and point-s...

  5. A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy.

    Directory of Open Access Journals (Sweden)

    Ken Coppieters

    Full Text Available Type 1 diabetes (T1D is characterized by the immune-mediated destruction of beta cells in the pancreas. Little is known about the in vivo dynamic interactions between T cells and beta cells or the kinetic behavior of other immune cell subsets in the pancreatic islets. Utilizing multiphoton microscopy we have designed a technique that allows for the real-time visualization of diabetogenic T cells and dendritic cells in pancreatic islets in a live animal, including their interplay with beta cells and the vasculature. Using a custom designed stage, the pancreas was surgically exposed under live conditions so that imaging of islets under intact blood pressure and oxygen supply became possible. We demonstrate here that this approach allows for the tracking of diabetogenic leukocytes as well as vascularization phenotype of islets and accumulation of dendritic cells in islets during diabetes pathogenesis. This technique should be useful in mapping crucial kinetic events in T1D pathogenesis and in testing the impact of immune based interventions on T cell migration, extravasation and islet destruction.

  6. The effect of polyunsaturated fatty acids on the homeostasis of yolk lipoprotein in C. elegans examined by CARS and two-photon excitation fluorescence (TPE-F) microscopy

    Science.gov (United States)

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Lin, Yi-Chun; Ma, Tian-Hsiang; Lo, Szecheng J.; Chang, Ta-Chau

    2016-03-01

    Yolk lipoprotein constitutes the major source of energy and the materials for synthesizing signaling factors for the development of oocytes and embryos in C. elegans. Polyunsaturated fatty acids (PUFAs) packed in yolk lipoprotein have been recently recognized as critical molecules for fertilization and reproduction.1 However, the relation between PUFAs and the homeostasis of yolk lipoprotein is not clear. Here we use coherent anti-Stokes Raman scattering (CARS) microscopy and two-photon excitation fluorescence (TPE-F) microscopy to examine the transportation of yolk lipoprotein. We demonstrate that CARS microscopy is a more sensitive method than the traditional Nile Red staining method in probing the abnormal accumulation of yolk lipoprotein in the body cavity of C. elegans. It is found that the accumulation of yolk lipoprotein is a time-dependent process. In addition, a negative correlation (r = -0.955) between reproductive aging and abnormal accumulation of yolk lipoprotein is established. We further examine wild-type, fat-1, and fat-2 worms with or without the expression of GFP-tagged yolk lipoprotein (VIT-2-GFP). Our data reveal that PUFAs have a positive effect on the synthesis and endocytosis of yolk lipoprotein, confirming the model proposed by Edmonds et al.2

  7. Sample preparation method for scanning force microscopy

    CERN Document Server

    Jankov, I R; Szente, R N; Carreno, M N P; Swart, J W; Landers, R

    2001-01-01

    We present a method of sample preparation for studies of ion implantation on metal surfaces. The method, employing a mechanical mask, is specially adapted for samples analysed by Scanning Force Microscopy. It was successfully tested on polycrystalline copper substrates implanted with phosphorus ions at an acceleration voltage of 39 keV. The changes of the electrical properties of the surface were measured by Kelvin Probe Force Microscopy and the surface composition was analysed by Auger Electron Spectroscopy.

  8. Scanning probe microscopy at video-rate

    Directory of Open Access Journals (Sweden)

    Georg Schitter

    2008-01-01

    Full Text Available Recent results have demonstrated the feasibility of video-rate scanning tunneling microscopy and video-rate atomic force microscopy. The further development of this technology will enable the direct observation of many dynamic processes that are impossible to observe today with conventional Scanning Probe Microscopes (SPMs. Examples are atom and molecule diffusion processes, the motion of molecular motors, real-time film growth, and chemical or catalytic reactions. Video-rate scanning probe technology might also lead to the extended application of SPMs in industry, e.g. for process control. In this paper we discuss the critical aspects that have to be taken into account for improving the imaging speed of SPMs. We point out the required instrumentation efforts, give an overview of the state of the art in high-speed scanning technology and discuss the required future developments for imaging at video-rates.

  9. Enhancement of two-photon photoluminescence and SERS for low-coverage gold films

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Frydendahl, Christian

    2016-01-01

    Electromagnetic field enhancement (FE) effects occurring in thin gold films 3-12-nm are investigated with two-photon photoluminescence (TPL) and Raman scanning optical microscopies. The samples are characterized using scanning electron microscopy images and linear optical spectroscopy. TPL images...

  10. Label-free imaging of brain and brain tumor specimens with combined two-photon excited fluorescence and second harmonic generation microscopy

    Science.gov (United States)

    Jiang, Liwei; Wang, Xingfu; Wu, Zanyi; Du, Huiping; Wang, Shu; Li, Lianhuang; Fang, Na; Lin, Peihua; Chen, Jianxin; Kang, Dezhi; Zhuo, Shuangmu

    2017-10-01

    Label-free imaging techniques are gaining acceptance within the medical imaging field, including brain imaging, because they have the potential to be applied to intraoperative in situ identifications of pathological conditions. In this paper, we describe the use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy in combination for the label-free detection of brain and brain tumor specimens; gliomas. Two independently detecting channels were chosen to subsequently collect TPEF/SHG signals from the specimen to increase TPEF/SHG image contrasts. Our results indicate that the combined TPEF/SHG microscopic techniques can provide similar rat brain structural information and produce a similar resolution like conventional H&E staining in neuropathology; including meninges, cerebral cortex, white-matter structure corpus callosum, choroid plexus, hippocampus, striatum, and cerebellar cortex. It can simultaneously detect infiltrating human brain tumor cells, the extracellular matrix collagen fiber of connective stroma within brain vessels and collagen depostion in tumor microenvironments. The nuclear-to-cytoplasmic ratio and collagen content can be extracted as quantitative indicators for differentiating brain gliomas from healthy brain tissues. With the development of two-photon fiberscopes and microendoscope probes and their clinical applications, the combined TPEF and SHG microcopy may become an important multimodal, nonlinear optical imaging approach for real-time intraoperative histological diagnostics of residual brain tumors. These occur in various brain regions during ongoing surgeries through the method of simultaneously identifying tumor cells, and the change of tumor microenvironments, without the need for the removal biopsies and without the need for tissue labelling or fluorescent markers.

  11. Semiconductor Surface Characterization by Scanning Probe Microscopies

    Science.gov (United States)

    2001-01-01

    potentiometry (STP)8 and ballistic electron emission microscopy (BEEM)9 which allow mapping of lateral surface potential and local subsurface Schottky...A.P.Fein. "Tunneling Spectroscopy of the Si(1 1 1)2xl Surface", Surf.Sci. 181, 295- 306, 1987. 8. P.Muralt, D.W.Pohl, "Scanning tunneling potentiometry

  12. Energetic materials research using scanning electron microscopy

    NARCIS (Netherlands)

    Elshout, J.J.M.H. van den; Duvalois, W.; Benedetto, G.L. Di; Bouma, R.H.B.; Heijden, A.E.D.M. van der

    2016-01-01

    A key-technique for the research of energetic materials is scanning electron microscopy. In this paper several examples are given of characterization studies on energetic materials, including a solid composite propellant formulation. Results of the characterization of energetic materials using scann

  13. Electrochemical gating in scanning electrochemical microscopy

    NARCIS (Netherlands)

    Ahonen, P.; Ruiz, V.; Kontturi, K.; Liljeroth, P.; Quinn, B.M.

    2008-01-01

    We demonstrate that scanning electrochemical microscopy (SECM) can be used to determine the conductivity of nanoparticle assemblies as a function of assembly potential. In contrast to conventional electron transport measurements, this method is unique in that electrical connection to the film is not

  14. Phosphogypsum surface characterisation using scanning electron microscopy

    Directory of Open Access Journals (Sweden)

    Rajković Miloš B.

    2003-01-01

    Full Text Available This paper presents the results of application of Scanning Electron Microscopy (SEM to examinations of the samples of natural gypsum and phosphogypsum. Phosphogypsum has a well developed crystalline structure, and appear in two polymorphous forms, of rombic and hexagonal shape crystals. Natural gypsum has a poorly crystalline structure. The differences in crystalline structure influence the chemical behavior of these row materials.

  15. Energetic materials research using scanning electron microscopy

    NARCIS (Netherlands)

    Elshout, J.J.M.H. van den; Duvalois, W.; Benedetto, G.L. Di; Bouma, R.H.B.; Heijden, A.E.D.M. van der

    2016-01-01

    A key-technique for the research of energetic materials is scanning electron microscopy. In this paper several examples are given of characterization studies on energetic materials, including a solid composite propellant formulation. Results of the characterization of energetic materials using

  16. Environmental scanning electron microscopy in cell biology.

    Science.gov (United States)

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

    2013-01-01

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

  17. Two-photon physics

    Energy Technology Data Exchange (ETDEWEB)

    Bardeen, W.A.

    1981-10-01

    A new experimental frontier has recently been opened to the study of two photon processes. The first results of many aspects of these reactions are being presented at this conference. In contrast, the theoretical development of research ito two photon processes has a much longer history. This talk reviews the many different theoretical ideas which provide a detailed framework for our understanding of two photon processes.

  18. Nanoscale thermometry by scanning thermal microscopy

    Science.gov (United States)

    Menges, Fabian; Riel, Heike; Stemmer, Andreas; Gotsmann, Bernd

    2016-07-01

    Measuring temperature is a central challenge in nanoscience and technology. Addressing this challenge, we report the development of a high-vacuum scanning thermal microscope and a method for non-equilibrium scanning probe thermometry. The microscope is built inside an electromagnetically shielded, temperature-stabilized laboratory and features nanoscopic spatial resolution at sub-nanoWatt heat flux sensitivity. The method is a dual signal-sensing technique inferring temperature by probing a total steady-state heat flux simultaneously to a temporally modulated heat flux signal between a self-heated scanning probe sensor and a sample. Contact-related artifacts, which so far limit the reliability of nanoscopic temperature measurements by scanning thermal microscopy, are minimized. We characterize the microscope's performance and demonstrate the benefits of the new thermometry approach by studying hot spots near lithographically defined constrictions in a self-heated metal interconnect.

  19. [Pili annulati. A scanning electron microscopy study].

    Science.gov (United States)

    Lalević-Vasić, B; Polić, D

    1988-01-01

    A case of ringed hair studied by light and electron microscopy is reported. The patient, a 20-year old girl, had been presenting with the hair abnormality since birth. At naked eye examination the hairs were dry, 6 to 7 cm long, and they showed dull and shining areas giving the scalp hair a scintillating appearance (fig. 1). Several samples of hair were taken and examined by light microscopy under white and polarized light. Hair shafts and cryo-fractured surfaces were examined by scanning electron microscopy. RESULTS. 1. Light microscopy. Lesions were found in every hair examined. There were abnormal, opaque and fusiform areas alternating with normal areas all along the hair shaft (fig. 2). The abnormal areas resulted from intracortical air-filled cavities. Fractures similar to those of trichorrhexis nodosa were found in the opaque areas of the distal parts of the hairs. 2. Scanning electron microscopy. A. Hair shaft surface. The abnormal areas showed a longitudinal, "curtain-like" folding of the cuticular cells which had punctiform depressions on their surface and worn free edges (fig. 4, 5, 6); trichorrhexis-type fractures were seen in the distal parts of the hair shafts (fig. 7, 8). Normal areas regularly presented with longitudinal, superficial, short and non-systematized depressions (fig. 9); the cuticular cells were worn, and there were places where the denuded cortex showed dissociated cortical fibres (fig. 10).(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Evaluation of Injured Axons Using Two-Photon Excited Fluorescence Microscopy after Spinal Cord Contusion Injury in YFP-H Line Mice.

    Science.gov (United States)

    Horiuchi, Hideki; Oshima, Yusuke; Ogata, Tadanori; Morino, Tadao; Matsuda, Seiji; Miura, Hiromasa; Imamura, Takeshi

    2015-07-13

    Elucidation of the process of degeneration of injured axons is important for the development of therapeutic modules for the treatment of spinal cord injuries. The aim of this study was to establish a method for time-lapse observation of injured axons in living animals after spinal cord contusion injury. YFP (yellow fluorescent protein)-H transgenic mice, which we used in this study, express fluorescence in their nerve fibers. Contusion damage to the spinal cord at the 11th vertebra was performed by IH (Infinite Horizon) impactor, which applied a pressure of 50 kdyn. The damaged spinal cords were re-exposed during the observation period under anesthesia, and then observed by two-photon excited fluorescence microscopy, which can observe deep regions of tissues including spinal cord axons. No significant morphological change of injured axons was observed immediately after injury. Three days after injury, the number of axons decreased, and residual axons were fragmented. Seven days after injury, only fragments were present in the damaged tissue. No hind-limb movement was observed during the observation period after injury. Despite the immediate paresis of hind-limbs following the contusion injury, the morphological degeneration of injured axons was delayed. This method may help clarification of pathophysiology of axon degeneration and development of therapeutic modules for the treatment of spinal cord injury.

  1. Tip-modulation scanned gate microscopy.

    Science.gov (United States)

    Wilson, Neil R; Cobden, David H

    2008-08-01

    We introduce a technique that improves the sensitivity and resolution and eliminates the nonlocal background of scanned gate microscopy (SGM). In conventional SGM, a voltage bias is applied to the atomic force microscope tip and the sample conductance is measured as the tip is scanned. In the new technique, which we call tip-modulation SGM (tmSGM), the biased tip is oscillated and the induced oscillation of the sample conductance is measured. Applied to single-walled carbon nanotube network devices, tmSGM gives sharp, low-noise and background-free images.

  2. Scanning Electron Microscopy Sample Preparation and Imaging.

    Science.gov (United States)

    Nguyen, Jenny Ngoc Tran; Harbison, Amanda M

    2017-01-01

    Scanning electron microscopes allow us to reach magnifications of 20-130,000× and resolve compositional and topographical images with intense detail. These images are created by bombarding a sample with electrons in a focused manner to generate a black and white image from the electrons that bounce off of the sample. The electrons are detected using positively charged detectors. Scanning electron microscopy permits three-dimensional imaging of desiccated specimens or wet cells and tissues by using variable pressure chambers. SEM ultrastructural analysis and intracellular imaging supplement light microscopy for molecular profiling of prokaryotes, plants, and mammals. This chapter demonstrates how to prepare and image samples that are (a) desiccated and conductive, (b) desiccated and nonconductive but coated with an electron conductive film using a gold sputter coater, and (c) wet and maintained in a hydrated state using a Deben Coolstage.

  3. Scanning electron microscopy of superficial white onychomycosis*

    Science.gov (United States)

    de Almeida Jr., Hiram Larangeira; Boabaid, Roberta Oliveira; Timm, Vitor; Silva, Ricardo Marques e; de Castro, Luis Antonio Suita

    2015-01-01

    Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis. PMID:26560225

  4. Manipulating atoms using scanning probe microscopy

    OpenAIRE

    Norris, Andrew Christopher

    2009-01-01

    Manipulating species using Scanning Probe Microscopy (SPM) is an important discipline in the field of bottom-up fabrication, which facilitates nano-mechanics and electron dynamics investigations. Previous low temperature manipulation highlights include the construction of quantum dots, nano-wires and quantum corrals, all of which began in 1989 with Don Eigler’s iconic writing of I-B-M using 35 xenon atoms. Since then, the field has developed, and we now push, pull, hop, excite, desorb, rotate...

  5. Investigation into scanning tunnelling luminescence microscopy

    CERN Document Server

    Manson-Smith, S K

    2001-01-01

    This work reports on the development of a scanning tunnelling luminescence (STL) microscope and its application to the study of Ill-nitride semiconductor materials used in the production of light emitting devices. STL microscopy is a technique which uses the high resolution topographic imaging capabilities of the scanning tunnelling microscope (STM) to generate high resolution luminescence images. The STM tunnelling current acts as a highly localised source of electrons (or holes) which generates luminescence in certain materials. Light generated at the STM tunnelling junction is collected concurrently with the height variation of the tunnelling probe as it is scanned across a sample surface, producing simultaneous topographic and luminescence images. Due to the very localised excitation source, high resolution luminescence images can be obtained. Spectroscopic resolution can be obtained by using filters. Additionally, the variation of luminescence intensity with tunnel current and with bias voltage can provi...

  6. Interferometric Synthetic Aperture Microscopy: Computed Imaging for Scanned Coherent Microscopy

    Directory of Open Access Journals (Sweden)

    Stephen A. Boppart

    2008-06-01

    Full Text Available Three-dimensional image formation in microscopy is greatly enhanced by the use of computed imaging techniques. In particular, Interferometric Synthetic Aperture Microscopy (ISAM allows the removal of out-of-focus blur in broadband, coherent microscopy. Earlier methods, such as optical coherence tomography (OCT, utilize interferometric ranging, but do not apply computed imaging methods and therefore must scan the focal depth to acquire extended volumetric images. ISAM removes the need to scan the focus by allowing volumetric image reconstruction from data collected at a single focal depth. ISAM signal processing techniques are similar to the Fourier migration methods of seismology and the Fourier reconstruction methods of Synthetic Aperture Radar (SAR. In this article ISAM is described and the close ties between ISAM and SAR are explored. ISAM and a simple strip-map SAR system are placed in a common mathematical framework and compared to OCT and radar respectively. This article is intended to serve as a review of ISAM, and will be especially useful to readers with a background in SAR.

  7. A method to investigate radial glia cell behavior using two-photon time-lapse microscopy in an ex vivo model of spinal cord development.

    Directory of Open Access Journals (Sweden)

    Janelle M.P. Pakan

    2014-04-01

    Full Text Available The mammalian central nervous system (CNS develops from multipotent progenitor cells, which proliferate and differentiate into the various cell types of the brain and spinal cord. Despite the wealth of knowledge from progenitor cell culture studies, there is a significant lack of understanding regarding dynamic progenitor cell behavior over the course of development. This is in part due to shortcomings in the techniques available to study these processes in living tissues as they are occurring. In order to investigate cell behavior under physiologically relevant conditions we established an ex vivo model of the developing rat spinal cord. This method allows us to directly observe specific populations of cells ex vivo in real time and over extended developmental periods as they undergo proliferation, migration and differentiation in the CNS. Previous investigations of progenitor cell behavior have been limited in either spatial or temporal resolution (or both due to the necessity of preserving tissue viability and avoiding phototoxic effects of fluorescent imaging. The method described here overcomes these obstacles. Using two-photon and confocal microscopy and transfected organotypic spinal cord slice cultures we have undertaken detailed imaging of a unique population of neural progenitors, radial glial cells. This method uniquely enables analysis of large populations as well as individual cells; ultimately resulting in a 4D dataset of progenitor cell behavior for up to seven days during embryonic development. This approach can be adapted to study a variety of cell populations at different stages of development using appropriate promoter driven fluorescent protein expression. The ability to control the tissue micro-environment makes this ex vivo method a powerful tool to elucidate the underlying molecular mechanisms regulating cell behavior during embryonic development.

  8. Two-photon STED spectral determination for a new V-shaped organic fluorescent probe with efficient two-photon absorption.

    Science.gov (United States)

    Belfield, Kevin D; Bondar, Mykhailo V; Morales, Alma R; Padilha, Lazaro A; Przhonska, Olga V; Wang, Xuhua

    2011-10-24

    Two-photon stimulated emission depletion (STED) cross sections were determined over a broad spectral range for a novel two-photon absorbing organic molecule, representing the first such report. The synthesis, comprehensive linear photophysical, two-photon absorption (2PA), and stimulated emission properties of a new fluorene-based compound, (E)-2-{3-[2-(7-(diphenylamino)-9,9-diethyl-9H-fluoren-2-yl)vinyl]-5-methyl-4-oxocyclohexa-2,5-dienylidene} malononitrile (1), are presented. Linear spectral parameters, including excitation anisotropy and fluorescence lifetimes, were obtained over a broad range of organic solvents at room temperature. The degenerate two-photon absorption (2PA) spectrum of 1 was determined with a combination of the direct open-aperture Z-scan and relative two-photon-induced fluorescence methods using 1 kHz femtosecond excitation. The maximum value of the 2PA cross section ~1700 GM was observed in the main, long wavelength, one-photon absorption band. One- and two-photon stimulated emission spectra of 1 were obtained over a broad spectral range using a femtosecond pump-probe technique, resulting in relatively high two-photon stimulated emission depletion cross sections (~1200 GM). A potential application of 1 in bioimaging was demonstrated through one- and two-photon fluorescence microscopy images of HCT 116 cells incubated with micelle-encapsulated dye.

  9. Scanning Electron Microscopy of the Presbylarynx.

    Science.gov (United States)

    Gonçalves, Tatiana Maria; Dos Santos, Daniela Carvalho; Pessin, Adriana Bueno Benito; Martins, Regina Helena Garcia

    2016-06-01

    To describe the findings on the presbylarynx under scanning electron microscopy. Cadaver study. Universidade Estadual Paulista (Botucatu, São Paulo, Brazil). Sixteen vocal folds were removed during necropsies and distributed into 2 age groups: control (n = 8; aged 30-50 years) and elderly (n = 8; aged 75-92 years). The right vocal fold was dissected, fixed in glutaraldehyde 2.5%, and prepared for scanning electron microscopy. The thickness of the epithelium was measured using a scandium morphometric digital program. In the control group, the epithelium had 5 to 7 overlapped cell layers, rare desquamation cells, and little undulation with protruding intercellular junctions. The lamina propria showed a uniform network of collagen and elastic fibers in the superficial layer. A dense network of collagen was identified in the deeper layer. In the elderly group, the epithelium was atrophic (2-3 cells), with more desquamation cells and intercellular junctions delimited by deep sulci. The epithelial thickness was lower in elderly than in controls (mean [SD], 221.64 [145.90] µm vs 41.79 [21.40] µm, respectively). The lamina propria had a dense and irregular distribution of collagen and elastic fibers in the superficial layer. In the deep layers, the collagen fibers formed a true fibrotic and rigid skeleton. Scanning electron microscopy identified several changes in the elderly larynx, differentiating it from the controls. These alterations are probably related to the aging process of the vocal folds. However, the exact interpretation of these findings requires additional studies, even to the molecular level, having the fibroblasts as targets. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.

  10. Traceable long range scanning tunneling microscopy

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Dimensionally correct and directly traceable measurement is not feasible with conventional scanning tunneling microscopy (STMs) due to severe hysteresis and non-linearity of the commonly applied piezo tube scanners and the very short range.By integrating a custom made probing system based on tunneling current measurement into a commercially available and laser-interferometrically position controlled positioning system, an STM with a principal measuring range of 25 mm×25 mm×5 mm and traceable position measurement has been set-up and tested.

  11. Scanning probe microscopy of protein nanowires

    Science.gov (United States)

    Walsh, Kathleen Ann

    The bacterium Geobacter sulfurreducens grows electrically-conductive pili, which act as protein nanowires, in order to transfer electrons from the cell to electron acceptors in its environment when direct charge transfer through the cell membrane is not feasible. Understanding the electronic structure of the pili can provide insight into fundamental processes of electron transfer in biological systems. This study investigated the electronic structure of these protein nanowires using the toolbox of scanning probe microscopy, specifically scanning tunneling microscopy and point tunneling spectroscopy. These measurements were performed at 77 K and at room temperature. The measured data are compared to theoretical calculations. Density of states measurements using tunneling spectroscopy show that these pili act as narrow-gap biological semiconductors at 77 K. The onset of nonzero density of states remains within the metabolically-relevant voltage range. At room temperature, spectroscopy of the pili retains a gap-like structure, but this pseudogap is raised to a nonzero density of states at even the smallest applied voltages. These pilus nanowires also exhibit a distinct spatial dependence of the density of states across the breadth of the pili.

  12. Pixel timing correction in time-lapsed calcium imaging using point scanning microscopy.

    Science.gov (United States)

    Boiroux, Dimitri; Oke, Yoshihiko; Miwakeichi, Fumikazu; Oku, Yoshitaka

    2014-11-30

    In point scanning imaging, data are acquired by sequentially scanning each pixel of a predetermined area. This way of scanning leads to time delays between pixels, especially for lower scanning speed or large scanned areas. Therefore, experiments are often performed at lower framerates in order to ensure a sufficient signal-to-noise ratio, even though framerates above 30 frames per second are technically feasible. For these framerates, we suggest that it becomes crucial to correct the time delay between image pixels prior to analyses. In this paper, we apply temporal interpolation (or pixel timing correction) for calcium imaging in two-photon microscopy as an example of fluorescence imaging. We present and compare three interpolation methods (linear, Lanczos and cubic B-spline). We test these methods on a simulated network of coupled bursting neurons at different framerates. In this network, we introduce a time delay to simulate a scanning by point scanning microscopy. We also assess these methods on actual microscopic calcium imaging movies recorded at usual framerates. Our numerical results suggest that point scanning microscopy imaging introduces statistically significant time delays between image pixels at low frequency. However, we demonstrate that pixel timing correction compensates for these time delays, regardless of the used interpolation method.

  13. Correlative photoactivated localization and scanning electron microscopy.

    Directory of Open Access Journals (Sweden)

    Benjamin G Kopek

    Full Text Available The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM and scanning electron microscope (SEM system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

  14. Correlative photoactivated localization and scanning electron microscopy.

    Science.gov (United States)

    Kopek, Benjamin G; Shtengel, Gleb; Grimm, Jonathan B; Clayton, David A; Hess, Harald F

    2013-01-01

    The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers.

  15. Nanoscale resolution immersion scanning thermal microscopy

    CERN Document Server

    Tovee, Peter D

    2013-01-01

    Nanoscale thermal properties are becoming of extreme importance for modern electronic circuits that dissipate increasing power on the length scale of few tens of nanometers, and for chemical and physical properties sensors and biosensors using nanoscale sized features. While Scanning Thermal Microscopy (SThM) is known for its ability to probe thermal properties and heat generation with nanoscale resolution, until today it was perceived impossible to use it in the liquid environment due to dominating direct heat exchange between microfabricated thermal probe and surrounding liquid that would deteriorate spatial resolution. Nonetheless, our theoretical analysis of SThM in liquids showed that for certain design of SThM probe with resistive heater located near the probe tip, their thermal signal is only moderately affected, by less than half on immersion in a dodecane environment. More significantly, its spatial resolution, surprisingly, would remain practically unaffected, and the thermal contact between the tip...

  16. Scanning electron microscopy of molluscum contagiosum*

    Science.gov (United States)

    de Almeida Jr, Hiram Larangeira; Abuchaim, Martha Oliveira; Schneider, Maiko Abel; Marques, Leandra; de Castro, Luis Antônio Suíta

    2013-01-01

    Molluscum contagiosum is a disease caused by a poxvirus. It is more prevalent in children up to 5 years of age. There is a second peak of incidence in young adults. In order to examine its ultrastructure, three lesions were curetted without disruption, cut transversely with a scalpel, and routinely processed for scanning electron microscopy (SEM). The oval structure of molluscum contagiosum could be easily identified. In its core, there was a central umbilication and just below this depression, there was a keratinized tunnel. Under higher magnification, a proliferation similar to the epidermis was seen. Moreover, there were areas of cells disposed like a mosaic. Under higher magnification, rounded structures measuring 0.4 micron could be observed at the end of the keratinized tunnel and on the surface of the lesion. PMID:23539009

  17. Resolution and contrast enhancement in laser scanning microscopy using dark beam imaging.

    Science.gov (United States)

    Dehez, Harold; Piché, Michel; De Koninck, Yves

    2013-07-01

    Laser scanning microscopy allows for three-dimensional imaging of cells with molecular specific labeling. However the spatial resolution of optical microscopy is fundamentally limited by the diffraction of light. In the last two decades many techniques have been introduced to enhance the resolution of laser scanning microscopes. However most of these techniques impose strong constraints on the specimen or rely on complex optical systems. These constraints limit the applicability of resolution improvement to various imaging modalities and sample types. To overcome these limitations, we introduce here a novel approach, which we called Switching LAser Mode (SLAM) microscopy, to enhance resolution and contrast in laser scanning microscopy. SLAM microscopy relies on subtracting images obtained with dark and bright modes, and exploits the smaller dimensions of the dark spot of the azimuthally polarized TE 01 mode. With this approach, resolution is improved by a factor of two in confocal microscopy. The technique is not based on complex nonlinear processes and thus requires laser power similar to that used in conventional imaging, minimizing photo-damage. The flexibility of the approach enables retrofitting in commercial confocal and two-photon microscopes and opens avenues for resolution enhancement in fluorescence-independent microscopy.

  18. Radio-frequency scanning tunnelling microscopy.

    Science.gov (United States)

    Kemiktarak, U; Ndukum, T; Schwab, K C; Ekinci, K L

    2007-11-01

    The scanning tunnelling microscope (STM) relies on localized electron tunnelling between a sharp probe tip and a conducting sample to attain atomic-scale spatial resolution. In the 25-year period since its invention, the STM has helped uncover a wealth of phenomena in diverse physical systems--ranging from semiconductors to superconductors to atomic and molecular nanosystems. A severe limitation in scanning tunnelling microscopy is the low temporal resolution, originating from the diminished high-frequency response of the tunnel current readout circuitry. Here we overcome this limitation by measuring the reflection from a resonant inductor-capacitor circuit in which the tunnel junction is embedded, and demonstrate electronic bandwidths as high as 10 MHz. This approximately 100-fold bandwidth improvement on the state of the art translates into fast surface topography as well as delicate measurements in mesoscopic electronics and mechanics. Broadband noise measurements across the tunnel junction using this radio-frequency STM have allowed us to perform thermometry at the nanometre scale. Furthermore, we have detected high-frequency mechanical motion with a sensitivity approaching approximately 15 fm Hz(-1/2). This sensitivity is on par with the highest available from nanoscale optical and electrical displacement detection techniques, and the radio-frequency STM is expected to be capable of quantum-limited position measurements.

  19. Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy.

    Directory of Open Access Journals (Sweden)

    Henry Pinkard

    Full Text Available Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM, the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.

  20. Spatiotemporal Rank Filtering Improves Image Quality Compared to Frame Averaging in 2-Photon Laser Scanning Microscopy.

    Science.gov (United States)

    Pinkard, Henry; Corbin, Kaitlin; Krummel, Matthew F

    2016-01-01

    Live imaging of biological specimens using optical microscopy is limited by tradeoffs between spatial and temporal resolution, depth into intact samples, and phototoxicity. Two-photon laser scanning microscopy (2P-LSM), the gold standard for imaging turbid samples in vivo, has conventionally constructed images with sufficient signal-to-noise ratio (SNR) generated by sequential raster scans of the focal plane and temporal integration of the collected signals. Here, we describe spatiotemporal rank filtering, a nonlinear alternative to temporal integration, which makes more efficient use of collected photons by selectively reducing noise in 2P-LSM images during acquisition. This results in much higher SNR while preserving image edges and fine details. Practically, this allows for at least a four fold decrease in collection times, a substantial improvement for time-course imaging in biological systems.

  1. Measurement of the Two-photon Absorption Coefficient of Gallium Phosphide (GaP) Using a Dispersion-minimized Sub-10 Femtosecond Z-scan Measurement System

    Science.gov (United States)

    2012-09-01

    samples of semiconductors and thin films of organic materials on substrates. The use of dispersive media in the optical path has been eliminated...other semiconductors such as cadmium sulfide (CdS) and zinc selenide (ZnSe). We will also begin measuring the two-photon absorption coefficient for...organic thin - film materials deposited on various substrates. 15 6. References 1. Sheik-Bahae, M.; Said, A. A.; Van Stryland, E. W. High

  2. Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber.

    Science.gov (United States)

    Nguyen, Kayla X; Holtz, Megan E; Richmond-Decker, Justin; Muller, David A

    2016-08-01

    A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

  3. Applications of orientation mapping by scanning and transmission electron microscopy

    DEFF Research Database (Denmark)

    Juul Jensen, D.

    1997-01-01

    The potentials of orientation mapping techniques (in the following referred to as OIM) for studies of thermomechanical processes are analysed. Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) based OIM techniques are considered. Among the thermomechanical processes...

  4. Scanned probe microscopy for thin film superconductor development

    Energy Technology Data Exchange (ETDEWEB)

    Moreland, J. [National Institute of Standards and Technology, Boulder, CO (United States)

    1996-12-31

    Scanned probe microscopy is a general term encompassing the science of imaging based on piezoelectric driven probes for measuring local changes in nanoscale properties of materials and devices. Techniques like scanning tunneling microscopy, atomic force microscopy, and scanning potentiometry are becoming common tools in the production and development labs in the semiconductor industry. The author presents several examples of applications specific to the development of high temperature superconducting thin films and thin-film devices.

  5. Bias modulated scanning ion conductance microscopy.

    Science.gov (United States)

    McKelvey, Kim; Perry, David; Byers, Joshua C; Colburn, Alex W; Unwin, Patrick R

    2014-04-01

    Nanopipets are versatile tools for nanoscience, particularly when used in scanning ion conductance microscopy (SICM) to determine, in a noncontact manner, the topography of a sample. We present a new method, applying an oscillating bias between a quasi-reference counter electrode (QRCE) in the SICM nanopipet probe and a second QRCE in the bulk solution, to generate a feedback signal to control the distance between the end of a nanopipet and a surface. Both the amplitude and phase of the oscillating ion current, induced by the oscillating bias and extracted using a phase-sensitive detector, are shown to be sensitive to the probe-surface distance and are used to provide stable feedback signals. The phase signal is particularly sensitive at high frequencies of the oscillating bias (up to 30 kHz herein). This development eliminates the need to physically oscillate the probe to generate an oscillating ion current feedback signal, as needed for conventional SICM modes. Moreover, bias modulation allows a feedback signal to be generated without any net ion current flow, ensuring that any polarization of the quasi reference counter electrodes, electro-osmotic effects, and perturbations of the supporting electrolyte composition are minimized. Both feedback signals, magnitude and phase, are analyzed through approach curve measurements to different surfaces at a range of distinct frequencies and via impedance measurements at different distances from a surface. The bias modulated response is readily understood via a simple equivalent circuit model. Bias modulated (BM)-SICM is compared to conventional SICM imaging through measurements of substrates with distinct topographical features and yields equivalent results. Finally, BM-SICM with both amplitude and phase feedback is used for topographical imaging of subtle etch features in a calcite crystal surface. The 2 modes yield similar results, but phase-detection opens up the prospect of faster imaging.

  6. Comparative study of electron microscopy and scanning probe microscopy in photosynthetic research

    OpenAIRE

    MATĚNOVÁ, Martina

    2009-01-01

    The aim of this study is to compare the ability of transmission electron microscopy, scanning electron microscopy and atomic force microscopy to visualize individual protein complexes. The principle of electron microscopy and atomic force microscopy is explained. For comparision of these methods well characterized photosynthetic complexes LH1, LH2, PSI and PSII were selected.

  7. Two-photon excited ultraviolet photoluminescence of zinc oxide nanorods.

    Science.gov (United States)

    Zhu, Guangping; Xu, Chunxiang; Zhu, Jing; Lu, Changgui; Cui, Yiping; Sun, Xiaowei

    2008-11-01

    High density zinc oxide nanorods with uniform size were synthesized on (100) silicon substrate by vapor-phase transport method. The scanning electron microscopy images reveal that the nanorods have an average diameter of about 400 nm. The X-ray diffraction pattern demonstrates the wurtzite crystalline structure of the ZnO nanorods growing along [0001] direction. The single-photon excited photoluminescence presents a strong ultraviolet emission band at 394 nm and a weak visible emission band at 600 nm. When the ZnO nanorods were respectively pumped by various wavelength lasers from 520 nm to 700 nm, two-photon excited ultraviolet photoluminescence was observed. The dependence of the two-photon excited photoluminescence intensity on the excitation wavelength and power was investigated in detail.

  8. EDITORIAL: Scanning probe microscopy: a visionary development Scanning probe microscopy: a visionary development

    Science.gov (United States)

    Demming, Anna

    2013-07-01

    The development of scanning probe microscopy repositioned modern physics. When Rohrer and Binnig first used electronic tunnelling effects to image atoms and quantum states they did more than pin down theoretical hypotheses to real-world observables; the scanning tunnelling microscope fed imaginations, prompting researchers to consider new directions and possibilities [1]. As Rohrer once commented, 'We could show that you can easily manipulate or position something small in space with an accuracy of 10 pm.... When you can do that, you simply have ideas of what you can do' [2]. The development heralded a cavalry of scanning probe techniques—such as atomic force microscopy (AFM) [3-5], scanning near-field optical microscopy (SNOM) [6-8] and Kelvin probe force microscopy (KPFM) [9, 10]—that still continue to bring nanomaterials and nanoscale phenomena into fresh focus. Not long after the development of scanning tunnelling microscopy, Binnig, Quate and Gerber collaborating in California in the US published work on a new type of microscope also capable of atomic level resolution [3]. The original concept behind scanning tunnelling microscopy uses electrical conductance, which places substantial limitations on the systems that it can image. Binnig, Quate and Gerber developed the AFM to 'feel' the topology of surfaces like the needle of an old fashioned vinyl player. In this way insulators could be imaged as well. The development of a force modulation mode AFM extended the tool's reach to soft materials making images of biological samples accessible with the technique [4]. There have now been a number of demonstrations of image capture at rates that allow dynamics at the nanoscale to be tracked in real time, opening further possibilities in applications of the AFM as described in a recent review by Toshio Ando at Kanazawa University [5]. Researchers also found a way to retrieve optical information at 'super-resolution' [6, 7]. Optical microscopy provides spectral

  9. Collective electronic effects in scanning probe microscopy

    Science.gov (United States)

    Passian, Ali

    The surface plasmon dispersion relations are calculated for a metal coated dielectric probe above a dielectric half space with and without metal coating. Employing prolate spheroidal coordinate system this configuration was modeled as confocal single-sheeted hyperboloids of revolution superimposed on planar domains. The involved media are characterized by frequency dependent, spatially local dielectric functions. Due to subwavelength dimensions of the region of interest, nonretarded electrodynamics is utilized to derive exact analytical expressions describing the resonant surface modes. The dispersion relations are studied as functions of the parameter that defines the hyperboloidal boundaries of the tip and the corresponding coating, and as functions of the involved coating thicknesses. Both parallel and perpendicular polarizations are considered. The results are simulated numerically and limiting cases are discussed with comparison to the Cartesian thin foil case. Using this new type of probe-substrate configuration, the surface plasmon coupling mechanism is investigated experimentally utilizing a scanning probe microscope, and the signal strength acquired by the probe is measured as a function of the distance between the probe and the sample. This is repeated at three different wavelengths of the incident p-polarized photons used to stimulate surface plasmons in the thin metal foil. The results are compared with the theory. Utilizing the prolate spheroidal coordinate system, the related and relevant problem of the Coulomb interaction of a dielectric probe tip with a uniform field existing above a semiinfinite, homogeneous dielectric substrate was studied. This is of interest in atomic force microscopy when the sample surface is electrically charged. The induced polarization surface charge density and the field distribution at the bounding surface of the dielectric medium with the geometry of a single-sheeted hyperboloid of revolution located above the dielectric

  10. Correction of depth-induced spherical aberration for deep observation using two-photon excitation fluorescence microscopy with spatial light modulator.

    Science.gov (United States)

    Matsumoto, Naoya; Inoue, Takashi; Matsumoto, Akiyuki; Okazaki, Shigetoshi

    2015-07-01

    We demonstrate fluorescence imaging with high fluorescence intensity and depth resolution in which depth-induced spherical aberration (SA) caused by refractive-index mismatch between the medium and biological sample is corrected. To reduce the impact of SA, we incorporate a spatial light modulator into a two-photon excitation fluorescence microscope. Consequently, when fluorescent beads in epoxy resin were observed with this method of SA correction, the fluorescence signal of the observed images was ∼27 times higher and extension in the direction of the optical axes was ∼6.5 times shorter at a depth of ∼890 μm. Thus, the proposed method increases the depth observable at high resolution. Further, our results show that the method improved the fluorescence intensity of images of the fluorescent beads and the structure of a biological sample.

  11. Scanning transmission electron microscopy imaging and analysis

    CERN Document Server

    Pennycook, Stephen J

    2011-01-01

    Provides the first comprehensive treatment of the physics and applications of this mainstream technique for imaging and analysis at the atomic level Presents applications of STEM in condensed matter physics, materials science, catalysis, and nanoscience Suitable for graduate students learning microscopy, researchers wishing to utilize STEM, as well as for specialists in other areas of microscopy Edited and written by leading researchers and practitioners

  12. Automated Scanning Electron Microscopy Analysis of Sampled Aerosol

    DEFF Research Database (Denmark)

    Bluhme, Anders Brostrøm; Kling, Kirsten; Mølhave, Kristian

    development of an automated software-based analysis of aerosols using Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM) coupled with Energy-Dispersive X-ray Spectroscopy (EDS). The automated analysis will be capable of providing both detailed physical and chemical single...

  13. Quantitative imaging of fibrotic and morphological changes in liver of non-alcoholic steatohepatitis (NASH) model mice by second harmonic generation (SHG) and auto-fluorescence (AF) imaging using two-photon excitation microscopy (TPEM).

    Science.gov (United States)

    Yamamoto, Shin; Oshima, Yusuke; Saitou, Takashi; Watanabe, Takao; Miyake, Teruki; Yoshida, Osamu; Tokumoto, Yoshio; Abe, Masanori; Matsuura, Bunzo; Hiasa, Yoichi; Imamura, Takeshi

    2016-12-01

    Non-alcoholic steatohepatitis (NASH) is a common liver disorder caused by fatty liver. Because NASH is associated with fibrotic and morphological changes in liver tissue, a direct imaging technique is required for accurate staging of liver tissue. For this purpose, in this study we took advantage of two label-free optical imaging techniques, second harmonic generation (SHG) and auto-fluorescence (AF), using two-photon excitation microscopy (TPEM). Three-dimensional ex vivo imaging of tissues from NASH model mice, followed by image processing, revealed that SHG and AF are sufficient to quantitatively characterize the hepatic capsule at an early stage and parenchymal morphologies associated with liver disease progression, respectively.

  14. Exploring control parameters of two photon processes in solutions

    Indian Academy of Sciences (India)

    Debabrata Goswami; Amit Nag

    2012-01-01

    Two-photon microscopy depends extensively on the two-photon absorption cross-sections of biologically relevant chromophores. High repetition rate (HRR) lasers are essential in multiphoton microscopy for generating satisfactory signal to noise at low average powers. However, HRR lasers generate thermal distortions in samples even with the slightest single photon absorption. We use an optical chopper with HRR lasers to intermittently `blank’ irradiation and effectively minimize thermal effects to result in a femtosecond z-scan setup that precisely measures the two-photon absorption (TPA) cross-sections of chromophores. Though several experimental factors impact such TPA measurements, a systematic effort to modulate and influence TPA characteristics is yet to evolve. Here, we present the effect of several control parameters on the TPA process that are independent of chromophore characteristics for femtosecond laser pulse based measurements; and demonstrate how the femtosecond laser pulse repetition rate, chromophore environment and incident laser polarization can become effective control parameters for such nonlinear optical properties.

  15. An endolithic microbial community in dolomite rock in central Switzerland: characterization by reflection spectroscopy, pigment analyses, scanning electron microscopy, and laser scanning microscopy.

    Science.gov (United States)

    Horath, T; Neu, T R; Bachofen, R

    2006-04-01

    A community of endolithic microorganisms dominated by phototrophs was found as a distinct band a few millimeters below the surface of bare exposed dolomite rocks in the Piora Valley in the Alps. Using in situ reflectance spectroscopy, we detected chlorophyll a (Chl a), phycobilins, carotenoids, and an unknown type of bacteriochlorophyll-like pigment absorbing in vivo at about 720 nm. In cross sections, the data indicated a defined distribution of different groups of organisms perpendicular to the rock surface. High-performance liquid chromatography analyses of pigments extracted with organic solvents confirmed the presence of two types of bacteriochlorophylls besides chlorophylls and various carotenoids. Spherical organisms of varying sizes and small filaments were observed in situ with scanning electron microscopy and confocal laser scanning microscopy (one- and two-photon technique). The latter allowed visualization of the distribution of phototrophic microorganisms by the autofluorescence of their pigments within the rock. Coccoid cyanobacteria of various sizes predominated over filamentous ones. Application of fluorescence-labeled lectins demonstrated that most cyanobacteria were embedded in an exopolymeric matrix. Nucleic acid stains revealed a wide distribution of small heterotrophs. Some biological structures emitting a green autofluorescence remain to be identified.

  16. Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tong Yongpeng [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China)], E-mail: yongpengt@yahoo.com.cn; Li Changming [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Liang Feng [Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen Jianmin [Shenzhen Municipal Hospital for Chronic Disease Control and Prevention, Guangdong 518020 (China); Zhang Hong; Liu Guoqing; Sun Huibin [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China); Luong, John H.T. [Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, H4P 2R2 (Canada)

    2008-12-15

    Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al{sub 2}O{sub 3} and TiO{sub 2}) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl{sub 2}) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al{sub 2}O{sub 3} and TiO{sub 2} nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe{sub 2}O{sub 3} nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

  17. Combined frequency modulated atomic force microscopy and scanning tunneling microscopy detection for multi-tip scanning probe microscopy applications

    Science.gov (United States)

    Morawski, Ireneusz; Spiegelberg, Richard; Korte, Stefan; Voigtländer, Bert

    2015-12-01

    A method which allows scanning tunneling microscopy (STM) tip biasing independent of the sample bias during frequency modulated atomic force microscopy (AFM) operation is presented. The AFM sensor is supplied by an electronic circuit combining both a frequency shift signal and a tunneling current signal by means of an inductive coupling. This solution enables a control of the tip potential independent of the sample potential. Individual tip biasing is specifically important in order to implement multi-tip STM/AFM applications. An extensional quartz sensor (needle sensor) with a conductive tip is applied to record simultaneously topography and conductivity of the sample. The high resonance frequency of the needle sensor (1 MHz) allows scanning of a large area of the surface being investigated in a reasonably short time. A recipe for the amplitude calibration which is based only on the frequency shift signal and does not require the tip being in contact is presented. Additionally, we show spectral measurements of the mechanical vibration noise of the scanning system used in the investigations.

  18. Characterization of Biopolymer Surfaces Using Scanning Microscopies

    Science.gov (United States)

    1989-11-15

    microscopic images of M’, Frommer JE, Foster JS (1989) adsorbed serum albumin on highly oriented Contrast mechanism for resolving organic pyrolyti: graphite J...Colloid Xncerface molecules with tunneling microscopy .re. 1Z:, 650-653. rature ._1, 137-139. Foster JS, Frommer JE (1938) Stemmer A, Rechelt R

  19. SCANNING TUNNELING MICROSCOPY STUDIES ON OPTICAL DISC

    Institute of Scientific and Technical Information of China (English)

    徐磊; 顾冬红; 等

    1994-01-01

    Scanning tunneling microscope(STM) is used to investigate the optical dise.The areas with and without data stampers are all observedcarefully.Three-dimensional images of the disc surface clearly demonstrate the period.depth of the grooves and the shape of data stampers.Some phenomena of STM imaging are also discussed.

  20. Automated spherical aberration correction in scanning confocal microscopy

    NARCIS (Netherlands)

    Yoo, H.W.; Royen, M.E.; van Cappellen, W.A.; Houtsmuller, A.B.; Verhaegen, M.H.G.; Schitter, G.

    2014-01-01

    Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy

  1. Simultaneous two-photon imaging and photo-stimulation with structured light illumination.

    Science.gov (United States)

    Dal Maschio, Marco; Difato, Francesco; Beltramo, Riccardo; Blau, Axel; Benfenati, Fabio; Fellin, Tommaso

    2010-08-30

    Holographic microscopy is increasingly recognized as a promising tool for the study of the central nervous system. Here we present a "holographic module", a simple optical path that can be combined with commercial scanheads for simultaneous imaging and uncaging with structured two-photon light. The present microscope is coupled to two independently tunable lasers and has two principal configurations: holographic imaging combined with galvo-steered uncaging and holographic uncaging combined with conventional scanning imaging. We applied this flexible system for simultaneous two-photon imaging and photostimulation of neuronal cells with complex light patterns, opening new perspectives for the study of brain function in situ and in vivo.

  2. Full information acquisition in scanning probe microscopy and spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jesse, Stephen; Belianinov, Alex; Kalinin, Sergei V.; Somnath, Suhas

    2017-04-04

    Apparatus and methods are described for scanning probe microscopy and spectroscopy based on acquisition of full probe response. The full probe response contains valuable information about the probe-sample interaction that is lost in traditional scanning probe microscopy and spectroscopy methods. The full probe response is analyzed post data acquisition using fast Fourier transform and adaptive filtering, as well as multivariate analysis. The full response data is further compressed to retain only statistically significant components before being permanently stored.

  3. Non-linear imaging and characterization of atherosclerotic arterial tissue using combined two photon fluorescence, second-harmonic generation and CARS microscopy

    Science.gov (United States)

    Cicchi, Riccardo; Matthäus, Christian; Meyer, Tobias; Lattermann, Annika; Dietzek, Benjamin; Brehm, Bernhard R.; Popp, Jürgen; Pavone, Francesco S.

    2014-02-01

    Atherosclerosis is among the most widespread cardiovascular diseases and one of the leading cause of death in the Western World. Characterization of arterial tissue in atherosclerotic condition is extremely interesting from the diagnostic point of view. Routinely used diagnostic methods, such as histopathological examination, are limited to morphological analysis of the examined tissues, whereas an exhaustive characterization requires a morpho-functional approach. Multimodal non-linear microscopy has the potential to bridge this gap by providing morpho-functional information on the examined tissues in a label-free way. Here we employed multiple non-linear microscopy techniques, including CARS, TPF, and SHG to provide intrinsic optical contrast from various tissue components in both arterial wall and atherosclerotic plaques. CARS and TPF microscopy were used to respectively image lipid depositions within plaques and elastin in the arterial wall. Cholesterol deposition in the lumen and collagen in the arterial wall were selectively imaged by SHG microscopy and distinguished by forward-backward SHG ratio. Image pattern analysis allowed characterizing collagen organization in different tissue regions. Different values of fiber mean size, distribution and anisotropy are calculated for lumen and media prospectively allowing for automated classification of atherosclerotic lesions. The presented method represents a promising diagnostic tool for evaluating atherosclerotic tissue and has the potential to find a stable place in clinical setting as well as to be applied in vivo in the near future.

  4. Reducing Photobleaching in STED Microscopy with Higher Scanning Speed

    CERN Document Server

    Wu, Yong

    2014-01-01

    Photobleaching is a major limitation of super-resolution STED microscopy. We show that the photobleaching rate in STED microscopy is slowed down by scanning with a higher linear speed, enabled by the large field of view in our custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching is more remarkable at higher levels of depletion laser irradiance. With a depletion irradiance of 0.4 GW/cm$^2$ (time average), we were able to slow down the photobleaching of the Atto 647N dye by 80% with 8-fold faster scanning. Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data qualitatively agree with a theoretical model. Our results encourage further increasing linear scanning speed for photobleaching reduction in STED microscopy.

  5. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    NARCIS (Netherlands)

    Yoo, H.W.

    2015-01-01

    Confocal laser scanning microscopy (CLSM) is considered as one of the major advancements in microscopy in the last century and is widely accepted as a 3D fluorescence imaging tool for biological studies. For the emerging biological questions CLSM requires fast imaging to detect rapid biological proc

  6. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    NARCIS (Netherlands)

    Yoo, H.W.

    2015-01-01

    Confocal laser scanning microscopy (CLSM) is considered as one of the major advancements in microscopy in the last century and is widely accepted as a 3D fluorescence imaging tool for biological studies. For the emerging biological questions CLSM requires fast imaging to detect rapid biological

  7. Super-resolution scanning laser microscopy through virtually structured detection

    OpenAIRE

    Lu, Rong-Wen; Wang, Ben-Quan; Zhang, Qiu-Xiang; Yao, Xin-Cheng

    2013-01-01

    High resolution microscopy is essential for advanced study of biological structures and accurate diagnosis of medical diseases. The spatial resolution of conventional microscopes is light diffraction limited. Structured illumination has been extensively explored to break the diffraction limit in wide field light microscopy. However, deployable application of the structured illumination in scanning laser microscopy is challenging due to the complexity of the illumination system and possible ph...

  8. Information Acquisition & Processing in Scanning Probe Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kalinin, Sergei V [ORNL; Jesse, Stephen [ORNL; Proksch, Roger [Asylum Research, Santa Barbara, CA

    2008-01-01

    Much of the imaging and spectroscopy capabilities of the existing 20,000+ scanning probe microscopes worldwide relies on specialized data processing that links the microsecond (and sometimes faster) time scale of cantilever motion to the millisecond (and sometimes slower) time scale of image acquisition and feedback. In most SPMs, the cantilever is excited to oscillate sinusoidally and the time-averaged amplitude and/or phase are used as imaging or control signals. Traditionally, the step of converting the rapid motion of the cantilever into an amplitude or phase is performed by phase sensitive homodyne or phase-locked loop detection. The emergence of fast configurable data processing electronics in last several years has allowed the development of non-sinusoidal data acquisition and processing methods. Here, we briefly review the principles and limitations of phase sensitive detectors and discuss some of the emergent technologies based on rapid spectroscopic measurements in frequency- and time domains.

  9. Scanning electron microscopy of bacteria Tetrasphaera duodecadis.

    Science.gov (United States)

    Arroyo, E; Enríquez, L; Sánchez, A; Ovalle, M; Olivas, A

    2014-01-01

    This study reports the characterization of the Tetrasphaera duodecadis bacteria and the techniques used therein. In order to evaluate the morphological characteristics of the T. duodecadis bacteria scanning electron microscope (SEM) was used throughout its different growth stages. These microorganisms were grown in vitamin B12 broths with 1% tryptone, 0.2% yeast extract, and 0.1% glucose. The turbidimetric method was employed for the determination of bacterial concentration and growth curve. The SEM results show small agglomerates of 0.8 ± 0.05 µm during the lag phase, and rod-like shapes during the exponential phase with similar shapes in the stationary phase.

  10. Scanning electron microscopy of Dalkon Shield tails.

    Science.gov (United States)

    Bank, H L; Williamson, H O

    1983-09-01

    Scanning electron micrographs of Dalkon Shield tails removed from asymptomatic patients show a variety of microbes and debris throughout their entire length. Apparently, even in undamaged tails, bacterial flora thrive in the protein-rich environment within the multifilament tail. The presence of microbes in the portion of the tail beyond the double knot indicates that an alternative mechanism of microbial transport can occur. Since transient endometritis often occurs immediately after insertion of intrauterine devices, microbes may come in contact with both exposed ends of the multifilament tail and be drawn into the tail by capillary action from the uterine environment down the tail toward the double knot as well as upward from the vagina. Such microorganisms could serve as an inoculum for infection.

  11. Scanning SQUID microscopy for magnetic flux systems

    CERN Document Server

    Suzuki, J I; Hata, Y

    2003-01-01

    Recently, vortices confined into micro-scale superconductors with shapes like a disk, triangle, square, etc., have attracted much attention because of the quantum phase transition of the self-organized vortex arrangement occurring within such geometrical constraints. Such a transition can be observed using a scanning SQUID microscope with high spatial resolution. We have successfully improved spatial resolution by incorporating a microfabrication technique that reduces both the size of the pick-up coil of the micro DC-SQUID and the standoff distance between the pick-up coil and the sample surface. Using this microscope, we have studied vortex arrangements in micro-scale superconductors made of Nb and YBa sub 2 Cu sub 3 O sub 7 sub - subdelta films with various sizes and geometrical shapes. A peculiar oscillating behavior of diamagnetic magnetization corresponding to the particular vortex state was observed.

  12. Preparation of platinum/iridium scanning probe microscopy tips

    DEFF Research Database (Denmark)

    Sørensen, Alexis Hammer; Hvid, U.; Mortensen, M.W.

    1999-01-01

    for the production of sharp tips. After being etched the tips are ready for use in scanning tunneling microscopes, or they may be bent to form integrated tip/cantilever systems in ordinary commercial atomic force microscopes, being applicable as tapping mode tips and as electrostatic force microscopy tips. ©1999......We report on the development of an etching setup for use in the preparation of platinum/iridium tips for atomic force microscopy and scanning electrostatic force microscopy as well as scanning tunneling microscopy. The etching process is based on a two step electrochemical procedure. The first step....... This mechanism is based on the formation of oxygen and hydrogen at the platinum/iridium electrode when the potential is above the dissociation potential of water (~ 1.23 V) and storage of these products interstitially in the outer layers of the platinum wire. This leads to "microexplosions" that detach fragments...

  13. Two photon physics. Personal recollection

    CERN Document Server

    Ginzburg, Ilya F

    2015-01-01

    The term two--photon processes is used for the reactions in which some system of particles is produced in collision of two photons, either real or virtual. In the study of these processes our main goal was to suggest approach, allowing to extract from the data information on proper two--photon process separating it from mechanism which responsible for the production of photons. Here I present my view for history of two--photon physics. I don't try to give complete review, concentrating mainly on works of our team (which cover essential part of the topic) and some colleagues. My citation is strongly incomplete. I cite here only papers which were essential in our understanding of the problems. The choice of presented details is the result of my discussions with Gleb Kotkin and Valery Serbo. 1. Prehistory. 2. Two photon processes at e^+e^- colliders. 3. Photon colliders. 4. Notes on physical program.

  14. Open Source Scanning Probe Microscopy Control Software Package Gxsm

    Energy Technology Data Exchange (ETDEWEB)

    Zahl P.; Wagner, T.; Moller, R.; Klust, A.

    2009-08-10

    Gxsm is a full featured and modern scanning probe microscopy (SPM) software. It can be used for powerful multidimensional image/data processing, analysis, and visualization. Connected toan instrument, it is operating many different avors of SPM, e.g., scanning tunneling microscopy(STM) and atomic force microscopy (AFM) or in general two-dimensional multi channel data acquisition instruments. The Gxsm core can handle different data types, e.g., integer and oating point numbers. An easily extendable plug-in architecture provides many image analysis and manipulation functions. A digital signal processor (DSP) subsystem runs the feedback loop, generates the scanning signals and acquires the data during SPM measurements. The programmable Gxsm vector probe engine performs virtually any thinkable spectroscopy and manipulation task, such as scanning tunneling spectroscopy (STS) or tip formation. The Gxsm software is released under the GNU general public license (GPL) and can be obtained via the Internet.

  15. Vector sensor for scanning SQUID microscopy

    Science.gov (United States)

    Dang, Vu The; Toji, Masaki; Thanh Huy, Ho; Miyajima, Shigeyuki; Shishido, Hiroaki; Hidaka, Mutsuo; Hayashi, Masahiko; Ishida, Takekazu

    2017-07-01

    We plan to build a novel 3-dimensional (3D) scanning SQUID microscope with high sensitivity and high spatial resolution. In the system, a vector sensor consists of three SQUID sensors and three pick-up coils realized on a single chip. Three pick-up coils are configured in orthogonal with each other to measure the magnetic field vector of X, Y, Z components. We fabricated some SQUID chips with one uniaxial pick-up coil or three vector pick-up coils and carried out fundamental measurements to reveal the basic characteristics. Josephson junctions (JJs) of sensors are designed to have the critical current density J c of 320 A/cm2, and the critical current I c becomes 12.5 μA for the 2.2μm × 2.2μm JJ. We carefully positioned the three pickup coils so as to keep them at the same height at the centers of all three X, Y and Z coils. This can be done by arranging them along single line parallel to a sample surface. With the aid of multilayer technology of Nb-based fabrication, we attempted to reduce an inner diameter of the pickup coils to enhance both sensitivity and spatial resolution. The method for improving a spatial resolution of a local magnetic field image is to employ an XYZ piezo-driven scanner for controlling the positions of the pick-up coils. The fundamental characteristics of our SQUID sensors confirmed the proper operation of our SQUID sensors and found a good agreement with our design parameters.

  16. HelioScan: a software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility.

    Science.gov (United States)

    Langer, Dominik; van 't Hoff, Marcel; Keller, Andreas J; Nagaraja, Chetan; Pfäffli, Oliver A; Göldi, Maurice; Kasper, Hansjörg; Helmchen, Fritjof

    2013-04-30

    Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community.

  17. Electron-beam-assisted Scanning Tunneling Microscopy Of Insulating Surfaces

    CERN Document Server

    Bullock, E T

    2000-01-01

    Insulating materials are widely used in electronic devices. Bulk insulators and insulating films pose unique challenges for high resolution study since most commonly used charged particle surface analysis techniques are incompatible with insulating surfaces and materials. A, method of performing scanning tunneling microscopy (STM) on insulating surfaces has been investigated. The method is referred to as electron-beam assisted scanning tunneling microscopy (e-BASTM). It is proposed that by coupling the STM and the scanning electron microscopy (SEM) as one integrated device, that insulating materials may be studied, obtaining both high spatial resolution, and topographic and electronic resolution. The premise of the technique is based on two physical consequences of the interaction of an energetic electron beam (PE) with a material. First, when an electron beam is incident upon a material, low level material electrons are excited into conduction band states. For insulators, with very high secondary electron yi...

  18. Optomechatronics Design and Control for Confocal Laser Scanning Microscopy

    OpenAIRE

    Yoo, H W

    2015-01-01

    Confocal laser scanning microscopy (CLSM) is considered as one of the major advancements in microscopy in the last century and is widely accepted as a 3D fluorescence imaging tool for biological studies. For the emerging biological questions CLSM requires fast imaging to detect rapid biological processes and aberration-corrected imaging to localize the targeted biomolecule precisely through optical disturbances by specimen. In this thesis, optomechatronics design and control are discussed for...

  19. System and method for compressive scanning electron microscopy

    Science.gov (United States)

    Reed, Bryan W

    2015-01-13

    A scanning transmission electron microscopy (STEM) system is disclosed. The system may make use of an electron beam scanning system configured to generate a plurality of electron beam scans over substantially an entire sample, with each scan varying in electron-illumination intensity over a course of the scan. A signal acquisition system may be used for obtaining at least one of an image, a diffraction pattern, or a spectrum from the scans, the image, diffraction pattern, or spectrum representing only information from at least one of a select subplurality or linear combination of all pixel locations comprising the image. A dataset may be produced from the information. A subsystem may be used for mathematically analyzing the dataset to predict actual information that would have been produced by each pixel location of the image.

  20. Transmissive liquid-crystal device correcting primary coma aberration and astigmatism in laser scanning microscopy

    Science.gov (United States)

    Tanabe, Ayano; Hibi, Terumasa; Ipponjima, Sari; Matsumoto, Kenji; Yokoyama, Masafumi; Kurihara, Makoto; Hashimoto, Nobuyuki; Nemoto, Tomomi

    2016-03-01

    Laser scanning microscopy allows 3D cross-sectional imaging inside biospecimens. However, certain aberrations produced can degrade the quality of the resulting images. We previously reported a transmissive liquid-crystal device that could compensate for the predominant spherical aberrations during the observations, particularly in deep regions of the samples. The device, inserted between the objective lens and the microscope revolver, improved the image quality of fixed-mouse-brain slices that were observed using two-photon excitation laser scanning microscopy, which was originally degraded by spherical aberration. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism, motivated by the fact that these asymmetric aberrations can also often considerably deteriorate image quality, even near the sample surface. The device's performance was evaluated by observing fluorescent beads using single-photon excitation laser scanning microscopy. The fluorescence intensity in the image of the bead under a cover slip tilted in the y-direction was increased by 1.5 times after correction by the device. Furthermore, the y- and z-widths of the imaged bead were reduced to 66% and 65%, respectively. On the other hand, for the imaged bead sucked into a glass capillary in the longitudinal x-direction, correction with the device increased the fluorescence intensity by 2.2 times compared to that of the aberrated image. In addition, the x-, y-, and z-widths of the bead image were reduced to 75%, 53%, and 40%, respectively. Our device successfully corrected several asymmetric aberrations to improve the fluorescent signal and spatial resolution, and might be useful for observing various biospecimens.

  1. A dynamic scanning method based on signal-statistics for scanning electron microscopy.

    Science.gov (United States)

    Timischl, F

    2014-01-01

    A novel dynamic scanning method for noise reduction in scanning electron microscopy and related applications is presented. The scanning method dynamically adjusts the scanning speed of the electron beam depending on the statistical behavior of the detector signal and gives SEM images with uniform and predefined standard deviation, independent of the signal value itself. In the case of partially saturated images, the proposed method decreases image acquisition time without sacrificing image quality. The effectiveness of the proposed method is shown and compared to the conventional scanning method and median filtering using numerical simulations.

  2. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum's magnetosome chains.

    Science.gov (United States)

    Keutner, Christoph; von Bohlen, Alex; Berges, Ulf; Espeter, Philipp; Schneider, Claus M; Westphal, Carsten

    2014-10-01

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  3. Microstress contrast in scanning electron acoustic microscopy of ceramics

    Science.gov (United States)

    Cantrell, John H.; Qian, Menglu

    1991-01-01

    A mathematical model of image contrast in scanning electron acoustic microscopy (SEAM) due to the effect of residual stresses in materials is presented. It is found that in regions near the ends of the radial cracks induced by Vickers indentation the SEAM micrographs reveal a rather large variation of the acoustic output signal.

  4. Preparation of Articular Cartilage Specimens for Scanning Electron Microscopy.

    Science.gov (United States)

    Stupina, T A

    2016-08-01

    We developed and adapted a technology for preparation of articular cartilage specimens for scanning electron microscopy. The method includes prefixation processing, fixation, washing, and dehydration of articular cartilage specimens with subsequent treatment in camphene and air-drying. The technological result consists in prevention of deformation of the articular cartilage structures. The method is simpler and cheaper than the known technologies.

  5. Band excitation method applicable to scanning probe microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jesse, Stephen; Kalinin, Sergei V.

    2017-01-03

    Scanning probe microscopy may include a method for generating a band excitation (BE) signal and simultaneously exciting a probe at a plurality of frequencies within a predetermined frequency band based on the excitation signal. A response of the probe is measured across a subset of frequencies of the predetermined frequency band and the excitation signal is adjusted based on the measured response.

  6. CONFOCAL LASER SCANNING MICROSCOPY OF RAT FOLLICLE DEVELOPMENT

    Science.gov (United States)

    This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pr...

  7. Ultrafast terahertz scanning tunneling microscopy with atomic resolution

    DEFF Research Database (Denmark)

    Jelic, Vedran; Iwaszczuk, Krzysztof; Nguyen, Peter H.

    2016-01-01

    We demonstrate that ultrafast terahertz scanning tunneling microscopy (THz-STM) can probe single atoms on a silicon surface with simultaneous sub-nanometer and sub-picosecond spatio-temporal resolution. THz-STM is established as a new technique for exploring high-field non-equilibrium tunneling...

  8. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1998-01-01

    Scanning Electron Microscopy provides a description of the physics of electron-probe formation and of electron-specimen interations The different imaging and analytical modes using secondary and backscattered electrons, electron-beam-induced currents, X-ray and Auger electrons, electron channelling effects, and cathodoluminescence are discussed to evaluate specific contrasts and to obtain quantitative information

  9. Scanning tunneling microscopy III theory of STM and related scanning probe methods

    CERN Document Server

    Güntherodt, Hans-Joachim

    1996-01-01

    Scanning Tunneling Microscopy III provides a unique introduction to the theoretical foundations of scanning tunneling microscopy and related scanning probe methods. The different theoretical concepts developed in the past are outlined, and the implications of the theoretical results for the interpretation of experimental data are discussed in detail. Therefore, this book serves as a most useful guide for experimentalists as well as for theoreticians working in the filed of local probe methods. In this second edition the text has been updated and new methods are discussed.

  10. Writing silica structures in liquid with scanning transmission electron microscopy.

    Science.gov (United States)

    van de Put, Marcel W P; Carcouët, Camille C M C; Bomans, Paul H H; Friedrich, Heiner; de Jonge, Niels; Sommerdijk, Nico A J M

    2015-02-04

    Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position.

  11. Cryo scanning electron microscopy of Plasmodium falciparum-infected erythrocytes.

    Science.gov (United States)

    Hempel, Casper

    2017-07-01

    Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite-stage parasites and schizonts introduce numerous small protrusions on the erythrocyte surface, called knobs. Current methods for studying these knobs include atomic force microscopy and electron microscopy. Standard electron microscopy methods rely on chemical fixation and dehydration modifying cell size. Here, a novel method is presented using rapid freezing and scanning electron microscopy under cryogenic conditions allowing for high resolution and magnification of erythrocytes. This novel technique can be used for precise estimates of knob density and for studies on cytoadhesion. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  12. Scanning near field microwave microscopy based on an active resonator

    Science.gov (United States)

    Qureshi, Naser; Kolokoltsev, Oleg; Ordonez-Romero, Cesar Leonardo

    2014-03-01

    A large number of recent implementations of near field scanning microwave microscopy (NFSMM) have been based on the perturbation of a resonant cavity connected to a sharp scanning probe. In this work we present results from an alternative approach: the perturbation of a microwave source connected to a scanning tip. Based on a yittrium iron garnet (YIG) cavity ring resonator this scanning probe system has a quality factor greater than 106, which allows us to detect very small frequency shifts, which translates to a very high sensitivity in sample impedance measurements. Using a selection of representative semiconductor, metal and biological samples we show how this approach leads to unusually high sensitivity and spatial resolution. Work supported by a grant from PAPIIT, UNAM 104513.

  13. Scanning conductance microscopy investigations on fixed human chromosomes

    DEFF Research Database (Denmark)

    Clausen, Casper Hyttel; Lange, Jacob Moresco; Jensen, Linda Boye;

    2008-01-01

    Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device...... optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value,for the dielectric constant of different human chromosomes....

  14. Insights into the regulation of transcription by scanning force microscopy.

    Science.gov (United States)

    Dame, R T; Wyman, C; Goosen, N

    2003-12-01

    The scanning force microscope (SFM) is a valuable tool for the structural analysis of complexes between protein(s) and DNA. In recent years the application of scanning force microscopy to the field of transcription regulation has been reported in numerous studies. Using this technique, novel insights could be obtained into the architecture and dynamics of complexes, which are relevant to the transcription process and the mechanisms by which this process is regulated. In this article an overview is given of SFM studies addressing, in particular, topics in the field of transcription in prokaryotic organisms.

  15. Nanochannel alignment analysis by scanning transmission ion microscopy

    DEFF Research Database (Denmark)

    Rajta, I.; Gál, G.A.B.; Szilasi, S.Z.

    2010-01-01

    In this paper a study on the ion transmission ratio of a nanoporous alumina sample is presented. The sample was investigated by scanning transmission ion microscopy (STIM) with different beam sizes. The hexagonally close-packed AlO nanocapillary array, realized as a suspended membrane of 15 νm...... domain. However, for larger beam scanning areas (sampling multiple domains) the transmission ratio was reduced to 5%. The STIM analysis over an area larger than the typical domain size revealed an overall capillary angular spread of ∼ 2°....

  16. Scanning microscopies of superconductors at very low temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Crespo, V.; Maldonado, A.; Galvis, J.A.; Kulkarni, P. [Laboratorio de Bajas Temperaturas, Departamento de Fisica de la Materia Condensada, Instituto de Ciencia de Materiales Nicolas Cabrera, Universidad Autonoma de Madrid, E-28049 Madrid (Spain); Guillamon, I. [Laboratorio de Bajas Temperaturas, Departamento de Fisica de la Materia Condensada, Instituto de Ciencia de Materiales Nicolas Cabrera, Universidad Autonoma de Madrid, E-28049 Madrid (Spain); H.H. Wills Physics Laboratory, University of Bristol, Tyndall Avenue, Bristol BS8 1TL (United Kingdom); Rodrigo, J.G. [Laboratorio de Bajas Temperaturas, Departamento de Fisica de la Materia Condensada, Instituto de Ciencia de Materiales Nicolas Cabrera, Universidad Autonoma de Madrid, E-28049 Madrid (Spain); Suderow, H., E-mail: hermann.suderow@uam.es [Laboratorio de Bajas Temperaturas, Departamento de Fisica de la Materia Condensada, Instituto de Ciencia de Materiales Nicolas Cabrera, Universidad Autonoma de Madrid, E-28049 Madrid (Spain); Vieira, S. [Laboratorio de Bajas Temperaturas, Departamento de Fisica de la Materia Condensada, Instituto de Ciencia de Materiales Nicolas Cabrera, Universidad Autonoma de Madrid, E-28049 Madrid (Spain); Banerjee, S. [Department of Physics, Indian Institute of Technology, Kanpur 208 016, UP (India); Rodiere, P. [Institut Neel, CNRS/UJF, 25 Avenue des Martyrs, B.P. 166, 38042 Grenoble Cedex 9 (France)

    2012-09-15

    We discuss basics of Scanning Tunneling Microscopy and Spectroscopy (STM/S) of the superconducting state with normal and superconducting tips. We present a new method to measure the local variations in the Andreev reflection amplitude between a superconducting tip and the sample. This method is termed Scanning Andreev Reflection Spectroscopy (SAS). We also briefly discuss vortex imaging with STM/S under an applied current through the sample, and show the vortex lattice as a function of the angle between the magnetic field and sample's surface.

  17. Phase modulation mode of scanning ion conductance microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Zhang, Changlin [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Lianqing, E-mail: lqliu@sia.cn, E-mail: gli@engr.pitt.edu; Wang, Yuechao; Yang, Yang [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Li, Guangyong, E-mail: lqliu@sia.cn, E-mail: gli@engr.pitt.edu [Department of Electrical and Computer Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 (United States)

    2014-08-04

    This Letter reports a phase modulation (PM) mode of scanning ion conductance microscopy. In this mode, an AC current is directly generated by an AC voltage between the electrodes. The portion of the AC current in phase with the AC voltage, which is the current through the resistance path, is modulated by the tip-sample distance. It can be used as the input of feedback control to drive the scanner in Z direction. The PM mode, taking the advantages of both DC mode and traditional AC mode, is less prone to electronic noise and DC drift but maintains high scanning speed. The effectiveness of the PM mode has been proven by experiments.

  18. Development of first ever scanning probe microscopy capabilities for plutonium

    Science.gov (United States)

    Beaux, Miles F.; Cordoba, Miguel Santiago; Zocco, Adam T.; Vodnik, Douglas R.; Ramos, Michael; Richmond, Scott; Moore, David P.; Venhaus, Thomas J.; Joyce, Stephen A.; Usov, Igor O.

    2017-04-01

    Scanning probe microscopy capabilities have been developed for plutonium and its derivative compounds. Specifically, a scanning tunneling microscope and an atomic force microscope housed in an ultra-high vacuum system and an inert atmosphere glove box, respectively, were prepared for the introduction of small non-dispersible δ-Pu coupons. Experimental details, procedures, and preliminary imaging of δ-Pu coupons are presented to demonstrate the functionality of these new capabilities. These first of a kind capabilities for plutonium represent a significant step forward in the ability to characterize and understand plutonium surfaces with high spatial resolution.

  19. Axial scanning in confocal microscopy employing adaptive lenses (CAL).

    Science.gov (United States)

    Koukourakis, Nektarios; Finkeldey, Markus; Stürmer, Moritz; Leithold, Christoph; Gerhardt, Nils C; Hofmann, Martin R; Wallrabe, Ulrike; Czarske, Jürgen W; Fischer, Andreas

    2014-03-10

    In this paper we analyze the capability of adaptive lenses to replace mechanical axial scanning in confocal microscopy. The adaptive approach promises to achieve high scan rates in a rather simple implementation. This may open up new applications in biomedical imaging or surface analysis in micro- and nanoelectronics, where currently the axial scan rates and the flexibility at the scan process are the limiting factors. The results show that fast and adaptive axial scanning is possible using electrically tunable lenses but the performance degrades during the scan. This is due to defocus and spherical aberrations introduced to the system by tuning of the adaptive lens. These detune the observation plane away from the best focus which strongly deteriorates the axial resolution by a factor of ~2.4. Introducing balancing aberrations allows addressing these influences. The presented approach is based on the employment of a second adaptive lens, located in the detection path. It enables shifting the observation plane back to the best focus position and thus creating axial scans with homogeneous axial resolution. We present simulated and experimental proof-of-principle results.

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Focused ion beam scanning electron microscopy in biology.

    Science.gov (United States)

    Kizilyaprak, C; Daraspe, J; Humbel, B M

    2014-06-01

    Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB-SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three-dimensional data, FIB-SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block-face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo-) transmission electron microscopy. Here, we will present an overview of the development of FIB-SEM and discuss a few points about sample preparation and imaging.

  2. Higgs Decay to Two Photons

    OpenAIRE

    Marciano, William J.; Zhang, Cen; Willenbrock, Scott

    2011-01-01

    The amplitude for Higgs decay to two photons is calculated in renormalizable and unitary gauges using dimensional regularization at intermediate steps. The result is finite, gauge independent, and in agreement with previously published results. The large Higgs mass limit is examined using the Goldstone-boson equivalence theorem as a check on the use of dimensional regularization and to explain the absence of decoupling.

  3. Applications of scanning probe microscopy in intrinsically conducting polymer research

    Institute of Scientific and Technical Information of China (English)

    YANG Tao; NIU Li; LI Zhuang; DONG Shaojun

    2007-01-01

    The applications of scanning probe microscopy(SPM)in intrinsically conducting polymer research is briefly reviewed,including morphology observation,nanofabrication,microcosmic electrical property measurements,electrochemistry researches,in-situ measurements of film thickness change,and so on.At the same time,some important variations of SPM and the related techniques are briefly introduced.Finally,the future development of SPM in the study of intrinsically conducting polymers is prospected.

  4. SCANNING ELECTRON MICROSCOPY STUDY OF FILLED SILICONE RUBBER

    Institute of Scientific and Technical Information of China (English)

    LI Yufu; YANG Qiyun; LI Guangliang

    1988-01-01

    The fracture surfaces of a number of silicone vulcanizates were investigated by the use of scanning electron microscopy (SEM). It was found that the difference in the presence and absence of filler, the variation of its surface modification as well as the history of thermal aging of the vulcanizates, all of these factors made difference in surface morphology of the fractured surface. This was correlated with the strength of the vulcanizates. The reinforcing effect of filler and the process of fracture were discussed.

  5. Single-nanoparticle-terminated tips for scanning probe microscopy.

    Science.gov (United States)

    Vakarelski, Ivan U; Higashitani, Ko

    2006-03-28

    We have developed a wet-chemistry procedure to attach a 10-40 nm colloidal gold nanoparticle to the top of a scanning probe microscopy (SPM) probe tip, making experiments of single nanoparticle interaction possible. This procedure of particle attachment is flexible and can be modified to attach nanoparticles of different kinds and sizes. The single-nanoparticle-terminated tips also have potential in various other applications, such as probes of enhanced sensitivity for optical and magnetic modes SPM.

  6. Scanning gate microscopy of ultra clean carbon nanotube quantum dots

    OpenAIRE

    Xue, Jiamin; Dhall, Rohan; Cronin, Stephen B.; LeRoy, Brian J.

    2015-01-01

    We perform scanning gate microscopy on individual suspended carbon nanotube quantum dots. The size and position of the quantum dots can be visually identified from the concentric high conductance rings. For the ultra clean devices used in this study, two new effects are clearly identified. Electrostatic screening creates non-overlapping multiple sets of Coulomb rings from a single quantum dot. In double quantum dots, by changing the tip voltage, the interactions between the quantum dots can b...

  7. Active nanocharacterization of nanofunctional materials by scanning tunneling microscopy

    OpenAIRE

    FUJITA, Daisuke; Sagisaka, Keisuke

    2008-01-01

    Recent developments in the application of scanning tunneling microscopy (STM) to nanofabrication and nanocharacterization are reviewed. The main focus of this paper is to outline techniques for depositing and manipulating nanometer-scale structures using STM tips. Firstly, the transfer of STM tip material through the application of voltage pulses is introduced. The highly reproducible fabrication of metallic silver nanodots and nanowires is discussed. The mechanism is thought to be spontaneou...

  8. Abrasion of 6 dentifrices measured by vertical scanning interference microscopy

    Science.gov (United States)

    PASCARETTI-GRIZON, Florence; MABILLEAU, Guillaume; CHAPPARD, Daniel

    2013-01-01

    Objectives The abrasion of dentifrices is well recognized to eliminate the dental plaque. The aims of this study were to characterize the abrasive powders of 6 dentifrices (3 toothpastes and 3 toothpowders) and to measure the abrasion on a test surface by Vertical Scanning Interference microscopy (VSI). Material and Methods Bright field and polarization microscopy were used to identify the abrasive particles on the crude dentifrices and after prolonged washes. Scanning electron microscopy and microanalysis characterized the shape and nature of the particles. Standardized and polished blocks of poly(methylmethacrylate) were brushed with a commercial electric toothbrush with the dentifrices. VSI quantified the mean roughness (Ra) and illustrated in 3D the abraded areas. Results Toothpastes induced a limited abrasion. Toothpowders induced a significantly higher roughness linked to the size of the abrasive particles. One powder (Gencix® produced a high abrasion when used with a standard testing weight. However, the powder is based on pumice particles covered by a plant homogenate that readily dissolves in water. When used in the same volume, or after dispersion in water, Ra was markedly reduced. Conclusion Light and electron microscopy characterize the abrasive particles and VSI is a new tool allowing the analysis of large surface of abraded materials. PMID:24212995

  9. Confocal laser scanning microscopy image correlation for nanoparticle flow velocimetry

    CERN Document Server

    Jun, Brian; Yang, Haisheng; Main, Russell; Vlachos, Pavlos

    2016-01-01

    We present a new particle image correlation technique for resolving nanoparticle flow velocity using confocal laser scanning microscopy (CLSM). The two primary issues that complicate nanoparticle scanning laser image correlation (SLIC) based velocimetry are (1) the use of diffusion dominated nanoparticles as flow tracers, which introduce a random decorrelating error into the velocity estimate, and (2) the effects of the scanning laser image acquisition, which introduces a bias error. To date, no study has quantified these errors or demonstrated a means to deal with them in SLIC velocimetry. In this work, we build upon the robust phase correlation (RPC) and existing methods of SLIC to quantify and mitigate these errors. First, we implement an ensemble RPC instead of using an ensemble standard cross correlation, and develop an SLIC optimal filter that maximizes the correlation strength in order to reliably and accurately detect the correlation peak representing the most probable average displacement of the nano...

  10. Two-photon light-sheet nanoscopy by fluorescence fluctuation correlation analysis

    Science.gov (United States)

    Chen, Xuanze; Zong, Weijian; Li, Rongqin; Zeng, Zhiping; Zhao, Jia; Xi, Peng; Chen, Liangyi; Sun, Yujie

    2016-05-01

    Advances in light-sheet microscopy have enabled the fast three-dimensional (3D) imaging of live cells and bulk specimens with low photodamage and phototoxicity. Combining light-sheet illumination with super-resolution imaging is expected to resolve subcellular structures. Actually, such kind of super-resolution light-sheet microscopy was recently demonstrated using a single-molecule localization algorithm. However, the imaging depth and temporal resolution of this method are limited owing to the requirements of precise single molecule localization and reconstruction. In this work, we present two-photon super-resolution light-sheet imaging via stochastic optical fluctuation imaging (2PLS-SOFI), which acquires high spatiotemporal resolution and excellent optical sectioning ability. 2PLS-SOFI is based on non-linear excitation of fluctuation/blinking probes using our recently developed fast two-photon three-axis digital scanned light-sheet microscope (2P3A-DSLM), which enables both deep penetration and thin sheet of light. Overall, 2PLS-SOFI demonstrates up to 3-fold spatial resolution enhancement compared with conventional two-photon light-sheet (2PLS) microscopy and about 40-fold temporal resolution enhancement compared with individual molecule localization-selective plane illumination microscopy (IML-SPIM). Therefore, 2PLS-SOFI is promising for 3D long-term, deep-tissue imaging with high spatiotemporal resolution.

  11. Surface sensitivity effects with local probe scanning Auger-scanning electron microscopy

    NARCIS (Netherlands)

    Van Agterveld, DTL; Palasantzas, G; De Hosson, JTM; Bentley, J; Allen, C; Dahmen, U; Petrov,

    2001-01-01

    Ultra-high-vacuum segregation studies on in-situ fractured Cu-Sb alloys were performed in terms of nanometer scale scanning Auger/Electron microscopy. S contamination leads to the formation Of Cu2S precipitates which, upon removal due to fracture, expose pits with morphology that depends on the prec

  12. Surface sensitivity effects with local probe scanning Auger-scanning electron microscopy

    NARCIS (Netherlands)

    Van Agterveld, DTL; Palasantzas, G; De Hosson, JTM; Bentley, J; Allen, C; Dahmen, U; Petrov,

    2001-01-01

    Ultra-high-vacuum segregation studies on in-situ fractured Cu-Sb alloys were performed in terms of nanometer scale scanning Auger/Electron microscopy. S contamination leads to the formation Of Cu2S precipitates which, upon removal due to fracture, expose pits with morphology that depends on the

  13. Correlative Analysis of Immunoreactivity in Confocal Laser-Scanning Microscopy and Scanning Electron Microscopy with Focused Ion Beam Milling

    Directory of Open Access Journals (Sweden)

    Takahiro eSonomura

    2013-02-01

    Full Text Available Three-dimensional reconstruction of ultrastructure of rat brain with minimal effort has recently been realized by scanning electron microscopy combined with focused ion beam milling (FIB-SEM. Because application of immunohistochemical staining to electron microscopy has a great advantage in that molecules of interest are specifically localized in ultrastructures, we here tried to apply immunocytochemistry to FIB-SEM and correlate immunoreactivity in confocal laser-scanning microcopy (CF-LSM with that in FIB-SEM. The dendrites of medium-sized spiny neurons in rat neostriatum were visualized with a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion, and thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2. After detecting the sites of terminals apposed to the dendrites in CF-LSM, GFP and VGluT2 immunoreactivities were further developed for electron microscopy by the immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB methods, respectively. In the contrast-inverted FIB-SEM images, silver precipitation and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were easily recognizable as in the images of transmission electron microscopy. In the sites of interest, some appositions were revealed to display synaptic specialization of asymmetric type. The present method is thus useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connection in the central neural circuit.

  14. Interaction of poxvirus intracellular mature virion proteins with the TPR domain of kinesin light chain in live infected cells revealed by two-photon-induced fluorescence resonance energy transfer fluorescence lifetime imaging microscopy.

    Science.gov (United States)

    Jeshtadi, Ananya; Burgos, Pierre; Stubbs, Christopher D; Parker, Anthony W; King, Linda A; Skinner, Michael A; Botchway, Stanley W

    2010-12-01

    Using two-photon-induced fluorescence lifetime imaging microscopy, we corroborate an interaction (previously demonstrated by yeast two-hybrid domain analysis) of full-length vaccinia virus (VACV; an orthopoxvirus) A36 protein with the cellular microtubule motor protein kinesin. Quenching of enhanced green fluorescent protein (EGFP), fused to the C terminus of VACV A36, by monomeric red fluorescent protein (mDsRed), fused to the tetratricopeptide repeat (TPR) domain of kinesin, was observed in live chicken embryo fibroblasts infected with either modified vaccinia virus Ankara (MVA) or wild-type fowlpox virus (FWPV; an avipoxvirus), and the excited-state fluorescence lifetime of EGFP was reduced from 2.5 ± 0.1 ns to 2.1 ± 0.1 ns due to resonance energy transfer to mDsRed. FWPV does not encode an equivalent of intracellular enveloped virion surface protein A36, yet it is likely that this virus too must interact with kinesin to facilitate intracellular virion transport. To investigate possible interactions between innate FWPV proteins and kinesin, recombinant FWPVs expressing EGFP fused to the N termini of FWPV structural proteins Fpv140, Fpv168, Fpv191, and Fpv198 (equivalent to VACV H3, A4, p4c, and A34, respectively) were generated. EGFP fusions of intracellular mature virion (IMV) surface protein Fpv140 and type II membrane protein Fpv198 were quenched by mDsRed-TPR in recombinant FWPV-infected cells, indicating that these virion proteins are found within 10 nm of mDsRed-TPR. In contrast, and as expected, EGFP fusions of the IMV core protein Fpv168 did not show any quenching. Interestingly, the p4c-like protein Fpv191, which demonstrates late association with preassembled IMV, also did not show any quenching.

  15. Scanning electron microscopy: preparation and imaging for SEM.

    Science.gov (United States)

    Jones, Chris G

    2012-01-01

    Scanning electron microscopy (SEM) has been almost universally applied for the surface examination and characterization of both natural and man-made objects. Although an invasive technique, developments in electron microscopy over the years has given the microscopist a much clearer choice in how invasive the technique will be. With the advent of low vacuum SEM in the 1970s (The environmental cold stage, 1970) and environmental SEM in the late 1980s (J Microsc 160(pt. 1):9-19, 1989), it is now possible in some circumstances to examine samples without preparation. However, for the examination of biological tissue and cells it is still advisable to chemically fix, dehydrate, and coat samples for SEM imaging and analysis. This chapter aims to provide an overview of SEM as an imaging tool, and a general introduction to some of the methods applied for the preparation of samples.

  16. Scanning electron microscopy of primate chorionic villi following ultrasonic microdissection.

    Science.gov (United States)

    King, B F

    1991-01-01

    Villi from human, macaque and baboon placentae were subjected to ultrasonication after prolonged osmication, and examined by scanning electron microscopy. The technique was often successful in removing the overlying trophoblast and revealing expanses of the trophoblastic basal lamina, a conclusion corroborated by transmission electron microscopy. These preparations bore a remarkable similarity in appearance to microvascular cast preparations of the fetal vasculature. Relatively straight parallel tubules appeared to correspond in position to the location of fetal vessels in intermediate villi, whereas portions of the basal laminae of terminal villi were in the form of convoluted, branched cylinders similar to SEM images of fetal capillaries of terminal villi. The basal lamina did not have evidence of pores as has been described in some basal laminae.

  17. Integrated Confocal and Scanning Probe Microscopy for Biomedical Research

    Directory of Open Access Journals (Sweden)

    B.J. Haupt

    2006-01-01

    Full Text Available Atomic force microscopy (AFM continues to be developed, not only in design, but also in application. The new focus of using AFM is changing from pure material to biomedical studies. More frequently, it is being used in combination with other optical imaging methods, such as confocal laser scanning microscopy (CLSM and fluorescent imaging, to provide a more comprehensive understanding of biological systems. To date, AFM has been used increasingly as a precise micromanipulator, probing and altering the mechanobiological characteristics of living cells and tissues, in order to examine specific, receptor-ligand interactions, material properties, and cell behavior. In this review, we discuss the development of this new hybrid AFM, current research, and potential applications in diagnosis and the detection of disease.

  18. High resolution helium ion scanning microscopy of the rat kidney.

    Science.gov (United States)

    Rice, William L; Van Hoek, Alfred N; Păunescu, Teodor G; Huynh, Chuong; Goetze, Bernhard; Singh, Bipin; Scipioni, Larry; Stern, Lewis A; Brown, Dennis

    2013-01-01

    Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide

  19. High-speed Lissajous-scan atomic force microscopy: scan pattern planning and control design issues.

    Science.gov (United States)

    Bazaei, A; Yong, Yuen K; Moheimani, S O Reza

    2012-06-01

    Tracking of triangular or sawtooth waveforms is a major difficulty for achieving high-speed operation in many scanning applications such as scanning probe microscopy. Such non-smooth waveforms contain high order harmonics of the scan frequency that can excite mechanical resonant modes of the positioning system, limiting the scan range and bandwidth. Hence, fast raster scanning often leads to image distortion. This paper proposes analysis and design methodologies for a nonlinear and smooth closed curve, known as Lissajous pattern, which allows much faster operations compared to the ordinary scan patterns. A simple closed-form measure is formulated for the image resolution of the Lissajous pattern. This enables us to systematically determine the scan parameters. Using internal model controllers (IMC), this non-raster scan method is implemented on a commercial atomic force microscope driven by a low resonance frequency positioning stage. To reduce the tracking errors due to actuator nonlinearities, higher order harmonic oscillators are included in the IMC controllers. This results in significant improvement compared to the traditional IMC method. It is shown that the proposed IMC controller achieves much better tracking performances compared to integral controllers when the noise rejection performances is a concern.

  20. Integrated micro ring resonator displacement sensor for scanning probe microscopies

    Science.gov (United States)

    Kiyat, Isa; Kocabas, Coskun; Aydinli, Atilla

    2004-03-01

    We describe a novel displacement sensor for scanning probe microscopies using an integrated optical micro ring resonator. This device operates by means of monitoring the changes in the transmission spectrum of a high finesse micro ring resonator. Finite element method simulations were carried out to obtain the optimum sensor design and finite difference time domain simulation was used to obtain the transfer characteristics of micro ring resonators. Operation principles and sensitivity calculations are discussed in detail. To achieve high sensitivity, we have studied different types of ring resonator. The highest sensitivity is obtained in a race-track resonator. This new design should provide sensitivities as high as ~10-4 Å-1.

  1. Laser-scanning photoacoustic microscopy with ultrasonic phased array transducer

    OpenAIRE

    Zheng, Fan; Zhang, Xiangyang; Chiu, Chi Tat; Zhou, Bill L.; Shung, K. Kirk; Zhang, Hao F.; Jiao, Shuliang

    2012-01-01

    In this paper, we report our latest progress on proving the concept that ultrasonic phased array can improve the detection sensitivity and field of view (FOV) in laser-scanning photoacoustic microscopy (LS-PAM). A LS-PAM system with a one-dimensional (1D) ultrasonic phased array was built for the experiments. The 1D phased array transducer consists of 64 active elements with an overall active dimension of 3.2 mm × 2 mm. The system was tested on imaging phantom and mouse ear in vivo. Experimen...

  2. Contrast distortion induced by modulation voltage in scanning capacitance microscopy

    Science.gov (United States)

    Chang, M. N.; Hu, C. W.; Chou, T. H.; Lee, Y. J.

    2012-08-01

    With a dark-mode scanning capacitance microscopy (SCM), we directly observed the influence of SCM modulation voltage (MV) on image contrasts. For electrical junctions, an extensive modulated area induced by MV may lead to noticeable changes in the SCM signal phase and intensity, resulting in a narrowed junction image and a broadened carrier concentration profile. This contrast distortion in SCM images may occur even if the peak-to-peak MV is down to 0.3 V. In addition, MV may shift the measured electrical junction depth. The balance of SCM signals components explain these MV-induced contrast distortions.

  3. Oxygen-free in situ scanning tunnelling microscopy

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Ulstrup, Jens

    2007-01-01

    Scanning tunneling microscopy under full electrochemical potential control (in situ STM) has been used extensively as an efficient method to characterize microstructures at solid/liquid interfaces at the atomic and molecular levels. However, under ambient conditions oxygen may interfere in open......, the STM image quality of the monolayer structures has improved significantly in the absence of oxygen. Reasons for the drastic effects of dioxygen based on both chemical surface reactivity and electron scattering effects are discussed. These observations are important in general for STM of oxygen...

  4. High-resolution low-dose scanning transmission electron microscopy.

    Science.gov (United States)

    Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

  5. Measurement of dihedral angles by scanning electron microscopy.

    Science.gov (United States)

    Achutaramayya, G.; Scott, W. D.

    1973-01-01

    The extension of Hoover's (1971) technique to the case of dihedral-angle measurement is described. Dihedral angles are often determined by interferometry on thermally grooved grain boundaries to obtain information on relative interfacial energies. In the technique considered the measured angles approach the true angles as the tilt angle approaches 90 deg. It is pointed out that the scanning electron microscopy method provides a means of seeing the real root of a groove at a lateral magnification which is higher than that obtainable with interferometry.

  6. Measurement of dihedral angles by scanning electron microscopy.

    Science.gov (United States)

    Achutaramayya, G.; Scott, W. D.

    1973-01-01

    The extension of Hoover's (1971) technique to the case of dihedral-angle measurement is described. Dihedral angles are often determined by interferometry on thermally grooved grain boundaries to obtain information on relative interfacial energies. In the technique considered the measured angles approach the true angles as the tilt angle approaches 90 deg. It is pointed out that the scanning electron microscopy method provides a means of seeing the real root of a groove at a lateral magnification which is higher than that obtainable with interferometry.

  7. Transfer functions in collection scanning near-field optical microscopy

    DEFF Research Database (Denmark)

    Bozhevolnyi, Sergey I.; Vohnsen, Brian; Bozhevolnaya, Elena A.

    1999-01-01

    It is generally accepted that, if in collection near-field optical microscopy the probe-sample coupling can be disregarded, a fiber probe can be considered as a detector of the near-field intensity whose size can be accounted for via an intensity transfer function. We show that, in general...... are considered with respect to the relation between near-field optical images and the corresponding intensity distributions. Our conclusions are supported with numerical simulations and experimental results obtained by using a photon scanning tunneling microscope with an uncoated fiber tip....

  8. Multifrequency scanning probe microscopy study of nanodiamond agglomerates

    Science.gov (United States)

    Aravind, Vasudeva; Lippold, Stephen; Li, Qian; Strelcov, Evgheny; Okatan, Baris; Legum, Benjamin; Kalinin, Sergei; Clarion University Team; Oak Ridge National Laboratory Team

    Due to their rich surface chemistry and excellent mechanical properties and non-toxic nature, nanodiamond particles have found applications such as biomedicine, tribology and lubrication, targeted drug delivery systems, tissue scaffolds and surgical implants. Although single nanodiamond particles have diameters about 4-5nm, they tend to form agglomerates. While these agglomerates can be useful for some purposes, many applications of nanodiamonds require single particle, disaggregated nanodiamonds. This work is oriented towards studying forces and interactions that contribute to agglomeration in nanodiamonds. In this work, using multifrequency scanning probe microscopy techniques, we show that agglomerate sizes can vary between 50-100nm in raw nanodiamonds. Extremeties of particles and Interfaces between agglomerates show dissipative forces with scanning probe microscope tip, indicating agglomerates could act as points of increased adhesion, thus reducing lubricating efficiency when nanodiamonds are used as lubricant additives. This research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.

  9. Imaging Photon Lattice States by Scanning Defect Microscopy

    Science.gov (United States)

    Underwood, D. L.; Shanks, W. E.; Li, Andy C. Y.; Ateshian, Lamia; Koch, Jens; Houck, A. A.

    2016-04-01

    Microwave photons inside lattices of coupled resonators and superconducting qubits can exhibit surprising matterlike behavior. Realizing such open-system quantum simulators presents an experimental challenge and requires new tools and measurement techniques. Here, we introduce scanning defect microscopy as one such tool and illustrate its use in mapping the normal-mode structure of microwave photons inside a 49-site kagome lattice of coplanar waveguide resonators. Scanning is accomplished by moving a probe equipped with a sapphire tip across the lattice. This locally perturbs resonator frequencies and induces shifts of the lattice resonance frequencies, which we determine by measuring the transmission spectrum. From the magnitude of mode shifts, we can reconstruct photon field amplitudes at each lattice site and thus create spatial images of the photon-lattice normal modes.

  10. Scanning tunneling microscopy and spectroscopy studies of graphite edges

    CERN Document Server

    Niimi, Y; Kambara, H; Tagami, K; Tsukada, M; Fukuyama, H; Fukuyama, Hiroshi

    2004-01-01

    We studied experimentally and theoretically the electronic local density of states (LDOS) near single step edges at the surface of exfoliated graphite. In scanning tunneling microscopy measurements, we observed the $(\\sqrt{3} \\times \\sqrt{3}) R 30^{\\circ}$ and honeycomb superstructures both extending over 3$-$4 nm either from the zigzag or armchair edge. Calculations based on a density-functional derived non-orthogonal tight-binding model show that these superstructures can coexist if the two types of edge admix each other in real graphite step edges. Scanning tunneling spectroscopy measurements near the zigzag edge reveal a clear peak in the LDOS at an energy below the Fermi energy by 20 meV. No such a peak was observed near the armchair edge. We concluded that this peak corresponds to the "edge state" theoretically predicted for graphene ribbons, since a similar prominent LDOS peak due to the edge state is obtained by the first principles calculations.

  11. Quantitative phase imaging with scanning holographic microscopy: an experimental assesment

    Directory of Open Access Journals (Sweden)

    Tada Yoshitaka

    2006-11-01

    Full Text Available Abstract This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example, while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies.

  12. Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.

    Science.gov (United States)

    Sonomura, Takahiro; Furuta, Takahiro; Nakatani, Ikuko; Yamamoto, Yo; Unzai, Tomo; Matsuda, Wakoto; Iwai, Haruki; Yamanaka, Atsushi; Uemura, Masanori; Kaneko, Takeshi

    2013-01-01

    Recently, three-dimensional reconstruction of ultrastructure of the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for EM by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy (TEM) images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit.

  13. Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers

    Science.gov (United States)

    Ossola, Dario; Dorwling-Carter, Livie; Dermutz, Harald; Behr, Pascal; Vörös, János; Zambelli, Tomaso

    2015-12-01

    We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.

  14. Ultra-fast 3D scanning and holographic illumination in non-linear microscopy using acousto-optic deflectors

    Science.gov (United States)

    Akemann, Walther; Ventalon, Cathie; Léger, Jean-François; Mathieu, Benjamin; Dieudonné, Stéphane; Blochet, Baptiste; Gigan, Sylvain; Bourdieu, Laurent

    2017-04-01

    Decoding of information in the brain requires the imaging of large neuronal networks using e.g. two-photon microscopy (TPM). Fast control of the focus in 3D can be achieved with phase shaping of the light beam using acoustooptic deflectors (AODs). However, beam shaping using AODs is not straightforward because of non-stationary of acousto-optic diffraction. Here, we demonstrated a new stable AOD-based phase modulator, which operates at a rate of up to about hundred kHz. It provides opportunity for 3D scanning in TPM with the possibility to correct aberrations independently for every focus position or to achieve refocusing of scattered photons in rapidly decorrelating tissues.

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

    Science.gov (United States)

    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.

  16. Two-Photon Flow Cytometry

    Science.gov (United States)

    Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

    2004-01-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

  17. Enhanced two-photon absorption using true thermal light

    CERN Document Server

    Jechow, Andreas; Kurzke, Henning; Heuer, Axel; Menzel, Ralf

    2013-01-01

    Two-photon excited fluorescence (TPEF) is a standard technique in modern microscopy but still affected by photo-damage of the probe. It was proposed that TPEF can be enhanced by using entangled photons, but has proven to be challenging. Recently it was shown that some features of entangled photons can be mimicked with thermal light, which finds application in ghost imaging, sub-wavelength lithography and metrology. Here, we utilize true thermal light from a super-luminescence diode to demonstrate enhanced TPEF compared to coherent light using two common fluorophores and luminescent quantum dots. We find that the two-photon absorption rate is directly proportional to the measured degree of second-order coherence, as predicted by theory. Our results show that photon bunching can be exploited in two-photon microscopy with the photon statistic providing a new degree of freedom.

  18. Sample heating system for spin-polarized scanning electron microscopy.

    Science.gov (United States)

    Kohashi, Teruo; Motai, Kumi

    2013-08-01

    A sample-heating system for spin-polarized scanning electron microscopy (spin SEM) has been developed and used for microscopic magnetization analysis at temperatures up to 500°C. In this system, a compact ceramic heater and a preheating operation keep the ultra-high vacuum conditions while the sample is heated during spin SEM measurement. Moreover, the secondary-electron collector, which is arranged close to the sample, was modified so that it is not damaged at high temperatures. The system was used to heat a Co(1000) single-crystal sample from room temperature up to 500°C, and the magnetic-domain structures were observed. Changes of the domain structures were observed around 220 and 400°C, and these changes are considered to be due to phase transitions of this sample.

  19. Ultramicrosensors based on transition metal hexacyanoferrates for scanning electrochemical microscopy

    Directory of Open Access Journals (Sweden)

    Maria A. Komkova

    2013-10-01

    Full Text Available We report here a way for improving the stability of ultramicroelectrodes (UME based on hexacyanoferrate-modified metals for the detection of hydrogen peroxide. The most stable sensors were obtained by electrochemical deposition of six layers of hexacyanoferrates (HCF, more specifically, an alternating pattern of three layers of Prussian Blue and three layers of Ni–HCF. The microelectrodes modified with mixed layers were continuously monitored in 1 mM hydrogen peroxide and proved to be stable for more than 5 h under these conditions. The mixed layer microelectrodes exhibited a stability which is five times as high as the stability of conventional Prussian Blue-modified UMEs. The sensitivity of the mixed layer sensor was 0.32 A·M−1·cm−2, and the detection limit was 10 µM. The mixed layer-based UMEs were used as sensors in scanning electrochemical microscopy (SECM experiments for imaging of hydrogen peroxide evolution.

  20. Laser-scanning photoacoustic microscopy with ultrasonic phased array transducer.

    Science.gov (United States)

    Zheng, Fan; Zhang, Xiangyang; Chiu, Chi Tat; Zhou, Bill L; Shung, K Kirk; Zhang, Hao F; Jiao, Shuliang

    2012-11-01

    In this paper, we report our latest progress on proving the concept that ultrasonic phased array can improve the detection sensitivity and field of view (FOV) in laser-scanning photoacoustic microscopy (LS-PAM). A LS-PAM system with a one-dimensional (1D) ultrasonic phased array was built for the experiments. The 1D phased array transducer consists of 64 active elements with an overall active dimension of 3.2 mm × 2 mm. The system was tested on imaging phantom and mouse ear in vivo. Experiments showed a 15 dB increase of the signal-to-noise ratio (SNR) when beamforming was employed compared to the images acquired with each single element. The experimental results demonstrated that ultrasonic phased array can be a better candidate for LS-PAM in high sensitivity applications like ophthalmic imaging.

  1. Conditioning of mealybug (Hemiptera: Pseudococcidae by Scanning Electron Microscopy.

    Directory of Open Access Journals (Sweden)

    Melissa Palma-Jiménez

    2015-06-01

    Full Text Available The aim of this work was to determine the methodology for an adequate conditioning for the cleaning of mealybugs specimens and its correct observation. This work was done in the laboratory of the Research Center in Microscopic Structures (CIEMIC of the University of Costa Rica, in 2012. Four types of methodologies were implemented, which evidenced a gradual improvement of the observation of the ultrastructures through the Scanning Electron Microscopy. Every process was described in detail. The best results were showed with 10% xylene (in some cases it was feasible using 95-100% ethanol. It allowed to remove the wax from the body of the insect, avoiding its collapse, and observing the specific ultrastructures of the individual. This approach will reduce the time and cost of future taxonomic research of mealybugs.

  2. Extracting Impurity Locations using Scanning Capacitance Microscopy Measurements

    Directory of Open Access Journals (Sweden)

    AGHAEI, S.

    2016-08-01

    Full Text Available In this article we investigate the possibility to use scanning capacitance microscopy (SCM for the 2-D and 3-D "atomistic" dopant profiling of semiconductor materials. For this purpose, we first analyze the effects of random dopant fluctuations (RDF on SCM measurements with nanoscale probes and show that the discrete and random locations of dopant impurities significantly affect the differential capacitance measured in SCM experiments if the dimension of the probe is below 50 nm. Then, we present an algorithm to compute the x, y, and z coordinates of the ionized impurities in the semiconductor material using a set of SCM measurements. The algorithm is based on evaluating the doping sensitivity functions of the differential capacitance and uses a gradient-based iterative method to compute the locations of dopants. Finally, we discuss a standard simulation case and show that we are able to successfully retrieve the locations of the ionized impurities using the proposed algorithm.

  3. Theory and application of scanning electron acoustic microscopy

    Science.gov (United States)

    Cantrell, John H.; Qian, Menglu; Chen, Ruiyi; Yost, William T.

    1992-01-01

    A three-dimensional theoretical model based on the application of the thermal conduction and Navier equations to a chopped electron beam incident on a disk specimen is used to obtain the particle displacement field in the specimen. The results lead to a consideration of the signal generation, spatial resolution, and contrast mechanisms in scanning electron acoustic microscopy (SEAM). The model suggests that the time-variant heat source produced by the beam chopping generates driving source, thermal wave, and acoustic wave displacements simultaneously in the specimen. Evidence of the correctness of the prediction is obtained from the mathematically similar problem of pulsed laser light injection into a tank of water. High speed Schlieren photographs taken following laser injection show the simultaneous evolution of thermal and acoustic waveforms. Examples of contrast reversal, stress-induced contrast, and acoustic zone contrast and resolution with SEAM are presented and explained in terms of the model features.

  4. Semiconductor characterization by scanning ion beam induced charge (IBIC) microscopy

    CERN Document Server

    Vittone, E; Olivero, P; Manfredotti, C; Jaksic, M; Giudice, A Lo; Fizzotti, F; Colombo, E

    2016-01-01

    The acronym IBIC (Ion Beam Induced Charge) was coined in early 1990's to indicate a scanning microscopy technique which uses MeV ion beams as probes to image the basic electronic properties of semiconductor materials and devices. Since then, IBIC has become a widespread analytical technique to characterize materials for electronics or for radiation detection, as testified by more than 200 papers published so far in peer-reviewed journals. Its success stems from the valuable information IBIC can provide on charge transport phenomena occurring in finished devices, not easily obtainable by other analytical techniques. However, IBIC analysis requires a robust theoretical background to correctly interpret experimental data. In order to illustrate the importance of using a rigorous mathematical formalism, we present in this paper a benchmark IBIC experiment aimed to test the validity of the interpretative model based on the Gunn's theorem and to provide an example of the analytical capability of IBIC to characteriz...

  5. Scanning transmission electron microscopy imaging dynamics at low accelerating voltages

    Energy Technology Data Exchange (ETDEWEB)

    Lugg, N.R. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Findlay, S.D. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Shibata, N. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); PRESTO, Japan Science and Technology Agency, Saitama 332-0012 (Japan); Mizoguchi, T. [Institute of Industrial Science, The University of Tokyo, Tokyo 153-8505 (Japan); D' Alfonso, A.J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Allen, L.J., E-mail: lja@unimelb.edu.au [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Ikuhara, Y. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Nanostructures Research Laboratory, Japan Fine Ceramic Center, Nagoya 456-8587 (Japan); WPI Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2011-07-15

    Motivated by the desire to minimize specimen damage in beam sensitive specimens, there has been a recent push toward using relatively low accelerating voltages (<100kV) in scanning transmission electron microscopy. To complement experimental efforts on this front, this paper seeks to explore the variations with accelerating voltage of the imaging dynamics, both of the channelling of the fast electron and of the inelastic interactions. High-angle annular-dark field, electron energy loss spectroscopic imaging and annular bright field imaging are all considered. -- Highlights: {yields} Both elastic and inelastic scattering in STEM are acceleration voltage dependent. {yields} HAADF, EELS and ABF imaging are assessed with a view to optimum imaging. {yields} Lower accelerating voltages improve STEM EELS contrast in very thin crystals. {yields} Higher accelerating voltages give better STEM EELS contrast in thicker crystals. {yields} At fixed resolution, higher accelerating voltage aids ABF imaging of light elements.

  6. Scanning electron microscopy of congenital corneal leukomas (Peters' anomaly).

    Science.gov (United States)

    Polack, F M; Graue, E L

    1979-08-01

    Specimens of three corneas in two patients with Peter's anomaly were obtained at the time of penetrating keratoplasty and studied by scanning and transmission electron microscopy. In one patient, the anomaly was monocular, and the endothelial surface showed a central defect in Descemet's layer with isolated rounded defects in the midperiphery. Fine collagenous material covered the posterior surface. The other two specimens were obtained from a patient with rubella syndrome without cataracts. The cornea showed malformation of Descemet's membrane with fibroblastic overgrowth on the endothelial layer. Epithelial-like cells and leukocytes were also found. The congenital central leukoma we believe was caused by adhesion of the pupillary membrane in our first patient, and possibly was inflammatory in our second patient.

  7. 2-photon laser scanning microscopy on native human cartilage

    Science.gov (United States)

    Martini, Joerg; Toensing, Katja; Dickob, Michael; Anselmetti, Dario

    2005-08-01

    Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 μm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2•107cm3. Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.

  8. Local deposition of anisotropic nanoparticles using scanning electrochemical microscopy (SECM).

    Science.gov (United States)

    Fedorov, Roman G; Mandler, Daniel

    2013-02-28

    We demonstrate localized electrodeposition of anisotropic metal nanoobjects, namely Au nanorods (GNR), on indium tin oxide (ITO) using scanning electrochemical microscopy (SECM). A gold microelectrode was the source of the gold ions whereby double pulse chronoamperometry was employed to generate initially Au seeds which were further grown under controlled conditions. The distance between the microelectrode and the ITO surface as well as the different experimental parameters (electrodeposition regime, solution composition and temperature) were optimized to produce faceted gold seeds with the required characteristics (size and distribution). Colloidal chemical synthesis was successfully exploited for better understanding the role of the surfactant and different additives in breaking the crystallographic symmetry and anisotropic growth of GNR. Experiments performed in a conventional three-electrode cell revealed the most appropriate electrochemical conditions allowing high yield synthesis of nanorods with well-defined shape as well as nanocubes and bipyramids.

  9. Scanning electron microscopy of ascospores of Debaryomyces and Saccharomyces.

    Science.gov (United States)

    Kurtzman, C P; Smiley, M J; Baker, F L

    1975-02-28

    Ascospores from species of Debaryomyces and the Torulaspora-group of Saccharomyces were examined by scanning electron microscopy. Ornamentation on ascospores of D. hansenii varied from short to long interconnected ridges or broad based, elongated conical protuberances. A spiral rigde system was detected on the ascospores of D. marama, but wart-like protuberances occurred on those of D. cantarelli, D. castellii, D. coudertii, D. formicarius, D. phaffii, D. vanriji and D. yarrowii. Ascospores of D. halotolerans did not have protuberances and the species appears to be identical with Pichia farinosa. Wart-like protuberances also were found on ascospores of S. delbrueckii, S. microellipsodes, S. rosei, S. inconspicuus, S. fermentati, S. montanus and S. vafer, but the ascospore surface of S. pretoriensis was covered by fine ridges. Short tapered ridges covered the ascospores of S. kloeckerianus.

  10. Scanning photoelectron microscopy using a pointed capillary probe

    Science.gov (United States)

    Mironov, B. N.; Cherkun, A. P.; Aseyev, S. A.; Chekalin, S. V.

    2017-08-01

    The possibilities of a new type of scanning probe microscopy (SPM) for two different samples are experimentally demonstrated. The method is based on the use of a pointed capillary, which can simultaneously act as a 'classical' SPM probe and also as a controlled thin channel for transporting charged particles emitted by the surface to the detector. In the experiment, photoelectrons pass through a dielectric hollow cone probe with an aperture radius of 1 μm and detected by microchannel plates at different points of the investigated conducting surface irradiated by the second harmonic of a femtosecond Ti : sapphire laser. As a result, the sample's surface profile is visualised with a subwavelength spatial resolution. This method makes it possible to control spatially localised beams of electrons, ions, neutral atoms (molecules) and soft X-ray radiation, as well as opens a possibility for research in the field of nanoscale photodesorption of molecular ions.

  11. Probing Individual Ice Nucleation Events with Environmental Scanning Electron Microscopy

    Science.gov (United States)

    Wang, Bingbing; China, Swarup; Knopf, Daniel; Gilles, Mary; Laskin, Alexander

    2016-04-01

    Heterogeneous ice nucleation is one of the processes of critical relevance to a range of topics in the fundamental and the applied science and technologies. Heterogeneous ice nucleation initiated by particles proceeds where microscopic properties of particle surfaces essentially control nucleation mechanisms. Ice nucleation in the atmosphere on particles governs the formation of ice and mixed phase clouds, which in turn influence the Earth's radiative budget and climate. Heterogeneous ice nucleation is still insufficiently understood and poses significant challenges in predictive understanding of climate change. We present a novel microscopy platform allowing observation of individual ice nucleation events at temperature range of 193-273 K and relative humidity relevant for ice formation in the atmospheric clouds. The approach utilizes a home built novel ice nucleation cell interfaced with Environmental Scanning Electron Microscope (IN-ESEM system). The IN-ESEM system is applied for direct observation of individual ice formation events, determining ice nucleation mechanisms, freezing temperatures, and relative humidity onsets. Reported microanalysis of the ice nucleating particles (INP) include elemental composition detected by the energy dispersed analysis of X-rays (EDX), and advanced speciation of the organic content in particles using scanning transmission x-ray microscopy with near edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS). The performance of the IN-ESEM system is validated through a set of experiments with kaolinite particles with known ice nucleation propensity. We demonstrate an application of the IN-ESEM system to identify and characterize individual INP within a complex mixture of ambient particles.

  12. Two-photon imaging of stem cells

    Science.gov (United States)

    Uchugonova, A.; Gorjup, E.; Riemann, I.; Sauer, D.; König, K.

    2008-02-01

    A variety of human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, dental pulpa stem cells) have been investigated by femtosecond laser 5D two-photon microscopy. Autofluorescence and second harmonic generation have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, NADH and flavoprotein fluorescence was detected in stem cells. Major emission peaks at 460nm and 530nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured using time-correlated single photon counting and spectral imaging. Differentiated stem cells produced the extracellular matrix protein collagen which was detected by SHG signals at 435 nm.

  13. Scanning electron microscopy of the neuropathology of murine cerebral malaria

    Directory of Open Access Journals (Sweden)

    Brenneis Christian

    2006-11-01

    Full Text Available Abstract Background The mechanisms leading to death and functional impairments due to cerebral malaria (CM are yet not fully understood. Most of the knowledge about the pathomechanisms of CM originates from studies in animal models. Though extensive histopathological studies of the murine brain during CM are existing, alterations have not been visualized by scanning electron microscopy (SEM so far. The present study investigates the neuropathological features of murine CM by applying SEM. Methods C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. When typical symptoms of CM developed perfused brains were processed for SEM or light microscopy, respectively. Results Ultrastructural hallmarks were disruption of vessel walls, parenchymal haemorrhage, leukocyte sequestration to the endothelium, and diapedesis of macrophages and lymphocytes into the Virchow-Robin space. Villous appearance of observed lymphocytes were indicative of activated state. Cerebral oedema was evidenced by enlargement of perivascular spaces. Conclusion The results of the present study corroborate the current understanding of CM pathophysiology, further support the prominent role of the local immune system in the neuropathology of CM and might expose new perspectives for further interventional studies.

  14. Three-Dimensional scanning transmission electron microscopy of biological specimens

    KAUST Repository

    De Jonge, Niels

    2010-01-18

    A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2-3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset. © 2010 Microscopy Society of America.

  15. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  16. Volume scanning electron microscopy for imaging biological ultrastructure.

    Science.gov (United States)

    Titze, Benjamin; Genoud, Christel

    2016-11-01

    Electron microscopy (EM) has been a key imaging method to investigate biological ultrastructure for over six decades. In recent years, novel volume EM techniques have significantly advanced nanometre-scale imaging of cells and tissues in three dimensions. Previously, this had depended on the slow and error-prone manual tasks of cutting and handling large numbers of sections, and imaging them one-by-one with transmission EM. Now, automated volume imaging methods mostly based on scanning EM (SEM) allow faster and more reliable acquisition of serial images through tissue volumes and achieve higher z-resolution. Various software tools have been developed to manipulate the acquired image stacks and facilitate quantitative analysis. Here, we introduce three volume SEM methods: serial block-face electron microscopy (SBEM), focused ion beam SEM (FIB-SEM) and automated tape-collecting ultramicrotome SEM (ATUM-SEM). We discuss and compare their capabilities, provide an overview of the full volume SEM workflow for obtaining 3D datasets and showcase different applications for biological research.

  17. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1985-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions, imaging modes, the interpretation of micrographs and the use of quantitative modes "in scanning electron microscopy (SEM). lt forms a counterpart to Transmission Electron Microscopy (Vol. 36 of this Springer Series in Optical Sciences) . The book evolved from lectures delivered at the University of Münster and from a German text entitled Raster-Elektronenmikroskopie (Springer-Verlag), published in collaboration with my colleague Gerhard Pfefferkorn. In the introductory chapter, the principles of the SEM and of electron­ specimen interactions are described, the most important imaging modes and their associated contrast are summarized, and general aspects of eiemental analysis by x-ray and Auger electron emission are discussed. The electron gun and electron optics are discussed in Chap. 2 in order to show how an electron probe of small diameter can be formed, how the elec­ tron beam can be blanked at high fre...

  18. Char porosity characterisation by scanning electron microscopy and image analysis

    Energy Technology Data Exchange (ETDEWEB)

    Soerensen, H.S.; Rosenberg, P.; Petersen, H.I.; Soerensen, L.H. [Danfoss A/S, Nordborg (Denmark)

    2000-09-01

    No significant change in either the morphotype composition or the macroporosity (pores {gt}5 {mu}m) in the 0-30 wt.% char burnout interval were revealed by reflected light microscopy or image analysis. Two high temperature char series from a Tertiary South American coal (C1) and a Permian Gondwana coal (C2) were therefore examined by scanning electron microscopy to provide information on the combustion process up to {approximately} 60 wt% char burnout. This study documents a significant mesopore ({approximately} 0.1-5 {mu}m) development on the fused chars in the burnout interval studied. A method to quantify the size and amount of the mesopores is described and both the parameters increased with increasing char burnout. Above a char burnout of {approximately} 30 wt% an increase in macroporosity was detected and ascribed to coalescence of mesopores to form large pores. Although the measurement of mesoporosity is restricted to fused chars, i.e. pores in fragments and the char morphotypes inertoid, fusinoid and solid could not be measured, the consideration of mesoporosity seems to be fundamental in understanding, evaluating and modelling combustion processes in the char burnout interval studied. 7 refs., 9 figs., 4 tabs.

  19. Scanning Surface Potential Microscopy of Spore Adhesion on Surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ida [University of Tennessee, Knoxville (UTK); Chung, Eunhyea [Georgia Institute of Technology; Kweon, Hyojin [Georgia Institute of Technology; Yiacoumi, Sotira [Georgia Institute of Technology; Tsouris, Costas [ORNL

    2012-01-01

    The adhesion of spores of Bacillus anthracis - the cause of anthrax and a likely biological threat - to solid surfaces is an important consideration in cleanup after an accidental or deliberate release. However, because of safety concerns, directly studying B. anthracis spores with advanced instrumentation is problematic. As a first step, we are examining the electrostatic potential of Bacillus thuringiensis (Bt), which is a closely related species that is often used as a simulant to study B. anthracis. Scanning surface potential microscopy (SSPM), also known as Kelvin probe force microscopy (KPFM), was used to investigate the influence of relative humidity (RH) on the surface electrostatic potential of Bt that had adhered to silica, mica, or gold substrates. AFM/SSPM side-by-side images were obtained separately in air, at various values of RH, after an aqueous droplet with spores was applied on each surface and allowed to dry before measurements. In the SSPM images, a negative potential on the surface of the spores was observed compared with that of the substrates. The surface potential decreased as the humidity increased. Spores were unable to adhere to a surface with an extremely negative potential, such as mica.

  20. [Two-photon excitation fluorescence of 5-ALA induced PpIX in DHL cells].

    Science.gov (United States)

    Huang, Zu-Fang; Chen, Rong; Li, Yong-Zeng; Chen, Guan-Nan; Chen, Xian-Ling; Feng, Shang-Yuan; Jia, Pei-Min

    2008-11-01

    Two-photon fluorescence microscopy is a novel imaging technique, which is primarily sensitive to a specimen's response coming from an in-focus plane, thus has low photo-bleaching and photo-damage to biological samples. 5-ALA induced production of PpIX in DHL cells was excited by 820 nm femtosecond laser; two-photon excitation fluorescence of single cell was obtained in Lambda mode of laser scanning confocal microscope. The specific fluorescence intensity of PpIX which accumulated in DHL cells was measured at 2, 4 and 10 mmol x L(-1) concentration of 5-ALA with different incubation time, which reflected the kinetics of 5-ALA accumulated in DHL cells. Accumulation of PpIX in DHL cells was a dynamic change process. Biphasic alterations of PpIX accumulation were noted: PpIX content enhanced with the increasing time and reached the maximal value around 3 h, however PpIX content decreased in the subsequent incubation time. Results indicate that two-photon fluorescence based on laser scanning microscope can be a useful technology for studying the kinetics of 5-ALA induced PpIX production in DHL cells and other leukemia cells.

  1. Confocal laser scanning microscopy in study of bone calcification

    Energy Technology Data Exchange (ETDEWEB)

    Nishikawa, Tetsunari, E-mail: tetsu-n@cc.osaka-dent.ac.jp [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Kokubu, Mayu; Kato, Hirohito [Department of Oral Pathology, Osaka Dental University, Osaka (Japan); Imai, Koichi [Department of Biomaterials, Osaka Dental University, Osaka (Japan); Tanaka, Akio [Department of Oral Pathology, Osaka Dental University, Osaka (Japan)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer High-magnification images with depth selection, and thin sections were observed using CLSM. Black-Right-Pointing-Pointer The direction and velocity of calcification of the bone was observed by administration of 2 fluorescent dyes. Black-Right-Pointing-Pointer In dog femora grafted with coral blocks, newly-formed bone was observed in the coral block space with a rough surface. Black-Right-Pointing-Pointer Twelve weeks after dental implant was grafted in dog femora, the space between screws was filled with newly-formed bones. - Abstract: Bone regeneration in mandible and maxillae after extraction of teeth or tumor resection and the use of rough surface implants in bone induction must be investigated to elucidate the mechanism of calcification. The calcified tissues are subjected to chemical decalcification or physical grinding to observe their microscopic features with light microscopy and transmission electron microscopy where the microscopic tissue morphology is significantly altered. We investigated the usefulness of confocal laser scanning microscopy (CLSM) for this purpose. After staggering the time of administration of calcein and alizarin red to experimental rats and dogs, rat alveolar bone and dog femur grafted with coral as scaffold or dental implants were observed with CLSM. In rat alveolar bone, the calcification of newly-formed bone and net-like canaliculi was observed at the mesial bone from the roots progressed at the rate of 15 {mu}m/day. In dog femur grafted with coral, newly-formed bones along the space of coral were observed in an orderly manner. In dog femur with dental implants, after 8 weeks, newly-formed bone proceeded along the rough surface of the implants. CLSM produced high-magnification images of newly-formed bone and thin sections were not needed.

  2. Morphological classification of bioaerosols from composting using scanning electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tamer Vestlund, A. [Institute for Energy and Resource Technology, Environmental Science and Technology Department, School of Applied Sciences, Cranfield University, Building 40, Bedfordshire MK43 0AL (United Kingdom); FIRA International Ltd., Maxwell Road, Stevenage, Herts SG1 2EW (United Kingdom); Al-Ashaab, R.; Tyrrel, S.F.; Longhurst, P.J.; Pollard, S.J.T. [Institute for Energy and Resource Technology, Environmental Science and Technology Department, School of Applied Sciences, Cranfield University, Building 40, Bedfordshire MK43 0AL (United Kingdom); Drew, G.H., E-mail: g.h.drew@cranfield.ac.uk [Institute for Energy and Resource Technology, Environmental Science and Technology Department, School of Applied Sciences, Cranfield University, Building 40, Bedfordshire MK43 0AL (United Kingdom)

    2014-07-15

    Highlights: • Bioaerosols were captured using the filter method. • Bioaerosols were analysed using scanning electron microscope. • Bioaerosols were classified on the basis of morphology. • Single small cells were found more frequently than aggregates and larger cells. • Smaller cells may disperse further than heavier aggregate structures. - Abstract: This research classifies the physical morphology (form and structure) of bioaerosols emitted from open windrow composting. Aggregation state, shape and size of the particles captured are reported alongside the implications for bioaerosol dispersal after release. Bioaerosol sampling took place at a composting facility using personal air filter samplers. Samples were analysed using scanning electron microscopy. Particles were released mainly as small (<1 μm) single, spherical cells, followed by larger (>1 μm) single cells, with aggregates occurring in smaller proportions. Most aggregates consisted of clusters of 2–3 particles as opposed to chains, and were <10 μm in size. No cells were attached to soil debris or wood particles. These small single cells or small aggregates are more likely to disperse further downwind from source, and cell viability may be reduced due to increased exposure to environmental factors.

  3. Scanning X-ray microscopy of superconductor/ferromagnet bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Stahl, Claudia; Ruoss, Stephen; Weigand, Markus; Schuetz, Gisela [Max Planck Institute for Intelligent Systems, Stuttgart (Germany); Zahn, Patrick; Bayer, Jonas [Max Planck Institute for Intelligent Systems, Stuttgart (Germany); Research Institute for Innovative Surfaces, FINO, Aalen University (Germany); Albrecht, Joachim [Research Institute for Innovative Surfaces, FINO, Aalen University (Germany)

    2016-07-01

    The magnetic flux distribution arising from a high-T{sub c} superconductor is detected and visualized with high spatial resolution using scanning x-ray microscopy (SXM). Therefore, we introduce a sensor layer, namely, an amorphous, soft-magnetic CoFeB cover layer. The magnetic stray fields of the supercurrents lead to a local reorientation of the magnetic moments in the ferromagnet, which is visualized using the large x-ray magnetic circular dichroism (XMCD) effect of the Co and Fe L3-edge. We show that the XMCD contrast in the sensor layer corresponds to the in-plane magnetic flux distribution of the superconductor and can hence be used to image magnetic structures in superconductors with high spatial resolution. Using the total electron yield (TEY) mode the surface structure and the magnetic domains can be imaged simultaneously and can be correlated. The measurements are carried out at our scanning x-ray microscope MAXYMUS at Bessy II, Berlin with the new low temperature setup.

  4. All-optical photoacoustic microscopy using a MEMS scanning mirror

    Science.gov (United States)

    Chen, Sung-Liang; Xie, Zhixing; Ling, Tao; Wei, Xunbin; Guo, L. Jay; Wang, Xueding

    2013-03-01

    It has been studied that a potential marker to obtain prognostic information about bladder cancer is tumor neoangiogenesis, which can be quantified by morphometric characteristics such as microvascular density. Photoacoustic microscopy (PAM) can render sensitive three-dimensional (3D) mapping of microvasculature, providing promise to evaluate the neoangiogenesis that is closely related to the diagnosis of bladder cancer. To ensure good image quality, it is desired to acquire bladder PAM images from its inside via the urethra, like conventional cystoscope. Previously, we demonstrated all-optical PAM systems using polymer microring resonators to detect photoacoustic signals and galvanometer mirrors for laser scanning. In this work, we build a miniature PAM system using a microelectromechanical systems (MEMS) scanning mirror, demonstrating a prototype of an endoscopic PAM head capable of high imaging quality of the bladder. The system has high resolutions of 17.5 μm in lateral direction and 19 μm in the axial direction at a distance of 5.4 mm. Images of printed grids and the 3D structure of microvasculature in animal bladders ex vivo by the system are demonstrated.

  5. Scanning Tunneling Microscopy of SILICON(100) 2 X 1

    Science.gov (United States)

    Hubacek, Jerome S.

    1992-01-01

    The Si(100) 2 x 1 surface, a technologically important surface in microelectronics and silicon molecular beam epitaxy (MBE), has been studied with the scanning tunneling microscope (STM) to attempt to clear up the controversy that surrounds previous studies of this surface. To this end, an ultra-high vacuum (UHV) STM/surface science system has been designed and constructed to study semiconductor surfaces. Clean Si(100) 2 x 1 surfaces have been prepared and imaged with the STM. Atomic resolution images probing both the filled states and empty states indicate that the surface consists of statically buckled dimer rows. With electronic device dimensions shrinking to smaller and smaller sizes, the Si-SiO_2 interface is becoming increasingly important and, although it is the most popular interface used in the microelectronics industry, little is known about the initial stages of oxidation of the Si(100) surface. Scanning tunneling microscopy has been employed to examine Si(100) 2 x 1 surfaces exposed to molecular oxygen in UHV. Ordered rows of bright and dark spots, rotated 45^circ from the silicon dimer rows, appear in the STM images, suggesting that the Si(100)-SiO_2 interface may be explained with a beta -cristobalite(100) structure rotated by 45^ circ on the Si(100) surface.

  6. Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

    Science.gov (United States)

    Seacor, Taylor; Howell, Carina

    2013-03-01

    Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

  7. Scanning probe microscopy investigation of complex-oxide heterostructures

    Science.gov (United States)

    Bi, Feng

    Advances in the growth of precisely tailored complex-oxide heterostructures have led to new emergent behavior and associated discoveries. One of the most successful examples consists of an ultrathin layer of LaAlO 3 (LAO) deposited on TiO2-terminated SrTiO3 (STO), where a high mobility quasi-two dimensional electron liquid (2DEL) is formed at the interface. Such 2DEL demonstrates a variety of novel properties, including field tunable metal-insulator transition, superconductivity, strong spin-orbit coupling, magnetic and ferroelectric like behavior. Particularly, for 3-unit-cell (3 u.c.) LAO/STO heterostructures, it was demonstrated that a conductive atomic force microscope (c-AFM) tip can be used to "write" or "erase" nanoscale conducting channels at the interface, making LAO/STO a highly flexible platform to fabricate novel nanoelectronics. This thesis is focused on scanning probe microscopy studies of LAO/STO properties. We investigate the mechanism of c-AFM lithography over 3 u.c. LAO/STO in controlled ambient conditions by using a vacuum AFM, and find that the water molecules dissociated on the LAO surface play a critical role during the c-AFM lithography process. We also perform electro-mechanical response measurements over top-gated LAO/STO devices. Simultaneous piezoresponse force microscopy (PFM) and capacitance measurements reveal a correlation between LAO lattice distortion and interfacial carrier density, which suggests that PFM could not only serve as a powerful tool to map the carrier density at the interface but also provide insight into previously reported frequency dependence of capacitance enhancement of top-gated LAO/STO structures. To study magnetism at the LAO/STO interface, magnetic force microscopy (MFM) and magnetoelectric force microscopy (MeFM) are carried out to search for magnetic signatures that depend on the carrier density at the interface. Results demonstrate an electronicallycontrolled ferromagnetic phase on top-gated LAO

  8. Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

    OpenAIRE

    Lawrence, J. R.; Swerhone, G. D. W.; Leppard, G. G.; T. Araki; Zhang, X.; West, M. M.; A. P. Hitchcock

    2003-01-01

    Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provid...

  9. Scanning Probe Microscopy at mK Temperatures *

    Science.gov (United States)

    Song, Young Jae

    2010-03-01

    Scanning probe microscopy has made significant advances with a wealth of new physics emerging as cryogenic instruments have been developed in the last decade allowing high resolution spectroscopic studies with spatial atomic resolution [1]. Most low temperature SPM instruments today operate at 4 K using liquid ^4He, with a few exceptions [2]. In this talk, we describe the next generation of ultra low temperature scanning probe microscope (SPM) with high magnetic field (15 T) capability operating at 10 mK using the circulation of a ^3He-^4He mixture in a dilution refrigerator (DR). With this system operating at 10 mK, we can extend the capability of scanning tunneling spectroscopy to higher energy resolution ( 3 μeV) for a range of applications in nanoscale systems. To achieve the design goal of mK operation for SPM applications we designed and constructed an ultra-high vacuum (UHV) SPM-compatible DR, an ultra-low temperature compatible SPM module, and extensive vibration isolation and RF shielding components. The DR was designed and constructed with features specific for UHV SPM applications, such as a Joule-Thomson (JT) condenser for lower noise operation. Noise measurements of the tunneling current show virtually no circulation-induced noise using the JT condenser, in contrast to noisy operation with a 1K pot. The custom-designed SPM module, with a three-axis position stage, is made from coin silver and ceramics for rigidity and thermal conduction in the mK regime. We also developed and constructed a low temperature current pre-amplifier, operating on the still at 650 mK, to circumvent problems due to long cable capacitances. Extensive noise measurements and first scanning measurements on graphene samples will be described. *In collaboration with Alexander F. Otte, Young Kuk, Phillip N. First, Walt A. de Heer, and Joseph A. Stroscio [1] D. L. Miller, et al., Science 324, 924 (2009) [2] A. J. Heinrich, et al., Science 306, 466 (2004)

  10. Microscopic techniques bridging between nanoscale and microscale with an atomically sharpened tip - field ion microscopy/scanning probe microscopy/ scanning electron microscopy.

    Science.gov (United States)

    Tomitori, Masahiko; Sasahara, Akira

    2014-11-01

    Over a hundred years an atomistic point of view has been indispensable to explore fascinating properties of various materials and to develop novel functional materials. High-resolution microscopies, rapidly developed during the period, have taken central roles in promoting materials science and related techniques to observe and analyze the materials. As microscopies with the capability of atom-imaging, field ion microscopy (FIM), scanning tunneling microscopy (STM), atomic force microscopy (AFM) and transmission electron microscopy (TEM) can be cited, which have been highly evaluated as methods to ultimately bring forward the viewpoint of reductionism in materials science. On one hand, there have been difficulties to derive useful and practical information on large (micro) scale unique properties of materials using these excellent microscopies and to directly advance the engineering for practical materials. To make bridges over the gap between an atomic scale and an industrial engineering scale, we have to develop emergence science step-by-step as a discipline having hierarchical structures for future prospects by combining nanoscale and microscale techniques; as promising ways, the combined microscopic instruments covering the scale gap and the extremely sophisticated methods for sample preparation seem to be required. In addition, it is noted that spectroscopic and theoretical methods should implement the emergence science.Fundamentally, the function of microscope is to determine the spatial positions of a finite piece of material, that is, ultimately individual atoms, at an extremely high resolution with a high stability. To define and control the atomic positions, the STM and AFM as scanning probe microscopy (SPM) have successfully demonstrated their power; the technological heart of SPM lies in an atomically sharpened tip, which can be observed by FIM and TEM. For emergence science we would like to set sail using the tip as a base. Meanwhile, it is significant

  11. Investigation of the Remineralization Effect Tnrough Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Damyanova Dobrinka M

    2016-05-01

    Full Text Available Background: Local fluoride varnishes have been widely used as a method of non-operative treatment and for caries preventive interventions for more than three decades. Purpose: Evaluation of the remineralization effect by means of electron microscopy of mineralization varnish - Clinpro ™ White Varnish with TCP (Tri-Calcium phosphate (3M. Materials and Methods: The material used is from 20 temporary intact teeth, extracted due to physiological change with permanent teeth, with a completely preserved structure and anatomy of crowns and fully physiologically resorbed roots. For the purposes of the study a scanning electron microscope JEOL JSM 6390 is used with an attachment for element analysis (EDS INCA of Oxford. Prepared samples are pre-coated with gold (cathode sputtering with apparatus JEOL JFC – 1200 to obtain a better contrast of the SEM image of early carious lesions on the smooth surfaces of the temporary teeth, with predilection for development of caries with a d1 threshold. For this purpose the two processes were monitored occurring continuously on the enamel surfacede- and remineralization. Performed was computer processing of the digital images. Results: There is presence of certain minerals deposited in the embossed enamel prisms after of remineralization. The chemical analysis established the presence of calcium (Ca2 + , around the organic matrix. Demineralised surface has pores present of around 1%, which is visible through the enamel on the surface of the deciduous teeth looking like filled and pores looking like partially covered, filled with newly formed and growing crystals. The crystals, which are hydroxylapatite, fluorapatite or fluorhydroxiapatite gradually connect, growing and forming mineral structure filling the microscopi defects and the pores from the demineralisation in the surface enamel prismless layer

  12. Preliminary Study of In Vivo Formed Dental Plaque Using Confocal Microscopy and Scanning Electron Microscopy

    Directory of Open Access Journals (Sweden)

    KA. Al-Salihi

    2009-12-01

    Full Text Available Objective: Confocal laser scanning microscopy (CLSM is relatively a new light microscopical imaging technique with a wide range of applications in biological sciences. The primary value of CLSM for the biologist is its ability to provide optical sections from athree-dimensional specimen. The present study was designed to assess the thickness and content of in vivo accumulated dental plaque using CLSM and scanning electron microscopy (SEM.Materials and Methods: Acroflat lower arch splints (acrylic appliance were worn by five participants for three days without any disturbance. The formed plaques were assessed using CLSM combined with vital fluorescence technique and SEM.Results: In this study accumulated dental plaque revealed varied plaque microflora vitality and thickness according to participant’s oral hygiene. The thickness of plaque smears ranged from 40.32 to 140.72 μm and 65.00 to 128.88 μm for live (vital and dead accumulated microorganisms, respectively. Meanwhile, the thickness of plaque on the appliance ranged from 101 μm to 653 μm. CLSM revealed both dead and vital bacteria on the surface of the dental plaque. In addition, SEM revealed layers of various bacterial aggregations in all dental plaques.Conclusion: This study offers a potent non-invasive tool to evaluate and assess the dental plaque biofilm, which is a very important factor in the development of dental caries.

  13. Simultaneous Nanomechanical and Electrochemical Mapping: Combining Peak Force Tapping Atomic Force Microscopy with Scanning Electrochemical Microscopy.

    Science.gov (United States)

    Knittel, Peter; Mizaikoff, Boris; Kranz, Christine

    2016-06-21

    Soft electronic devices play a crucial role in, e.g., neural implants as stimulating electrodes, transducers for biosensors, or selective drug-delivery. Because of their elasticity, they can easily adapt to their environment and prevent immunoreactions leading to an overall improved long-term performance. In addition, flexible electronic devices such as stretchable displays will be increasingly used in everyday life, e.g., for so-called electronic wearables. Atomic force microscopy (AFM) is a versatile tool to characterize these micro- and nanostructured devices in terms of their topography. Using advanced imaging techniques such as peak force tapping (PFT), nanomechanical properties including adhesion, deformation, and Young's modulus can be simultaneously mapped along with surface features. However, conventional AFM provides limited laterally resolved information on electrical or electrochemical properties such as the activity of an electrode array. In this study, we present the first combination of AFM with scanning electrochemical microscopy (SECM) in PFT mode, thereby offering spatially correlated electrochemical and nanomechanical information paired with high-resolution topographical data under force control (QNM-AFM-SECM). The versatility of this combined scanning probe approach is demonstrated by mapping topographical, electrochemical, and nanomechanical properties of gold microelectrodes and of gold electrodes patterned onto polydimethylsiloxane.

  14. Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

    Science.gov (United States)

    Blancett, Candace D; Fetterer, David P; Koistinen, Keith A; Morazzani, Elaine M; Monninger, Mitchell K; Piper, Ashley E; Kuehl, Kathleen A; Kearney, Brian J; Norris, Sarah L; Rossi, Cynthia A; Glass, Pamela J; Sun, Mei G

    2017-10-01

    A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Scanning near-field infrared microscopy on semiconductor structures

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, Rainer

    2011-01-15

    literature. While the structures of the first system were in the micrometer regime, the capability to probe buried nanostructures is demonstrated at a sample of indium arsenide quantum dots. Those dots are covered by a thick layer of gallium arsenide. For the first time ever, it is shown experimentally that transitions between electron states in single quantum dots can be investigated by near-field microscopy. By monitoring the near-field response of these quantum dots while scanning the wavelength of the incident light beam, it was possible to obtain characteristic near-field signatures of single dots. Near-field contrasts up to 30 % could be measured for resonant excitation of electrons in the conduction band of the indium arsenide dots. (orig.)

  16. 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

    Science.gov (United States)

    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

  17. 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

    Science.gov (United States)

    Russell, Matthew R G; 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; Collinson, Lucy M

    2017-01-01

    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. © 2017. Published by The Company of Biologists Ltd.

  18. Scanning SQUID microscopy of local superconductivity in inhomogeneous combinatorial ceramics.

    Science.gov (United States)

    Iranmanesh, Mitra; Stir, Manuela; Kirtley, John R; Hulliger, Jürg

    2014-11-24

    Although combinatorial solid-state chemistry promises to be an efficient way to search for new superconducting compounds, the problem of determining which compositions are strongly diamagnetic in a mixed-phase sample is challenging. By means of reactions in a system of randomly mixed starting components (Ca, Sr, Ba, La, Y, Pb, Bi, Tl, and Cu oxides), samples were produced that showed an onset of diamagnetic response above 115 K in bulk measurements. Imaging of this diamagnetic response in ceramic samples by scanning SQUID microscopy (SSM) revealed local superconducting areas with sizes down to as small as the spatial resolution of a few micrometers. In addition, locally formed superconducting matter was extracted from mixed-phase samples by magnetic separation. The analysis of single grains (d<80 μm) by X-ray diffraction, elemental analysis, and bulk SQUID measurements allowed Tl2Ca3Ba2Cu4O12, TlCaBaSrCu2O(7-δ), BaPb(0.5)Bi(0.25)Tl(0.25)O(3-δ), TlBa2Ca2Cu3O9, Tl2Ba2CaCu2O8, and YBa2Cu3O7 phases to be identified. SSM, in combination with other diagnostic techniques, is therefore shown to be a useful instrument to analyze inhomogeneous reaction products in the solid-state chemistry of materials showing magnetic properties.

  19. Non-thermal plasma mills bacteria: Scanning electron microscopy observations

    Energy Technology Data Exchange (ETDEWEB)

    Lunov, O., E-mail: lunov@fzu.cz; Churpita, O.; Zablotskii, V.; Jäger, A.; Dejneka, A. [Institute of Physics AS CR, Prague 18221 (Czech Republic); Deyneka, I. G.; Meshkovskii, I. K. [St. Petersburg State University of Information Technologies, Mechanics and Optics, St. Petersburg 197101 (Russian Federation); Syková, E. [Institute of Experimental Medicine AS CR, Prague 14220 (Czech Republic); Kubinová, Š. [Institute of Physics AS CR, Prague 18221 (Czech Republic); Institute of Experimental Medicine AS CR, Prague 14220 (Czech Republic)

    2015-02-02

    Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin–stained rat skin sections from plasma–treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.

  20. Band excitation method applicable to scanning probe microscopy

    Science.gov (United States)

    Jesse, Stephen; Kalinin, Sergei V

    2013-05-28

    Methods and apparatus are described for scanning probe microscopy. A method includes generating a band excitation (BE) signal having finite and predefined amplitude and phase spectrum in at least a first predefined frequency band; exciting a probe using the band excitation signal; obtaining data by measuring a response of the probe in at least a second predefined frequency band; and extracting at least one relevant dynamic parameter of the response of the probe in a predefined range including analyzing the obtained data. The BE signal can be synthesized prior to imaging (static band excitation), or adjusted at each pixel or spectroscopy step to accommodate changes in sample properties (adaptive band excitation). An apparatus includes a band excitation signal generator; a probe coupled to the band excitation signal generator; a detector coupled to the probe; and a relevant dynamic parameter extractor component coupled to the detector, the relevant dynamic parameter extractor including a processor that performs a mathematical transform selected from the group consisting of an integral transform and a discrete transform.

  1. Characterization of fragile nanostructures using scanning force microscopy

    Science.gov (United States)

    Buss, Michael Richard

    1998-12-01

    The possibility of using scanning force microscopy (SFM) to image monolayer arrays of nanometer diameter gold clusters on flat substrates has been investigated. A major difficulty in SFM studies of supported cluster arrays is the interaction force between the SFM probe and the clusters which results in the clusters moving on the substrate during imaging. The addition of a carbon nanotube to the tip of the SFM probe reduces this interaction force, resulting in stable, tapping mode height images of the supported cluster arrays. However, resolution of individual nanometer-size cluster in an array requires reduction of the endform radius of the nanotubes. In an attempt to reduce the effects of image dilation, the endform shape of the nanotube has been modified by spark etching. Also, metal atoms have been deposited onto the tip endform by sparking the tip of the nanotube by sparking the tip against a metal substrate and by electro-depositing metal onto the tip. Molecular dynamics (MD) simulations using embedded-atom-method (EAM) potentials have been used to model the compression behavior of nanometer-size gold clusters. The simulations examined the behavior of free and supported gold particles compressed with normal and shear forces. These results have been used to explain previously observed descrepencies between the calculated and measured elastic modulus of nanometer-size gold clusters.

  2. Energy dissipation measurements in frequency-modulated scanning probe microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Proksch, Roger [Asylum Research, Santa Barbara, CA (United States); Kalinin, Sergei V [Oak Ridge National Laboratory, Oak Ridge, TN (United States)

    2010-11-12

    Local dissipation measurements by scanning probe microscopy have attracted increasing interest as a method for probing energy losses and hysteretic phenomena due to magnetic, electrical, and structural transformations at the tip-surface junction. One challenge of this technique is the lack of a standard for ensuring quantification of the dissipation signal. In the following, we explored magnetic dissipation imaging of an yttrium-iron garnet (YIG) sample, using a number of similar but not identical cantilever probes. Typical frequency-dependent dispersion of the actuator-probe assembly commonly approached {+-} 1 part in 10{sup 3} Hz{sup -1}, much larger than the minimum detectable level of {+-} 1 part in 10{sup 5} Hz{sup -1}. This cantilever-dependent behavior results in a strong crosstalk between the conservative (frequency) and dissipative channels. This crosstalk was very apparent in the YIG dissipation images and in fact should be an inherent feature of single-frequency heterodyne detection schemes. It may also be a common effect in other dissipation imaging, even down to the atomic level, and in particular may be a significant issue when there are correlations between the conservative and dissipative components. On the other hand, we present a simple method for correcting for this effect. This correction technique resulted in self-consistent results for the YIG dissipation measurements and would presumably be effective for other systems as well.

  3. Scanning microwave microscopy technique for nanoscale characterization of magnetic materials

    Science.gov (United States)

    Joseph, C. H.; Sardi, G. M.; Tuca, S. S.; Gramse, G.; Lucibello, A.; Proietti, E.; Kienberger, F.; Marcelli, R.

    2016-12-01

    In this work, microwave characterization of magnetic materials using the scanning microwave microscopy (SMM) technique is presented. The capabilities of the SMM are employed for analyzing and imaging local magnetic properties of the materials under test at the nanoscale. The analyses are performed by acquiring both amplitude and phase of the reflected microwave signal. The changes in the reflection coefficient S11 are related to the local properties of the material under investigation, and the changes in its magnetic properties have been studied as a function of an external DC magnetic bias. Yttrium iron garnet (YIG) films deposited by RF sputtering and grown by liquid phase epitaxial (LPE) on gadolinium gallium garnet (GGG) substrates and permalloy samples have been characterized. An equivalent electromagnetic transmission line model is discussed for the quantitative analysis of the local magnetic properties. We also observed the hysteretic behavior of the reflection coefficient S11 with an external bias field. The imaging and spectroscopy analysis on the experimental results are evidently indicating the possibilities of measuring local changes in the intrinsic magnetic properties on the surface of the material.

  4. X-ray absorption measurement by scanning capacitance microscopy

    Science.gov (United States)

    Ishii, Masashi; Uchihashi, Takayuki

    2003-12-01

    This paper describes a demonstration of scanning capacitance microscopy (SCM) as a technique for measuring X-ray absorption fine structure (XAFS) in what is called the SCM-XAFS method. This method achieves the simultaneous analysis of the electrical and chemical characteristics of surface-trapping centers. In obtaining the XAFS spectrum of trapping centers, the method takes advantage of the fact that the X-ray-induced photoemission of a localized electron leads to a change in capacitance. When the Fermi level corresponds to the trapping level, the photoemission process is sensitively detected. Therefore, a specific trapping center may be selectively observed by controlling the bias. From SCM-XAFS measurements of a GaAs surface, we found that gallium oxide trapping centers capture electrons in the positive bias voltage region. Moreover, experimental findings that resonant intra-transition and resonant scattering of emitted photoelectrons enhance the SCM-XAFS signal at particular X-ray photon energy reveal the local density of states of the gallium oxide and the complex structure of the trapping centers.

  5. Materials characterisation by angle-resolved scanning transmission electron microscopy

    Science.gov (United States)

    Müller-Caspary, Knut; Oppermann, Oliver; Grieb, Tim; Krause, Florian F.; Rosenauer, Andreas; Schowalter, Marco; Mehrtens, Thorsten; Beyer, Andreas; Volz, Kerstin; Potapov, Pavel

    2016-11-01

    Solid-state properties such as strain or chemical composition often leave characteristic fingerprints in the angular dependence of electron scattering. Scanning transmission electron microscopy (STEM) is dedicated to probe scattered intensity with atomic resolution, but it drastically lacks angular resolution. Here we report both a setup to exploit the explicit angular dependence of scattered intensity and applications of angle-resolved STEM to semiconductor nanostructures. Our method is applied to measure nitrogen content and specimen thickness in a GaNxAs1‑x layer independently at atomic resolution by evaluating two dedicated angular intervals. We demonstrate contrast formation due to strain and composition in a Si- based metal-oxide semiconductor field effect transistor (MOSFET) with GexSi1‑x stressors as a function of the angles used for imaging. To shed light on the validity of current theoretical approaches this data is compared with theory, namely the Rutherford approach and contemporary multislice simulations. Inconsistency is found for the Rutherford model in the whole angular range of 16–255 mrad. Contrary, the multislice simulations are applicable for angles larger than 35 mrad whereas a significant mismatch is observed at lower angles. This limitation of established simulations is discussed particularly on the basis of inelastic scattering.

  6. Histological preparation of developing vestibular otoconia for scanning electron microscopy

    Science.gov (United States)

    Huss, D.; Dickman, J. D.

    2003-01-01

    The unique nature of vestibular otoconia as calcium carbonate biominerals makes them particularly susceptible to chemical deformation during histological processing. We fixed and stored otoconia from all three otolith endorgans of embryonic, hatchling and adult Japanese quail in glutaraldehyde containing either phosphate or non-phosphate buffers for varying lengths of time and processed them for scanning electron microscopy. Otoconia from all age groups and otolith endorgans processed in 0.1 M phosphate buffer (pH 7.4) showed abnormal surface morphology when compared to acetone fixed controls. Otoconia processed in 0.1 M sodium cacodylate or HEPES buffered artificial endolymph (pH 7.4) showed normal morphology that was similar to controls. The degree of otoconial deformation was directly related to the time exposed to phosphate buffer. Short duration exposure produced particulate deformations while longer exposures resulted in fused otoconia that formed solid sheets. Otoconial surface deformation and fusing was independent of the glutaraldehyde component of the histological processing. These findings should help vestibular researchers to develop appropriate histological processing protocols in future studies of otoconia.

  7. Scanning electron microscopy and roughness study of dental composite degradation.

    Science.gov (United States)

    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.

  8. Scanning electron microscopy of xiphinema, longidorus, and californidorus stylet morphology.

    Science.gov (United States)

    Cho, M R; Robbins, R T

    1990-04-01

    Stylet ultrastructure of five Xiphinema, four Longidorus, and three Californidorus species was compared by scanning electron microscopy. Morphological differences were seen in the odontophores and odontostyle bases between the genera and some of the species. All Xiphinema studied had well-developed odontophore flanges; the Longidorus species lacked flanges, except for weakly developed ones in L. diadecturus; and none of the Californidorus had flanges. Three sinuses were present in the odontophores of all species. The sinuses varied in length depending upon species. In Xiphinema and Californidorus the odontostyle bases had distinct overlapping collars, but in Longidorus the collars were absent except for L. diadecturus. The odontostyle-odontophore junction from a lateral view appeared as a slanted transverse line in all the species, but in a dorsal view of Xiphinema and Californidorus it was V-shaped. Dorsal longitudinal seams of the odontostyle and odontophore were observed in all the species. The dorsally located odontostyle aperture was ca. 1 mum from the anterior end in all species, except in one Longidorus sp. it was ca. 4 mum from the end.

  9. Bifurcation, chaos, and scan instability in dynamic atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Cantrell, John H., E-mail: john.h.cantrell@nasa.gov [Research Directorate, NASA Langley Research Center, Hampton, Virginia 23681 (United States); Cantrell, Sean A., E-mail: scantrell@nlsanalytics.com [NLS Analytics, LLC, 375 Dundee Road, Glencoe, Illinois 60022 (United States)

    2016-03-28

    The dynamical motion at any point on the cantilever of an atomic force microscope can be expressed quite generally as a superposition of simple harmonic oscillators corresponding to the vibrational modes allowed by the cantilever shape. Central to the dynamical equations is the representation of the cantilever-sample interaction force as a polynomial expansion with coefficients that account for the interaction force “stiffness,” the cantilever-to-sample energy transfer, and the displacement amplitude of cantilever oscillation. Renormalization of the cantilever beam model shows that for a given cantilever drive frequency cantilever dynamics can be accurately represented by a single nonlinear mass-spring model with frequency-dependent stiffness and damping coefficients [S. A. Cantrell and J. H. Cantrell, J. Appl. Phys. 110, 094314 (2011)]. Application of the Melnikov method to the renormalized dynamical equation is shown to predict a cascade of period doubling bifurcations with increasing cantilever drive force that terminates in chaos. The threshold value of the drive force necessary to initiate bifurcation is shown to depend strongly on the cantilever setpoint and drive frequency, effective damping coefficient, nonlinearity of the cantilever-sample interaction force, and the displacement amplitude of cantilever oscillation. The model predicts the experimentally observed interruptions of the bifurcation cascade for cantilevers of sufficiently large stiffness. Operational factors leading to the loss of image quality in dynamic atomic force microscopy are addressed, and guidelines for optimizing scan stability are proposed using a quantitative analysis based on system dynamical parameters and choice of feedback loop parameter.

  10. An overview on bioaerosols viewed by scanning electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Wittmaack, K. [GSF-National Research Centre for Environment and Health, Institute of Radiation Protection, 85758 Neuherberg (Germany)]. E-mail: wittmaack@gsf.de; Wehnes, H. [GSF-National Research Centre for Environment and Health, Institute of Pathology, 85758 Neuherberg (Germany); Heinzmann, U. [GSF-National Research Centre for Environment and Health, Institute of Pathology, 85758 Neuherberg (Germany); Agerer, R. [Ludwig-Maximilians University Munich, Department Biology, Biodiversity Research: Mycology, Menzinger Stasse 67, 80638 Munich (Germany)

    2005-06-15

    Bioaerosols suspended in ambient air were collected with single-stage impactors at a semiurban site in southern Germany during late summer and early autumn. Sampling was mostly carried out at a nozzle velocity of 35 m/s, corresponding to a minimum aerodynamic diameter (cut-off diameter) of aerosol particles of 0.8 {mu}m. The collected particles, sampled for short periods ({approx}15 min) to avoid pile-up, were characterized by scanning electron microscopy (SEM). The observed bioaerosols include brochosomes, fungal spores, hyphae, insect scales, hairs of plants and, less commonly, bacteria and epicuticular wax. Brochosomes, which serve as a highly water repellent body coating of leafhoppers, are hollow spheroids with diameters around 400 nm, resembling C{sub 60} or footballs (soccer balls). They are usually airborne not as individuals but in the form of large clusters containing up to 10,000 individual species or even more. Various types of spores and scales were observed, but assignment turned out be difficult due to the large number of fungi and insects from which they may have originated. Pollens were observed only once. The absence these presumably elastic particles suggests that they are frequently lost, at the comparatively high velocities, due to bounce-off from the nonadhesive impaction surfaces.

  11. Histological preparation of developing vestibular otoconia for scanning electron microscopy

    Science.gov (United States)

    Huss, D.; Dickman, J. D.

    2003-01-01

    The unique nature of vestibular otoconia as calcium carbonate biominerals makes them particularly susceptible to chemical deformation during histological processing. We fixed and stored otoconia from all three otolith endorgans of embryonic, hatchling and adult Japanese quail in glutaraldehyde containing either phosphate or non-phosphate buffers for varying lengths of time and processed them for scanning electron microscopy. Otoconia from all age groups and otolith endorgans processed in 0.1 M phosphate buffer (pH 7.4) showed abnormal surface morphology when compared to acetone fixed controls. Otoconia processed in 0.1 M sodium cacodylate or HEPES buffered artificial endolymph (pH 7.4) showed normal morphology that was similar to controls. The degree of otoconial deformation was directly related to the time exposed to phosphate buffer. Short duration exposure produced particulate deformations while longer exposures resulted in fused otoconia that formed solid sheets. Otoconial surface deformation and fusing was independent of the glutaraldehyde component of the histological processing. These findings should help vestibular researchers to develop appropriate histological processing protocols in future studies of otoconia.

  12. Scanning electron microscopy applied to seed-borne fungi examination.

    Science.gov (United States)

    Alves, Marcelo de Carvalho; Pozza, Edson Ampélio

    2009-07-01

    The aim of this study was to test the standard scanning electron microscopy (SEM) as a potential alternative to study seed-borne fungi in seeds, by two different conditions of blotter test and water restriction treatment. In the blotter test, seeds were subjected to conditions that enabled pathogen growth and expression, whereas the water restriction method consisted in preventing seed germination during the incubation period, resulting in the artificial inoculation of fungi. In the first condition, seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.), and cotton (Gossypium hirsutum L.) were submitted to the standard blotter test and then prepared and observed with SEM. In the second condition, seeds of cotton (G. hirsutum), soybean (Glycine max L.), and common bean (P. vulgaris L.) were, respectively, inoculated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum, and Colletotrichum lindemuthianum by the water restriction technique, followed by preparation and observation with SEM. The standard SEM methodology was adopted to prepare the specimens. Considering the seeds submitted to the blotter test, it was possible to identify Fusarium sp. on maize, C. gossypii var. cephalosporioides, and Fusarium oxysporum on cotton, Aspergillus flavus, Penicillium sp., Rhizopus sp., and Mucor sp. on common bean. Structures of C. gossypii var. cephalosporioides, C. truncatum, and C. lindemuthianum were observed in the surface of inoculated seeds. (c) 2009 Wiley-Liss, Inc.

  13. Thermal mapping of a scanning thermal microscopy tip.

    Science.gov (United States)

    Jóźwiak, Grzegorz; Wielgoszewski, Grzegorz; Gotszalk, Teodor; Kępiński, Leszek

    2013-10-01

    Scanning thermal microscopy (SThM) is a very promising technique for local investigation of temperature and thermal properties of nanostructures with great application potential in contemporary nanoelectronics and nanotechnology. In order to increase the localization of SThM measurements, the size of probes has recently substantially decreased, which results in novel types of SThM probes manufactured with the use of modern silicon microfabrication technology. Quantitative SThM measurements with these probes need methods, which enable to assess the quality of thermal contact between the probe and the investigated surface. In this paper we propose a tip thermal mapping (TThM) procedure, which is used to estimate experimentally the distribution of power dissipated by the tip of an SThM probe. We also show that the proposed power dissipation model explains the results of active-mode SThM measurements and that the TThM procedure is reversible for a given probe and sample. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Scanning Auger microscopy for high lateral and depth elemental sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, E., E-mail: eugenie.martinez@cea.fr [CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9 (France); Yadav, P. [CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9 (France); Bouttemy, M. [Institut Lavoisier de Versailles, 45 av. des Etats-Unis, 78035 Versailles Cedex (France); Renault, O.; Borowik, Ł.; Bertin, F. [CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9 (France); Etcheberry, A. [Institut Lavoisier de Versailles, 45 av. des Etats-Unis, 78035 Versailles Cedex (France); Chabli, A. [CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9 (France)

    2013-12-15

    Highlights: •SAM performances and limitations are illustrated on real practical cases such as the analysis of nanowires and nanodots. •High spatial elemental resolution is shown with the analysis of reference semiconducting Al{sub 0.7}Ga{sub 0.3}As/GaAs multilayers. •High in-depth elemental resolution is also illustrated. Auger depth profiling with low energy ion beams allows revealing ultra-thin layers (∼1 nm). •Analysis of cross-sectional samples is another effective approach to obtain in-depth elemental information. -- Abstract: Scanning Auger microscopy is currently gaining interest for investigating nanostructures or thin multilayers stacks developed for nanotechnologies. New generation Auger nanoprobes combine high lateral (∼10 nm), energy (0.1%) and depth (∼2 nm) resolutions thus offering the possibility to analyze the elemental composition as well as the chemical state, at the nanometre scale. We report here on the performances and limitations on practical examples from nanotechnology research. The spatial elemental sensitivity is illustrated with the analysis of Al{sub 0.7}Ga{sub 0.3}As/GaAs heterostructures, Si nanowires and SiC nanodots. Regarding the elemental in-depth composition, two effective approaches are presented: low energy depth profiling to reveal ultra-thin layers (∼1 nm) and analysis of cross-sectional samples.

  15. Alternating current scanning electrochemical microscopy with simultaneous fast-scan cyclic voltammetry.

    Science.gov (United States)

    Koch, Jason A; Baur, Melinda B; Woodall, Erica L; Baur, John E

    2012-11-06

    Fast-scan cyclic voltammetry (FSCV) is combined with alternating current scanning electrochemical microscopy (AC-SECM) for simultaneous measurements of impedance and faradaic current. Scan rates of 10-1000 V s(-1) were used for voltammetry, while a high-frequency (100 kHz), low-amplitude (10 mV rms) sine wave was added to the voltammetric waveform for the ac measurement. Both a lock-in amplifier and an analog circuit were used to measure the amplitude of the resultant ac signal. The effect of the added sine wave on the voltammetry at a carbon fiber electrode was investigated and found to have negligible effect. The combined FSCV and ac measurements were used to provide simultaneous chemical and topographical information about a substrate using a single carbon fiber probe. The technique is demonstrated in living cell culture, where cellular respiration and topography were simultaneously imaged without the addition of a redox mediator. This approach promises to be useful for the topographical and multidimensional chemical imaging of substrates.

  16. Holographic Two-Photon Induced Photopolymerization

    Data.gov (United States)

    Federal Laboratory Consortium — Holographic two-photon-induced photopolymerization (HTPIP) offers distinct advantages over conventional one-photon-induced photopolymerization and current techniques...

  17. Quantitative Scanning Transmission Electron Microscopy of Electronic and Nanostructured Materials

    Science.gov (United States)

    Yankovich, Andrew B.

    Electronic and nanostructured materials have been investigated using advanced scanning transmission electron microscopy (STEM) techniques. The first topic is the microstructure of Ga and Sb-doped ZnO. Ga-doped ZnO is a candidate transparent conducting oxide material. The microstructure of GZO thin films grown by MBE under different growth conditions and different substrates were examined using various electron microscopy (EM) techniques. The microstructure, prevalent defects, and polarity in these films strongly depend on the growth conditions and substrate. Sb-doped ZnO nanowires have been shown to be the first route to stable p-type ZnO. Using Z-contrast STEM, I have showed that an unusual microstructure of Sb-decorated head-to-head inversion domain boundaries and internal voids contain all the Sb in the nanowires and cause the p-type conduction. InGaN thin films and InGaN / GaN quantum wells (QW) for light emitting diodes are the second topic. Low-dose Z-contrast STEM, PACBED, and EDS on InGaN QW LED structures grown by MOCVD show no evidence for nanoscale composition variations, contradicting previous reports. In addition, a new extended defect in GaN and InGaN was discovered. The defect consists of a faceted pyramid-shaped void that produces a threading dislocation along the [0001] growth direction, and is likely caused by carbon contamination during growth. Non-rigid registration (NRR) and high-precision STEM of nanoparticles is the final topic. NRR is a new image processing technique that corrects distortions arising from the serial nature of STEM acquisition that previously limited the precision of locating atomic columns and counting the number of atoms in images. NRR was used to demonstrate sub-picometer precision in STEM images of single crystal Si and GaN, the best achieved in EM. NRR was used to measure the atomic surface structure of Pt nanoacatalysts and Au nanoparticles, which revealed new bond length variation phenomenon of surface atoms. In

  18. Automated Quantitative Rare Earth Elements Mineralogy by Scanning Electron Microscopy

    Science.gov (United States)

    Sindern, Sven; Meyer, F. Michael

    2016-09-01

    Increasing industrial demand of rare earth elements (REEs) stems from the central role they play for advanced technologies and the accelerating move away from carbon-based fuels. However, REE production is often hampered by the chemical, mineralogical as well as textural complexity of the ores with a need for better understanding of their salient properties. This is not only essential for in-depth genetic interpretations but also for a robust assessment of ore quality and economic viability. The design of energy and cost-efficient processing of REE ores depends heavily on information about REE element deportment that can be made available employing automated quantitative process mineralogy. Quantitative mineralogy assigns numeric values to compositional and textural properties of mineral matter. Scanning electron microscopy (SEM) combined with a suitable software package for acquisition of backscatter electron and X-ray signals, phase assignment and image analysis is one of the most efficient tools for quantitative mineralogy. The four different SEM-based automated quantitative mineralogy systems, i.e. FEI QEMSCAN and MLA, Tescan TIMA and Zeiss Mineralogic Mining, which are commercially available, are briefly characterized. Using examples of quantitative REE mineralogy, this chapter illustrates capabilities and limitations of automated SEM-based systems. Chemical variability of REE minerals and analytical uncertainty can reduce performance of phase assignment. This is shown for the REE phases parisite and synchysite. In another example from a monazite REE deposit, the quantitative mineralogical parameters surface roughness and mineral association derived from image analysis are applied for automated discrimination of apatite formed in a breakdown reaction of monazite and apatite formed by metamorphism prior to monazite breakdown. SEM-based automated mineralogy fulfils all requirements for characterization of complex unconventional REE ores that will become

  19. Engineering and Characterization of Collagen Networks Using Wet Atomic Force Microscopy and Environmental Scanning Electron Microscopy

    Science.gov (United States)

    Osborn, Jenna; Coffey, Tonya; Conrad, Brad; Burris, Jennifer; Hester, Brooke

    2014-03-01

    Collagen is an abundant protein and its monomers covalently crosslink to form fibrils which form fibers which contribute to forming macrostructures like tendon or bone. While the contribution is well understood at the macroscopic level, it is not well known at the fibril level. We wish to study the mechanical properties of collagen for networks of collagen fibers that vary in size and density. We present here a method to synthesize collagen networks from monomers and that allows us to vary the density of the networks. By using biotynilated collagen and a surface that is functionalized with avidin, we generate two-dimensional collagen networks across the surface of a silicon wafer. During network synthesis, the incubation time is varied from 30 minutes to 3 hours or temperature is varied from 25°C to 45°C. The two-dimensional collagen network created in the process is characterized using environmental atomic force microscopy (AFM) and scanning electron microscopy (SEM). The network density is measured by the number of strands in one frame using SPIP software. We expect that at body temperature (37°C) and with longer incubation times, the network density should increase.

  20. Scanning tunneling microscopy III theory of STM and related scanning probe methods

    CERN Document Server

    Güntherodt, Hans-Joachim

    1993-01-01

    While the first two volumes on Scanning Tunneling Microscopy (STM) and its related scanning probe (SXM) methods have mainly concentrated on intro­ ducing the experimental techniques, as well as their various applications in different research fields, this third volume is exclusively devoted to the theory of STM and related SXM methods. As the experimental techniques including the reproducibility of the experimental results have advanced, more and more theorists have become attracted to focus on issues related to STM and SXM. The increasing effort in the development of theoretical concepts for STM/SXM has led to considerable improvements in understanding the contrast mechanism as well as the experimental conditions necessary to obtain reliable data. Therefore, this third volume on STM/SXM is not written by theorists for theorists, but rather for every scientist who is not satisfied by just obtaining real­ space images of surface structures by STM/SXM. After a brief introduction (Chap. 1), N. D. Lang first co...

  1. Observation of the sweating in lipstick by scanning electron microscopy.

    Science.gov (United States)

    Seo, S Y; Lee, I S; Shin, H Y; Choi, K Y; Kang, S H; Ahn, H J

    1999-06-01

    The relationship between the wax matrix in lipstick and sweating has been investigated by observing the change of size and shape of the wax matrix due to sweating by Scanning Electron Microscopy (SEM). For observation by SEM, a lipstick sample was frozen in liquid nitrogen. The oil in the lipstick was then extracted in cold isopropanol (-70 degrees C) for 1-3 days. After the isopropanol was evaporated, the sample was sputtered with gold and examined by SEM. The change of wax matrix underneath the surface from fine, uniform structure to coarse, nonuniform structure resulted from the caking of surrounding wax matrix. The oil underneath the surface migrated to the surface of lipstick with sweating; consequently the wax matrix in that region was rearranged into the coarse matrix. In case of flamed lipstick, sweating was delayed and the wax matrix was much coarser than that of the unflamed one. The larger wax matrix at the surface region was good for including oil. The effect of molding temperature on sweating was also studied. As the molding temperature rose, sweating was greatly reduced and the size of the wax matrix increased. It was found that sweating was influenced by the compatibility of wax and oil. A formula consisting of wax and oil that have good compatibility has a tendency to reduce sweating and increase the size of the wax matrix. When pigments were added to wax and oil, the size of the wax matrix was changed, but in all cases sweating was increased due to the weakening of the binding force between wax and oil. On observing the thick membrane of wax at the surface of lipstick a month after molding it was also found that sweating was influenced by ageing. In conclusion, the structure of the wax matrix at the surface region of lipstick was changed with the process of flaming, molding temperature, compatibility of wax and oil, addition of pigment, and ageing. In most cases, as the size of the wax matrix was increased, sweating was reduced and delayed.

  2. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum’s magnetosome chains

    Energy Technology Data Exchange (ETDEWEB)

    Keutner, Christoph [Technische Univ. Dortmund, Dortmung (Germany); von Bohlen, Alex [Leibniz-Institut fur Analytische Wissenschaften, Dortmund (Germany); Berges, Ulf [Technische Univ. Dortmund, Dortmung (Germany); Espeter, Philipp [Technische Univ. Dortmund, Dortmung (Germany); Schneider, Claus M. [Peter Grunberg Institut, Julich (Germany); Westphal, Carsten [Technische Univ. Dortmund, Dortmung (Germany)

    2014-10-07

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  3. Synthesis of two carbazole-based dyes and application of two-photon initiating polymerization

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Two carbazole-based polymerization initiators possessing blue fluorescence emission have been synthesized via Wittig reaction in the solid phase at room temperature.Two-photon excited fluorescence(TPEF) spectra for them were investigated under 800 nm fs laser pulse and two-photon absorption cross sections were determined by the Z-scan technique.Then two-photon initiating polymerization(TPIP) microfabrication experiments were successfully carried out.Three-dimensional lattice and artificial defects were gained,indicating that they were viable candidates for the two-photon polymerization initiator in practical application of microfabrication.

  4. Synthesis of two carbazole-based dyes and application of two-photon initiating polymerization

    Institute of Scientific and Technical Information of China (English)

    HU RenTao; LU LiangFei; RUAN BanFeng; WANG Peng; ZHANG MingLiang; ZHOU HongPing; LI ShengLi; WU JieYing; TIAN YuPeng

    2009-01-01

    Two carbazole-based polymerization initiators possessing blue fluorescence emission have been synthesized via Wittig reaction in the solid phase at room temperature.Two-photon excited fluorescence (TPEF) spectra for them were investigated under 800 nm fs laser pulse and two-photon absorption cross sections were determined by the Z-scan technique.Then two-photon initiating polymerization (TPIP) microfabrication experiments were successfully carried out.Three-dimensional lattice and artificial defects were gained,indicating that they were viable candidates for the two-photon polymerization initiator in practical application of microfabrication.

  5. Two-photon absorption-induced photoacoustic imaging of Rhodamine B dyed polyethylene spheres using a femtosecond laser.

    Science.gov (United States)

    Langer, Gregor; Bouchal, Klaus-Dieter; Grün, Hubert; Burgholzer, Peter; Berer, Thomas

    2013-09-23

    In the present paper we demonstrate the possibility to image dyed solids, i.e. Rhodamine B dyed polyethylene spheres, by means of two-photon absorption-induced photoacoustic scanning microscopy. A two-photon luminescence image is recorded simultaneously with the photoacoustic image and we show that location and size of the photoacoustic and luminescence image match. In the experiments photoacoustic signals and luminescence signals are generated by pulses from a femtosecond laser. Photoacoustic signals are acquired with a hydrophone; luminescence signals with a spectrometer or an avalanche photo diode. In addition we derive the expected dependencies between excitation intensity and photoacoustic signal for single-photon absorption, two-photon absorption and for the combination of both. In order to verify our setup and evaluation method the theoretical predictions are compared with experimental results for liquid and solid specimens, i.e. a carbon fiber, Rhodamine B solution, silicon, and Rhodamine B dyed microspheres. The results suggest that the photoacoustic signals from the Rhodamine B dyed microspheres do indeed stem from two-photon absorption.

  6. Optical characterication of probes for photon scanning tunnelling microscopy

    DEFF Research Database (Denmark)

    Vohnsen, Brian; Bozhevolnyi, Sergey I.

    1999-01-01

    The photon scanning tunnelling microscope is a well-established member of the family of scanning near-field optical microscopes used for optical imaging at the sub-wavelength scale. The quality of the probes, typically pointed uncoated optical fibres, used is however difficult to evaluate...

  7. Second-harmonic scanning optical microscopy of semiconductor quantum dots

    DEFF Research Database (Denmark)

    Vohnsen, B.; Bozhevolnyi, S.I.; Pedersen, K.;

    2001-01-01

    Second-harmonic (SH) optical imaging of self-assembled InAlGaAs quantum dots (QD's) grown on a GaAs(0 0 1) substrate has been accomplished at room temperature by use of respectively a scanning far-field optical microscope in reflection mode and a scanning near-field optical microscope...

  8. Two-photon imaging through a multimode fiber

    CERN Document Server

    Morales-Delgado, Edgar E; Moser, Christophe

    2015-01-01

    In this work we demonstrate 3D imaging using two-photon excitation through a 20 cm long multimode optical fiber (MMF) of 350 micrometers diameter. The imaging principle is similar to single photon fluorescence through a MMF, except that a focused femtosecond pulse is delivered and scanned over the sample. In our approach, focusing and scanning through the fiber is accomplished by digital phase conjugation using mode selection by time gating with an ultra-fast reference pulse. The excited two-photon emission is collected through the same fiber. We demonstrate depth sectioning by scanning the focused pulse in a 3D volume over a sample consisting of fluorescent beads suspended in a polymer. The achieved resolution is 1 micrometer laterally and 15 micrometers axially. Scanning is performed over an 80x80 micrometers field of view. To our knowledge, this is the first demonstration of high-resolution three-dimensional imaging using two-photon fluorescence through a multimode fiber.

  9. Fano interference in two-photon transport

    Science.gov (United States)

    Xu, Shanshan; Fan, Shanhui

    2016-10-01

    We present a general input-output formalism for the few-photon transport in multiple waveguide channels coupled to a local cavity. Using this formalism, we study the effect of Fano interference in two-photon quantum transport. We show that the physics of Fano interference can manifest as an asymmetric spectral line shape in the frequency dependence of the two-photon correlation function. The two-photon fluorescence spectrum, on the other hand, does not exhibit the physics of Fano interference.

  10. Visualization of Microbial Biomarkers by Scanning Electron Microscopy

    Science.gov (United States)

    Wainwright, Norman R.; Allen, Carlton C.; Child, Alice

    2001-01-01

    . Fortunately, many antimicrobial defense systems of higher organisms require sensitive detection to combat microbial pathogens. We employ here the primitive immune system of the evolutionarily ancient horseshoe crab, Limulus polyphemus. This species relies on multi-enzyme signal amplification detection of cell wall molecules and they can be applied to the development of useful detectors of life. An extension of this work includes the visualization of microbial signatures by labeling LAL components with chromogenic or electron dense markers. The protein Limulus Anti-LPS Factor (LALF) has an extremely high affinity for LPS. By coupling LALF binding with colloidal gold labels we demonstrate a correlation of the structures visible by electron microscopy with biochemical evidence of microbial cell wall materials. Pure silica particles were mixed with cultures of E. coli (10(exp 6) cfu/mL). Samples were washed sequentially with buffered saline, LALF, antibody to LALF and finally colloidal gold-labeled Protein A. Negative controls were not exposed to E. coli but received identical treatment otherwise. Samples were coated with carbon and imaged on a JEOL JSM-840 scanning electron microscope with LaB6 source in the back scatter mode with the JEOL annular back scatter detector. 20 nm-scale black spots in this contrast-reversed image originate from electrons back-scattered by gold atoms. Negative controls did not give any signal. Future work will expand application of this technique to soil simulants and mineralized rock samples.

  11. Visualization of Microbial Biomarkers by Scanning Electron Microscopy

    Science.gov (United States)

    Wainwright, Norman R.; Allen, Carlton C.; Child, Alice

    2001-01-01

    . Fortunately, many antimicrobial defense systems of higher organisms require sensitive detection to combat microbial pathogens. We employ here the primitive immune system of the evolutionarily ancient horseshoe crab, Limulus polyphemus. This species relies on multi-enzyme signal amplification detection of cell wall molecules and they can be applied to the development of useful detectors of life. An extension of this work includes the visualization of microbial signatures by labeling LAL components with chromogenic or electron dense markers. The protein Limulus Anti-LPS Factor (LALF) has an extremely high affinity for LPS. By coupling LALF binding with colloidal gold labels we demonstrate a correlation of the structures visible by electron microscopy with biochemical evidence of microbial cell wall materials. Pure silica particles were mixed with cultures of E. coli (10(exp 6) cfu/mL). Samples were washed sequentially with buffered saline, LALF, antibody to LALF and finally colloidal gold-labeled Protein A. Negative controls were not exposed to E. coli but received identical treatment otherwise. Samples were coated with carbon and imaged on a JEOL JSM-840 scanning electron microscope with LaB6 source in the back scatter mode with the JEOL annular back scatter detector. 20 nm-scale black spots in this contrast-reversed image originate from electrons back-scattered by gold atoms. Negative controls did not give any signal. Future work will expand application of this technique to soil simulants and mineralized rock samples.

  12. Adiabatic following in two-photon transition

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.; Nayfeh, A.H.

    1977-01-01

    There has been much interest recently in coherent multiphoton transitions in many-level systems. The present work considers the effect of relaxation in the response of a three-level system to a smoothly varying, near-resonant, two-photon field. The relaxation-dependent contributions to the nonlinear refractive index are calculated. It is shown that the coherent interaction of two smoothly varying, near-resonant, two-photon pulses with a three-level system can be described by ''two-photon damped Bloch equations'' which are analogous to those for a one-photon transition in a two-level system except for the presence of a two-photon coupling and a frequency shift. 1 figure. (RWR)

  13. Two-Photon Physics in Hadronic Processes

    Energy Technology Data Exchange (ETDEWEB)

    Carl Carlson; Marc Vanderhaeghen

    2007-11-01

    Two-photon exchange contributions to elastic electron-scattering are reviewed. The apparent discrepancy in the extraction of elastic nucleon form factors between unpolarized Rosenbluth and polarization transfer experiments is discussed, as well as the understanding of this puzzle in terms of two-photon exchange corrections. Calculations of such corrections both within partonic and hadronic frameworks are reviewed. In view of recent spin-dependent electron scattering data, the relation of the two-photon exchange process to the hyperfine splitting in hydrogen is critically examined. The imaginary part of the two-photon exchange amplitude as can be accessed from the beam normal spin asymmetry in elastic electron-nucleon scattering is reviewed. Further extensions and open issues in this field are outlined.

  14. Sideband-Induced Two-Photon Transparency

    Institute of Scientific and Technical Information of China (English)

    CHENG Guang-Ling; HU Xiang-Ming

    2006-01-01

    @@ We show that it is possible to use a single sideband to induce two-photon transparency in a three-level cascade medium. The medium simultaneously absorbs two photons as a one-step process when the middle level is far off one-photon resonance. A resonant sideband coupling on the upper transition and the two-photon one-step process drive the medium into a trapped state, and the dominant component is the ground state. Thus almost all population is trapped in the ground state and the two-photon absorption is dramatically suppressed. We present a numerical calculation for arbitrary values of the atomic and field parameters and also provide an analytic description for the required conditions.

  15. Real-time high dynamic range laser scanning microscopy

    Science.gov (United States)

    Vinegoni, C.; Leon Swisher, C.; Fumene Feruglio, P.; Giedt, R. J.; Rousso, D. L.; Stapleton, S.; Weissleder, R.

    2016-04-01

    In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

  16. Real-time high dynamic range laser scanning microscopy.

    Science.gov (United States)

    Vinegoni, C; Leon Swisher, C; Fumene Feruglio, P; Giedt, R J; Rousso, D L; Stapleton, S; Weissleder, R

    2016-04-01

    In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

  17. A compact two photon light sheet microscope for applications in neuroscience

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2016-01-01

    We present a compact setup for two photon light sheet microscopy. By using pulsed Airy beam illumination we demonstrate eight-fold increase of the FOV compared to Gaussian light sheet with the same axial resolution....

  18. Correlations of two photons at hadron colliders

    OpenAIRE

    Kozlov, G. A.

    2011-01-01

    We study the Bose-Einstein correlations of two photons and their coherent properties that can provide the information about the space-time structure of the emitting source through the Higgs-boson decays into two photons. We argue that such an investigation could probe the Higgs-boson mass. The model is rather sensitive to the temperature of the environment and to the external distortion effect in medium.

  19. Platinum Acetylide Two-Photon Chromophores (Preprint)

    Science.gov (United States)

    2007-04-01

    the higher energy range that lead to its photodegradation . Secondly, because there is a quadratic dependence of two-photon absorption (2PA) on the...to either an electron donating amino- fluorenyl or electron withdrawing benzothiazolyl-fluorene that are themselves known as two-photon absorbing dyes ...groups in place of phenyl groups have shown a doubling of the intrinsic cr2value at 740 nm.40,41In this paper we describe novel platinum dyes that

  20. Surfaces of III-V semiconductors studied by scanning tunneling microscopy and scanning luminescence

    Science.gov (United States)

    Chizhov, Ilya Yu

    The investigation of semiconductor surfaces on an atomic scale is of key importance for research areas such as the growth of thin films and low-dimensional objects by epitaxial techniques. Surfaces of III-V semiconductors, especially GaAs(001) surface, hold a central position in semiconductor surface science due to their extremely wide use in the growth of heterostructure-based electronic and optoelectronic devices. This work describes the results of experimental studies of (001) surfaces of two III-V semiconductors, GaAs and InAs, using scanning tunneling microscopy (STM) and scanning tunneling luminescence (STL). For STL studies an optical detection system consisting of an optical spectrometer (replacable by a photomultiplier tube), electronics, an IBM PC computer and acquisition software has been added to an existing STM (Omicron). The system is capable of recording luminescence images of surfaces and the acquisition of optical spectra of STM-induced luminescence. Application of STL to GaAs(001) surfaces has revealed that atomic-scale features, such as steps, domain boundaries etc., do not give any contrast in luminescence images, while larger objects, such as arsenic islands, do produce a pronounced contrast. The work in STL has helped to identify several key problems that have to be solved in order to make STL a valuable analytical technique. The STM studies of reconstructions on the GaAs(001) surface, which has a fairly complicated phase diagram, have concentrated on two major phase transitions, from As-rich c(4× 4) to As-rich (2× 4) phase and from As-rich (2× 4) to Ga-rich (4× 2) phase. The first transition has been found to proceed through an intermediate (4× 3)/c(4× 6) phase which has been previously identified as having (2× 3) symmetry. The second transition has been found to involve the formation of (3× 6) and (4× 6) multi-domain phases. The local structure and composition of these phases have been analyzed in detail and a comprehensive dynamic

  1. Cytogenetic Characterization of the TM4 Mouse Sertoli Cell Line. II. Chromosome Microdissection, FISH, Scanning Electron Microscopy, and Confocal Laser Scanning Microscopy.

    Science.gov (United States)

    Schmid, Michael; Guttenbach, Martina; Steinlein, Claus; Wanner, Gerhard; Houben, Andreas

    2015-01-01

    The chromosomes and interphase cell nuclei of the permanent mouse Sertoli cell line TM4 were examined by chromosome microdissection, FISH, scanning electron microscopy, and confocal laser scanning microscopy. The already known marker chromosomes m1-m5 were confirmed, and 2 new large marker chromosomes m6 and m7 were characterized. The minute heterochromatic marker chromosomes m4 and m5 were microdissected and their DNA amplified by DOP-PCR. FISH of this DNA probe on TM4 metaphase chromosomes demonstrated that the m4 and m5 marker chromosomes have derived from the centromeric regions of normal telocentric mouse chromosomes. Ectopic pairing of the m4 and m5 marker chromosomes with the centromeric region of any of the other chromosomes (centromeric associations) was apparent in ∼60% of the metaphases. Scanning electron microscopy revealed DNA-protein bridges connecting the centromeric regions of normal chromosomes and the associated m4 and m5 marker chromosomes. Interphase cell nuclei of TM4 Sertoli cells did not exhibit the characteristic morphology of Sertoli cells in the testes of adult mice as shown by fluorescence microscopy and confocal laser scanning microscopy.

  2. Ultrastructure of Proechinophthirus zumpti (Anoplura, Echinophthiriidae by scanning electron microscopy

    Directory of Open Access Journals (Sweden)

    Dolores del Carmen Castro

    2002-09-01

    Full Text Available The ultrastructure of Proechinophthirus zumpti Werneck, 1955, mainly the external chorionic features of the egg, is described through electronic microscopy techniques. This species was first cited in Argentina, infesting Arctocephalus australis (Zimmermann, 1873. The morphological adaptations of adults and nymphs are described in both species of Proechinophthirus parasitic on Otariidae: P. fluctus (Ferris, 1916 and P. zumpti.

  3. Scanning Probe Microscopy at 650 °C in Air

    DEFF Research Database (Denmark)

    Hansen, Karin Vels; Jacobsen, Torben; Nørgaard, Anne-Mette

    2009-01-01

    The controlled atmosphere high temperature scanning probe microscope was designed to study the electrical properties of surfaces at elevated temperatures by using the probe as an electrode. The capability of a simultaneous acquisition of topographical and electrical data for the same surface area...

  4. Scanning Emitter Lifetime Imaging Microscopy for Spontaneous Emission Control

    DEFF Research Database (Denmark)

    Frimmer, Martin; Chen, Yuntian; Koenderink, A. Femius

    2011-01-01

    We report an experimental technique to map and exploit the local density of optical states of arbitrary planar nanophotonic structures. The method relies on positioning a spontaneous emitter attached to a scanning probe deterministically and reversibly with respect to its photonic environment while...

  5. Evaluation of the bleached human enamel by Scanning Electron Microscopy

    DEFF Research Database (Denmark)

    Miranda, Carolina Baptista; Pagani, Clovis; Benetti, Ana Raquel

    2005-01-01

    Since bleaching has become a popular procedure, the effect of peroxides on dental hard tissues is of great interest in research. Purpose: The aim of this in vitro study was to perform a qualitative analysis of the human enamel after the application of in-office bleaching agents, using Scanning El...

  6. Apparent Barrier Height in Scanning Tunneling Microscopy Revisited

    DEFF Research Database (Denmark)

    Olesen, L.; Brandbyge, Mads; Sørensen, Mads Reinholdt;

    1996-01-01

    The apparent barrier height phi(ap), that is, the rate of change of the logarithm of the conductance with tip-sample separation in a scanning tunneling microscope (STM), has been measured for Ni, Pt, and Au single crystal surfaces. The results show that phi(ap) is constant until point contact...

  7. Scanning electron microscopy of the male genitalia of Sarcophagidae (Diptera

    Directory of Open Access Journals (Sweden)

    Hugo de Souza Lopes

    1990-03-01

    Full Text Available The male genitalia of nine species of Sarcophagidae (Diptera - Goniophyto honsuensis Rohdendorf, 1962, Tricharaea brevicornis (Wiedemann, 1830, Chaetoravinia derelicta (Walker, 1852, Austrohartigia spinigena (Rondani, 1864, Chrysagria duodecimpunctata Townsend, 1935, Boettcheria bisetosa Parker, 1914, Lipoptilocnema lanei Townsend, 1934, L. crispina (Lopes, 1938 and Euboettcheria alvarengai Lopes & Tibana, 1982 - were examined by scanning electron microscope (SEM and the main morphological features are descirbed.

  8. Cryo scanning electron microscopy of Plasmodium falciparum-infected erythrocytes

    DEFF Research Database (Denmark)

    Hempel, Casper

    2017-01-01

    Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite-stage parasites and schizonts introduce numerous small protrusions on t...... microscopy under cryogenic conditions allowing for high resolution and magnification of erythrocytes. This novel technique can be used for precise estimates of knob density and for studies on cytoadhesion....

  9. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    Science.gov (United States)

    Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

    2016-05-01

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.

  10. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography.

    Science.gov (United States)

    Jesse, S; Chi, M; Belianinov, A; Beekman, C; Kalinin, S V; Borisevich, A Y; Lupini, A R

    2016-05-23

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called "big-data" methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.

  11. The use of laser scanning confocal microscopy (LSCM) in materials science.

    Science.gov (United States)

    Hovis, D B; Heuer, A H

    2010-12-01

    Laser scanning confocal microscopes are essential and ubiquitous tools in the biological, biochemical and biomedical sciences, and play a similar role to scanning electron microscopes in materials science. However, modern laser scanning confocal microscopes have a number of advantages for the study of materials, in addition to their obvious uses for high resolution reflected and transmitted light optical microscopy. In this paper, we provide several examples that exploit the laser scanning confocal microscope's capabilities of pseudo-infinite depth of field imaging, topographic imaging, photo-stimulated luminescence imaging and Raman spectroscopic imaging. © 2010 The Authors Journal of Microscopy © 2010 The Royal Microscopical Society.

  12. In Vivo Non Linear Optical (NLO) Imaging in Live Rabbit Eyes Using the Heidelberg Two-Photon Laser Ophthalmoscope

    Science.gov (United States)

    Hao, Ming; Flynn, Kevin; Nien-Shy, Chyong; Jester, Bryan E.; Winkler, Moritz; Brown, Donald J.; La Schiazza, Olivier; Bille, Josef; Jester, James V.

    2010-01-01

    Imaging of non-linear optical (NLO) signals generated from the eye using ultrafast pulsed lasers has been limited to the study of ex vivo tissues because of the use of conventional microscopes with slow scan speeds. The purpose of this study was to evaluate the ability of a novel, high scan rate ophthalmoscope to generate NLO signals using an attached femtosecond laser. NLO signals were generated and imaged in live, anesthetized albino rabbits using a newly designed Heidelberg Two-Photon Laser Ophthalmoscope with attached 25 mW femtosecond laser having a central wavelength of 780 nm, pulsewidth of 75 fs, and a repetition rate of 50 MHz. To assess two-photon excited fluorescent (TPEF) signal generation, cultured rabbit corneal fibroblasts (RCF) were first labeled by Blue-green fluorescent FluoSpheres (1 μm diameter) and then cells were micro-injected into the central cornea. Clumps of RCF cells could be detected by both reflectance and TPEF imaging at 6 hours after injection. By 6 days, RCF containing fluorescent microspheres confirmed by TPEF showed a more spread morphology and had migrated from the original injection site. Overall, this study demonstrates the potential of using NLO microscopy to sequentially detect TPEF signals from live, intact corneas. We conclude that further refinement of the Two-photon laser Ophthalmoscope should lead to the development of an important, new clinical instrument capable of detecting NLO signals from patient corneas. PMID:20558159

  13. Scanning SQUID microscopy in a cryogen-free refrigerator

    Science.gov (United States)

    Schaefer, Brian T.; Low, David; Prawiroatmodjo, Guenevere E. D. K.; Nangoi, J. Kevin; Kim, Jihoon; Nowack, Katja C.

    With helium prices rising and supply becoming increasingly uncertain, it has become attractive to use dry cryostats with cryocoolers rather than liquid helium to reach low temperatures. However, a cryocooler introduces vibrations at the sample stage, making scanning probe experiments more challenging. Here, we report our progress on a superconducting quantum interference device (SQUID) microscope implemented for the first time in a compact, cryogen-free 5 K system. Our microscope is designed to reach submicron spatial resolution and a flux sensitivity of approximately 1 μΦ0 /√{ Hz} , where Φ0 is the magnetic flux quantum. To enable height feedback while approaching and scanning samples, we mount the SQUID on a quartz tuning fork. Our system promises to meet the capabilities of similar systems implemented in helium cryostats.

  14. Low voltage scanning electron microscopy of interplanetary dust particles

    Science.gov (United States)

    Blake, D. F.; Bunch, T. E.; Reilly, T. W.; Brownlee, D. E.

    1987-01-01

    The resolution of available low-voltage SEM (LVSEM) models used in the characterization of interplanetary dust particles (IDPs) is limited by a number of factors including energy spread in the electron source, beam brightness, scanning electron detector geometry, and various lens aberrations. This paper describes an improved model of LVSEM which offers an increased resolution at low voltage. The improvements include a cold cathode FE source which has an extremely low inherent energy spread and high brightness, a second condenser lens to converge the beam and maintain an optimum aperture half-angle, and a detector optimized for low-voltage scanning-electron collection. To reduce lens aberrations, the specimen is immersed in the objective lens field. The features of several IDP samples observed using the images obtained with this LVSEM model are described.

  15. Simulation study of secondary electron images in scanning ion microscopy

    CERN Document Server

    Ohya, K

    2003-01-01

    The target atomic number, Z sub 2 , dependence of secondary electron yield is simulated by applying a Monte Carlo code for 17 species of metals bombarded by Ga ions and electrons in order to study the contrast difference between scanning ion microscopes (SIM) and scanning electron microscopes (SEM). In addition to the remarkable reversal of the Z sub 2 dependence between the Ga ion and electron bombardment, a fine structure, which is correlated to the density of the conduction band electrons in the metal, is calculated for both. The brightness changes of the secondary electron images in SIM and SEM are simulated using Au and Al surfaces adjacent to each other. The results indicate that the image contrast in SIM is much more sensitive to the material species and is clearer than that for SEM. The origin of the difference between SIM and SEM comes from the difference in the lateral distribution of secondary electrons excited within the escape depth.

  16. Low voltage scanning electron microscopy of interplanetary dust particles

    Science.gov (United States)

    Blake, D. F.; Bunch, T. E.; Reilly, T. W.; Brownlee, D. E.

    1987-01-01

    The resolution of available low-voltage SEM (LVSEM) models used in the characterization of interplanetary dust particles (IDPs) is limited by a number of factors including energy spread in the electron source, beam brightness, scanning electron detector geometry, and various lens aberrations. This paper describes an improved model of LVSEM which offers an increased resolution at low voltage. The improvements include a cold cathode FE source which has an extremely low inherent energy spread and high brightness, a second condenser lens to converge the beam and maintain an optimum aperture half-angle, and a detector optimized for low-voltage scanning-electron collection. To reduce lens aberrations, the specimen is immersed in the objective lens field. The features of several IDP samples observed using the images obtained with this LVSEM model are described.

  17. Near-field scanning microwave microscopy of microwave devices

    Science.gov (United States)

    Vlahacos, C. P.; Steinhauer, David E.; Dutta, S.; Anlage, S. M.; Wellstood, F. C.; Newman, H.

    1997-03-01

    We have developed a scanning microwave microscope which can presently image features with a spatial resolution of 10-100 μm in the frequency range 5-15 GHz.(C. P. Vlahacos, et al.), Appl. Phys. Lett. 69, 3272 (1996).^,(S. M. Anlage, et al.), IEEE. Trans. Appl. Supercond. (1997). The microscope consists of a resonant section of a coaxial cable which is terminated with a small-diameter open-ended coaxial probe. Images are made by scanning the sample under the probe while recording the induced near-field microwave voltage as a function of sample position. We will present images for several microwave devices, including an X-band microstrip planar ferrite circulator and a high-temperature superconducting microstrip YBa_2Cu_3O_7-δ resonator, and compare them to the calculated field profiles.

  18. Tomographic imaging and scanning thermal microscopy: thermal impedance tomography

    OpenAIRE

    2002-01-01

    The application of tomographic imaging techniques developed for medical applications to the data provided by the scanning thermal microscope will give access to true three-dimensional information on the thermal properties of materials on a mm length scale. In principle, the technique involves calculating and inverting a sensitivity matrix for a uniform isotropic material, collecting ordered data at several modulation frequencies, and multiplying the inverse of the matrix with the data vector....

  19. Nanomaterial datasets to advance tomography in scanning transmission electron microscopy

    OpenAIRE

    Levin, Barnaby D.A.; Padgett, Elliot; Chen, Chien-Chun; Scott, M C; Xu, Rui; Theis, Wolfgang; Jiang, Yi; Yang, Yongsoo; Ophus, Colin; Zhang, Haitao; Ha, Don-Hyung; Wang, Deli; Yu, Yingchao; Abruña, Hector D.; Robinson, Richard D.

    2016-01-01

    Electron tomography in materials science has flourished with the demand to characterize nanoscale materials in three dimensions (3D). Access to experimental data is vital for developing and validating reconstruction methods that improve resolution and reduce radiation dose requirements. This work presents five high-quality scanning transmission electron microscope (STEM) tomography datasets in order to address the critical need for open access data in this field. The datasets represent the cu...

  20. Effect of morphology and solvent on two-photon absorption of nano zinc oxide

    Energy Technology Data Exchange (ETDEWEB)

    Kavitha, M.K. [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India); Haripadmam, P.C.; Gopinath, Pramod; Krishnan, Bindu [Department of Physics, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India); John, Honey, E-mail: honey@iist.ac.in [Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram 695547, Kerala (India)

    2013-05-15

    Highlights: ► ZnO nanospheres and triangular structures synthesis by novel precipitation technique. ► The effect of precursor concentration on the size and shape of nano ZnO. ► Open aperture Z-scan measurements of the ZnO nanoparticle dispersions. ► Nanospheres exhibit higher two photon absorption coefficient than triangular nanostructures. ► Nanospheres dispersed in water exhibit higher two photon absorption coefficient than its dispersion in 2-propanol. - Abstract: In this paper, we report the effect of morphology and solvent on the two-photon absorption of nano zinc oxide. Zinc oxide nanoparticles in two different morphologies like nanospheres and triangular nanostructures are synthesized by novel precipitation technique and their two-photon absorption coefficient is measured using open aperture Z-scan technique. Experimental results show that the zinc oxide nanospheres exhibit higher two-photon absorption coefficient than the zinc oxide triangular nanostructures. The zinc oxide nanospheres dispersed in water exhibit higher two-photon absorption coefficient than that of its dispersion in 2-propanol. The zinc oxide nanospheres dispersed in water shows a decrease in two-photon absorption coefficient with an increase in on-axis irradiance. The result confirms the dependence of shape and solvent on the two-photon absorption of nano zinc oxide.

  1. Four-dimensional multi-site two-photon excitation

    CERN Document Server

    Daria, Vincent Ricardo; Bowman, Richard; Redman, Stephen; Bachor, Hans-A

    2009-01-01

    We report the first demonstration of dynamic and arbitrary multi-site two-photon excitation in three-dimensional (3D) space using the holographic projection method. Rapid temporal response (fourth dimension) is achieved through high-speed non-iterative and non-optimized calculation of the hologram using a video graphics accelerator board. We verify that the projected asymmetric spot configurations have sufficient spatiotemporal photon density for localized two-photon excitation. This system is a significant advance and ready for applications such as time-resolved 3D photolysis of complex biological cell and neuronal networks, 3D microscopy, non-linear micro-fabrication and volume holographic optical storage.

  2. High resolution imaging using scanning ion conductance microscopy with improved distance feedback control

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Nicholas Johnson; Victor Ostanin; Andrew Shevchuk; Liming Ying; Yuri Korchev; David Klenerman

    2008-01-01

    Microscopy is an essential technique for observation on living cells. There is currently great interest in applying scanning probe microscopy to image-living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high-resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third-order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high-resolution imaging.

  3. Modeling the Effect of Wave-front Aberrations in Fiber-based Scanning Optical Microscopy

    NARCIS (Netherlands)

    Verstraete, H.R.G.W.; Verhaegen, M.H.G.; Kalkman, J.

    2013-01-01

    In scanning microscopy and optical coherence tomography, aberrations of the wave-front cause a loss in intensity and resolution. Intensity and resolution are quantified using Fresnel propagation, Fraunhofer diffraction, and the calculation of overlap integrals.

  4. Scanning electron microscopy of acrothoracican cypris larvae (Crustacea, Thecostraca, Cirripedia, Acrothoracica, Lithoglyptidae)

    NARCIS (Netherlands)

    Kolbasov, Gregory A.; Høeg, Jens T.; Elfimov, Alexei S.

    1999-01-01

    Scanning electron microscopy was used to provide a full morphological description of cypris morphology in the acrothoracican species Lithoglyptes milis and L. habei (Lithoglyptidae). Special attention was given to lattice organs, antennules, thorax, thoracopods, abdomen, and furcal rami. Cypris larv

  5. Quantitative detection of gold nanoparticles on individual, unstained cancer cells by scanning electron microscopy

    NARCIS (Netherlands)

    Hartsuiker, L.; Es, van P.; Petersen, W.; Leeuwen, van T.G.; Terstappen, L.W.M.M.; Otto, C.

    2011-01-01

    Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample

  6. Quantitative detection of gold nanoparticles on individual, unstained cancer cells by Scanning Electron Microscopy

    NARCIS (Netherlands)

    Hartsuiker, Liesbeth; van Es, Peter; Petersen, Wilhelmina; van Leeuwen, Ton; Terstappen, Leonardus Wendelinus Mathias Marie; Otto, Cornelis

    2011-01-01

    Gold nanoparticles are rapidly emerging for use in biomedical applications. Characterization of the interaction and delivery of nanoparticles to cells through microscopy is important. Scanning electron microscopes have the intrinsic resolution to visualize gold nanoparticles on cells. A novel sample

  7. Voltammetric scanning electrochemical cell microscopy: dynamic imaging of hydrazine electro-oxidation on platinum electrodes

    NARCIS (Netherlands)

    Chen, C.-H.; Jacobse, L.; McKelvey, K.; Lai, S.C.S.; Koper, M.T.M.; Unwin, P.R.

    2015-01-01

    Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potenti

  8. Two-photon-excited autofluorescence and second-harmonic generation microscopy for the visualization of penetration of TiO2 and ZnO nanoparticles into human tooth tissue ex vivo

    Science.gov (United States)

    Trunina, Natalia A.; Popov, Alexey P.; Lademann, Jürgen; Tuchin, Valery V.; Myllylä, Risto; Darvin, Maxim E.

    2012-06-01

    Penetration of nanoparticles into tooth tissues is of significant interest in solving problems related to reduction of tooth sensitivity, enamel strengthening and restoration and cosmetic bleaching. In this work we demonstrate two-photonexcited autofluorescence and second-harmonic generation microscopy for visualization of penetration of TiO2 and ZnO nanoparticles into tooth tissues.

  9. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    Science.gov (United States)

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

  10. Scanning electron microscopy of erythropoietin-stimulated bone marrow

    Energy Technology Data Exchange (ETDEWEB)

    Leblond, P.F. (Hospital of St. Sacrement, Quebec); Chamberlain, J.K.; Weed, R.I.

    1975-01-01

    This work describes and illustrates the scanning electron microscopic modifications observed in the femoral bone marrow of normal mice 72 hours after a single injection of partly purified sheep erythropoietin and of mice afflicted with a chronic congenital hemolytic anemia analogous to the disease Hereditary Spherocytosis in man. In acordance with previous transmission electron microscopic studies, the observations are consistent with an effect of erythropoietin both on the frequency of cell migration across the normally intact marrow sinus endothelium and on the morphology of sinus adventitial cells. It is suggested that these ultrastructural modifications may be responsible for the greater patency of the marrow-blood barrier under erythropoietin stimulation.

  11. Scanning Electron Microscopy of Lagochilascaris minor Leiper, 1909 (Nematoda: Ascarididae

    Directory of Open Access Journals (Sweden)

    Lanfredi Reinalda Marisa

    1998-01-01

    Full Text Available Lagochilascaris minor Leiper, 1909 is a parasitic nematode with its biological cycle still unknown, even though it was found in humans, domestic and silvatic animals. Adult worms, collected by surgical drainage from a human patient from the State of Pará, Brazil, were micrographed using a scanning electron microscope. Morphological aspects of males and females such as cephalic structures, caudal papillae and cuticular patterns were analyzed and compared with the previous descriptions adding new data for the identification of this species.

  12. Scanning transmission electron microscopy: Albert Crewe's vision and beyond.

    Science.gov (United States)

    Krivanek, Ondrej L; Chisholm, Matthew F; Murfitt, Matthew F; Dellby, Niklas

    2012-12-01

    Some four decades were needed to catch up with the vision that Albert Crewe and his group had for the scanning transmission electron microscope (STEM) in the nineteen sixties and seventies: attaining 0.5Å resolution, and identifying single atoms spectroscopically. With these goals now attained, STEM developments are turning toward new directions, such as rapid atomic resolution imaging and exploring atomic bonding and electronic properties of samples at atomic resolution. The accomplishments and the future challenges are reviewed and illustrated with practical examples.

  13. [Scanning electron microscopy of heat-damaged bone tissue].

    Science.gov (United States)

    Harsanyl, L

    1977-02-01

    Parts of diaphyses of bones were exposed to high temperature of 200-1300 degrees C. Damage to the bone tissue caused by the heat was investigated. The scanning electron microscopic picture seems to be characteristic of the temperature applied. When the bones heated to the high temperature of 700 degrees C characteristic changes appear on the periostal surface, higher temperatura on the other hand causes damage to the compact bone tissue and can be observed on the fracture-surface. Author stresses the importance of this technique in the legal medicine and anthropology.

  14. Observation of diamond turned OFHC copper using Scanning Tunneling Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Grigg, D.A.; Russell, P.E.; Dow, T.A.

    1988-12-01

    Diamond turned OFHC copper samples have been observed within the past few months using the Scanning Tunneling Microscope. Initial results have shown evidence of artifacts which may be used to better understand the diamond turning process. The STM`s high resolution capability and three dimensional data representation allows observation and study of surface features unobtainable with conventional profilometry systems. Also, the STM offers a better quantitative means by which to analyze surface structures than the SEM. This paper discusses findings on several diamond turned OFHC copper samples having different cutting conditions. Each sample has been cross referenced using STM and SEM.

  15. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Science.gov (United States)

    Gary, Ramla; Carbone, Giovani; Petriashvili, Gia; De Santo, Maria Penelope; Barberi, Riccardo

    2016-01-01

    The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. PMID:26907286

  16. Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

    Directory of Open Access Journals (Sweden)

    Ramla Gary

    2016-02-01

    Full Text Available The gold nanoparticle (GNP aggregation growth induced by deoxyribonucleic acid (DNA is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA.

  17. A scanning tunneling microscopy tip with a stable atomic structure

    Science.gov (United States)

    Kim, Yeong-Cheol; Seidman, David N.

    2004-02-01

    A single stable adatom on a {110}-type plane of a tungsten tip is created via field-evaporation in a field-ion microscope (FIM) operating at room temperature. This single adatom has sufficient surface mobility at room temperature and migrates, in one-dimension, along a -type direction toward an edge of a {110}-type plane, due to the existence of an electric field gradient. The plane edge has a higher local electric field than its center, since it has a higher local geometric curvature. This result implies that the stable position of a single adatom during a scan of a scanning tunneling microscope (STM) tip on a surface is at the edge and not at the center of a {110}-type plane at room temperature. Therefore, the electron wave function of a tip is not symmetric and this fact should be taken into account in a careful analysis of STM images. Also a tip with a dislocation emerging at a {110}-type plane is suggested as an improved STM tip configuration, as the step at the surface, created by the intersection of the dislocation with it, is a perpetual source of single adatoms.

  18. Medical prototyping using two photon polymerization

    Directory of Open Access Journals (Sweden)

    Roger J Narayan

    2010-12-01

    Full Text Available Two photon polymerization involves nearly simultaneous absorption of ultrashort laser pulses for selective curing of photosensitive material. This process has recently been used to create small-scale medical devices out of several classes of photosensitive materials, such as acrylate-based polymers, organically-modified ceramic materials, zirconium sol-gels, and titanium-containing hybrid materials. In this review, the use of two photon polymerization for fabrication of several types of small-scale medical devices, including microneedles, artificial tissues, microfluidic devices, pumps, sensors, and valves, from computer models is described. Necessary steps in the development of two photon polymerization as a commercially viable medical device manufacturing method are also considered.

  19. Two Photon Couplings of Hybrid Mesons

    CERN Document Server

    Page, P R

    1996-01-01

    A new formalism is developed for the two photon production of hybrid mesons via intermediate hadronic decays. In an adiabatic and non--relativistic context with spin 1 pair creation we obtain the first absolute estimates of unmixed hybrid production strengths to be small (0.03 - 3 eV) in relation to experimental meson widths (0.1 - 5 keV). Within this context, two photon collisions therefore strongly discriminate between hybrid and conventional meson wave function components at BaBar, Cleo II, LEP2 and LHC, filtering out non--gluonic components. Decay widths of unmixed hybrids are tiny. The formalism also induces conventional meson two photon widths roughly in agreement with experiment.

  20. Scanning Probe Microscopy as a Tool Applied to Agriculture

    Science.gov (United States)

    Leite, Fabio Lima; Manzoli, Alexandra; de Herrmann, Paulo Sérgio Paula; Oliveira, Osvaldo Novais; Mattoso, Luiz Henrique Capparelli

    The control of materials properties and processes at the molecular level inherent in nanotechnology has been exploited in many areas of science and technology, including agriculture where nanotech methods are used in release of herbicides and monitoring of food quality and environmental impact. Atomic force microscopy (AFM) and related techniques are among the most employed nanotech methods, particularly with the possibility of direct measurements of intermolecular interactions. This chapter presents a brief review of the applications of AFM in agriculture that may be categorized into four main topics, namely thin films, research on nanomaterials and nanostructures, biological systems and natural fibers, and soils science. Examples of recent applications will be provided to give the reader a sense of the power of the technique and potential contributions to agriculture.

  1. Scanning electron microscopy analysis of experimental bone hacking trauma.

    Science.gov (United States)

    Alunni-Perret, Veronique; Muller-Bolla, Michèle; Laugier, Jean-Pierre; Lupi-Pégurier, Laurence; Bertrand, Marie-France; Staccini, Pascal; Bolla, Marc; Quatrehomme, Gérald

    2005-07-01

    The authors report on their macro- and microscopy study of bone lesions made by a sharp force instrument (a single blade knife), and a sharp-blunt instrument classified as a chopping weapon (a hatchet). The aim of this work was to attempt to identify the instrument by analyzing the general class characteristics of the cuts. Each weapon was used on human bones. The results indicate that macroscopic analysis is more problematic. The microscopic analysis assessed that characteristics examined were effective in distinguishing sharp from sharp-blunt injury to the bone. The microscope facilitates analysis unachievable with macroscopic methods, some three-dimensional characteristics not visible to the naked eye being clearly defined with its use. Emphasis has been placed on the value of SEM as an anthropologist's tool in bone lesion injuries.

  2. Local thermal conductivity of polycrystalline AlN ceramics measured by scanning thermal microscopy and complementary scanning electron microscopy techniques

    Institute of Scientific and Technical Information of China (English)

    Zhang Yue-Fei; Wang Li; R. Heiderhoff; A. K. Geinzer; Wei Bin; Ji Yuan; Han Xiao-Dong; L. J. Balk; Zhang Ze

    2012-01-01

    The local thermal conductivity of polycrystalline aluminum nitride (AlN) ceramics is measured and imaged by using a scanning thermal microscope (SThM) and complementary scanning electron microscope (SEM) based techniques at room temperature.The quantitative thermal conductivity for the AlN sample is gained by using a SThM with a spatial resolution of sub-micrometer scale through using the 3w method.A thermal conductivity of 308 W/m·K withingrains corresponding to that of high-purity single crystal AlN is obtained.The slight differences in thermal conduction between the adjacent grains are found to result from crystallographic misorientations,as demonstrated in the electron backscattered diffraction.A much lower thermal conductivity at the grain boundary is due to impurities and defects enriched in these sites,as indicated by energy dispersive X-ray spectroscopy.

  3. In-situ Scanning Transmission X-Ray Microscopy of Catalytic Solids and Related Nanomaterials

    NARCIS (Netherlands)

    de Groot, F.M.F.; de Smit, E.; van Schooneveld, M.M.; Aramburo, L.R.; Weckhuysen, B.M.

    2013-01-01

    The present status of in-situ scanning transmission X-ray microscopy (STXM) is reviewed, with an emphasis on the abilities of the STXM technique in comparison with electron microscopy. The experimental aspects and interpretation of X-ray absorption spectroscopy (XAS) are briefly introduced and the e

  4. Segmentation of scanning electron microscopy images from natural rubber samples with gold nanoparticles using starlet wavelets

    OpenAIRE

    de Siqueira, Alexandre Fioravante; Cabrera, Flavio Camargo [UNESP; Pagamisse, Aylton; Job,Aldo Eloizo

    2016-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our...

  5. Nanomaterial datasets to advance tomography in scanning transmission electron microscopy

    CERN Document Server

    Levin, Barnaby D A; Chen, Chien-Chun; Scott, M C; Xu, Rui; Theis, Wolfgang; Jiang, Yi; Yang, Yongsoo; Ophus, Colin; Zhang, Haitao; Ha, Don-Hyung; Wang, Deli; Yu, Yingchao; Abruna, Hector D; Robinson, Richard D; Ercius, Peter; Kourkoutis, Lena F; Miao, Jianwei; Muller, David A; Hovden, Robert

    2016-01-01

    Electron tomography in materials science has flourished with the demand to characterize nanoscale materials in three dimensions (3D). Access to experimental data is vital for developing and validating reconstruction methods that improve resolution and reduce radiation dose requirements. This work presents five high-quality scanning transmission electron microscope (STEM) tomography datasets in order to address the critical need for open access data in this field. The datasets represent the current limits of experimental technique, are of high quality, and contain materials with structural complexity. Included are tomographic series of a hyperbranched Co2P nanocrystal, platinum nanoparticles on a carbon nanofibre imaged over the complete 180{\\deg} tilt range, a platinum nanoparticle and a tungsten needle both imaged at atomic resolution by equal slope tomography, and a through-focal tilt series of PtCu nanoparticles. A volumetric reconstruction from every dataset is provided for comparison and development of p...

  6. A nanoscale gigahertz source realized with Josephson scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jäck, Berthold, E-mail: b.jaeck@fkf.mpg.de; Eltschka, Matthias; Assig, Maximilian; Etzkorn, Markus; Ast, Christian R. [Max-Planck-Institut für Festkörperforschung, 70569 Stuttgart (Germany); Hardock, Andreas [Institut für Theoretische Elektrotechnik, Technische Universität Hamburg-Harburg, 21079 Hamburg (Germany); Kern, Klaus [Max-Planck-Institut für Festkörperforschung, 70569 Stuttgart (Germany); Institut de Physique de la Matière Condensée, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne (Switzerland)

    2015-01-05

    Using the AC Josephson effect in the superconductor-vacuum-superconductor tunnel junction of a scanning tunneling microscope (STM), we demonstrate the generation of GHz radiation. With the macroscopic STM tip acting as a λ/4-monopole antenna, we first show that the atomic scale Josephson junction in the STM is sensitive to its frequency-dependent environmental impedance in the GHz regime. Further, enhancing Cooper pair tunneling via excitations of the tip eigenmodes, we are able to generate high-frequency radiation. We find that for vanadium junctions, the enhanced photon emission can be tuned from about 25 GHz to 200 GHz and that large photon flux in excess of 10{sup 20 }cm{sup −2} s{sup −1} is reached in the tunnel junction. These findings demonstrate that the atomic scale Josephson junction in an STM can be employed as a full spectroscopic tool for GHz frequencies on the atomic scale.

  7. Band Excitation in Scanning Probe Microscopy: Recognition and Functional Imaging

    Energy Technology Data Exchange (ETDEWEB)

    Jesse, Stephen [ORNL; Vasudevan, Dr. Rama [Oak Ridge National Laboratory (ORNL); Collins, Liam [University College, Dublin; Strelcov, Evgheni [ORNL; Okatan, Mahmut B [ORNL; Belianinov, Alex [ORNL; Baddorf, Arthur P [ORNL; Proksch, Roger [Asylum Research, Santa Barbara, CA; Kalinin, Sergei V [ORNL

    2014-01-01

    Field confinement at the junction between a biased scanning probe microscope s (SPM) tip and solid surface enables local probing of various bias-induced transformations such as polarization switching, ionic motion, or electrochemical reactions to name a few. The nanoscale size of the biased region is smaller or comparable to features like grain boundaries and dislocations, potentially allows for the study of kinetics and thermodynamics at the level of a single defect. In contrast to classical statistically averaged approaches, this allows one to link structure to functionality and deterministically decipher associated mesoscopic and atomistic mechanisms. Furthermore, this type of information can serve as a fingerprint of local material functionality, allowing for local recognition imaging. Here, current progress in multidimensional SPM techniques based on band-excitation time and voltage spectroscopies is illustrated, including discussions on data acquisition, dimensionality reduction, and visualization along with future challenges and opportunities for the field.

  8. Optimization of the imaging response of scanning microwave microscopy measurements

    Energy Technology Data Exchange (ETDEWEB)

    Sardi, G. M.; Lucibello, A.; Proietti, E.; Marcelli, R., E-mail: romolo.marcelli@imm.cnr.it [National Research Council, Institute for Microelectronics and Microsystems, Via del Fosso del Cavaliere 100, 00133 Rome (Italy); Kasper, M.; Gramse, G. [Biophysics Institute, Johannes Kepler University, Gruberstrasse 40, 4020 Linz (Austria); Kienberger, F. [Keysight Technologies Austria GmbH, Gruberstrasse 40, 4020 Linz (Austria)

    2015-07-20

    In this work, we present the analytical modeling and preliminary experimental results for the choice of the optimal frequencies when performing amplitude and phase measurements with a scanning microwave microscope. In particular, the analysis is related to the reflection mode operation of the instrument, i.e., the acquisition of the complex reflection coefficient data, usually referred as S{sub 11}. The studied configuration is composed of an atomic force microscope with a microwave matched nanometric cantilever probe tip, connected by a λ/2 coaxial cable resonator to a vector network analyzer. The set-up is provided by Keysight Technologies. As a peculiar result, the optimal frequencies, where the maximum sensitivity is achieved, are different for the amplitude and for the phase signals. The analysis is focused on measurements of dielectric samples, like semiconductor devices, textile pieces, and biological specimens.

  9. Moessbauer spectroscopy and scanning electron microscopy of the Murchison meteorite

    Science.gov (United States)

    Brown, Christopher L.; Oliver, Frederick W.; Hammond, Ernest C., Jr.

    1989-01-01

    Meteorites provide a wealth of information about the solar system's formation, since they have similar building blocks as the Earth's crust but have been virtually unaltered since their formation. Some stony meteorites contain minerals and silicate inclusions, called chondrules, in the matrix. Utilizing Moessbauer spectroscopy, we identified minerals in the Murchison meteorite, a carbonaceous chondritic meteorite, by the gamma ray resonance lines observed. Absorption patterns of the spectra were found due to the minerals olivine and phyllosilicate. We used a scanning electron microscope to describe the structure of the chondrules in the Murchison meteorite. The chondrules were found to be deformed due to weathering of the meteorite. Diameters varied in size from 0.2 to 0.5 mm. Further enhancement of the microscopic imagery using a digital image processor was used to describe the physical characteristics of the inclusions.

  10. Scanning electron microscopy of human cortical bone failure surfaces.

    Science.gov (United States)

    Braidotti, P; Branca, F P; Stagni, L

    1997-02-01

    Undecalcified samples extracted from human femoral shafts are fractured by bending and the fracture surfaces are examined with a scanning electron microscope (SEM). The investigation is performed on both dry and wet (hydrated with a saline solution) specimens. SEM micrographs show patterns in many respects similar to those observed in fractography studies of laminated fiber-reinforced synthetic composites. In particular, dry and wet samples behave like brittle and ductile matrix laminates, respectively. An analysis carried out on the basis of the mechanisms that dominate the fracture process of laminates shows that a reasonable cortical bone model is that of a laminated composite material whose matrix is composed of extracellular noncollagenous calcified proteins, and the reinforcement is constituted by the calcified collagen fiber system.

  11. Evaluation of the bleached human enamel by Scanning Electron Microscopy

    DEFF Research Database (Denmark)

    Miranda, Carolina Baptista; Pagani, Clovis; Benetti, Ana Raquel

    2005-01-01

    Since bleaching has become a popular procedure, the effect of peroxides on dental hard tissues is of great interest in research. Purpose: The aim of this in vitro study was to perform a qualitative analysis of the human enamel after the application of in-office bleaching agents, using Scanning......: 2h); G3- four 2-hour exposures to 35% carbamide peroxide (total exposure: 8h); G4- two applications of 35% hydrogen peroxide, which was light-activated with halogen lamp at 700mW/cm² during 7min and remained in contact with enamel for 20min (total exposure: 40min). All bleaching treatments adopted...... analysis performing gold sputter coating under vacuum and were examined using 15kV at 500x and 2000x magnification. Results: Morphological alterations on the enamel surface were similarly detected after bleaching with either 35% carbamide peroxide or 35% hydrogen peroxide. Surface porosities were...

  12. U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Prabhakaran, Ramprashad [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Joshi, Vineet V. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rhodes, Mark A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Schemer-Kohrn, Alan L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Guzman, Anthony D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lavender, Curt A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-10-01

    The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

  13. U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Prabhakaran, Ramprashad [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Joshi, Vineet V. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Rhodes, Mark A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Schemer-Kohrn, Alan L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Guzman, Anthony D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lavender, Curt A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-03-30

    The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

  14. Tribology studies of organic thin films by scanning force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Bar, G. [Albert-Ludwigs Univ., Freiburg (Germany). Freiburger Materialforschungszentrum; Rubin, S.; Parikh, A.N.; Swanson, B.I.; Zawodzinski, T.A. [Los Alamos National Lab., NM (United States)

    1996-06-01

    The use of organic thin films as lubricants on solid surfaces is important in many modern technologies including magnetic storage and micromachines. Langmuir-Blodgett (LB) films and self-assembled monolayers (SAMs) are attractive candidates for lubricant layers and for model studies of lubrication because of their strong adsorption to the surface. The recent interest on the properties of LB films and SAMs has been also motivated by their potential applications in sensors, non-linear optical devices, lithography and microelectronics. Using the micro-contact printing method the authors prepared patterned SAMs consisting of methyl-terminated alkanethiols of different chain lengths. The samples were characterized using lateral force microscopy (LFM) and the force modulation technique (FMT). In general, higher friction is observed over the short chain regions than over the long chain regions when a low or moderate load is applied to the SFM tip. For such cases the high friction (short chain) regions are also ``softer`` as measured by FMT. A high loads, a reversal of the image contrast is observed and the short chain regions show a lower friction than the long chain regions. This image contrast is reversible upon reduction of the applied load.

  15. Scanning electron microscopy investigations regarding Adonis vernalis L. flower morphology

    Directory of Open Access Journals (Sweden)

    Irina Neta GOSTIN

    2009-11-01

    Full Text Available The floral morphology of Adonis vernalis L. was observed with a scanning electron microscope (SEM. The investigations are important to clarify some taxonomical problems and also could provide useful diagnostic elements for the identification of this medicinal plant in powdered materials. All floral organs are initiated spirally and centripetally and develop centripetally. The petals (8-12 are shorter than the sepals (5-6 in early developmental stages. The petals are disposed on spiral (with 3-4 whorls. The stamens (numerous are unbranched and reach maturity centripetally; they are free of the perianth. The anther walls consisting of a single layer epidermis in the anther wall surrounding the sporagenous tissue, one row of endothecium, two to four rows of middle layer and one row of tapetum layer. In the anther walls, the tapetal cells, by glandular type, persist later in ontogenesis. Pollen grains are tricolpate with echinate surface. The gynoecium is multiple, apocarpous with distinct carpels. The carpels are ascidiate from the beginning. At the base of each carpel, numerousness short, unicellular, trichomes are present. The stigma differentiates as two crests along the ventral slit of the ovary. Each carpel contains a single ovule inside the ovary cavity. The mature ovule is anatropous, with two integuments. It is almost parallel to the funicle.

  16. A broadband toolbox for scanning microwave microscopy transmission measurements

    Science.gov (United States)

    Lucibello, Andrea; Sardi, Giovanni Maria; Capoccia, Giovanni; Proietti, Emanuela; Marcelli, Romolo; Kasper, Manuel; Gramse, Georg; Kienberger, Ferry

    2016-05-01

    In this paper, we present in detail the design, both electromagnetic and mechanical, the fabrication, and the test of the first prototype of a Scanning Microwave Microscope (SMM) suitable for a two-port transmission measurement, recording, and processing the high frequency transmission scattering parameter S21 passing through the investigated sample. The S21 toolbox is composed by a microwave emitter, placed below the sample, which excites an electromagnetic wave passing through the sample under test, and is collected by the cantilever used as the detector, electrically matched for high frequency measurements. This prototype enhances the actual capability of the instrument for a sub-surface imaging at the nanoscale. Moreover, it allows the study of the electromagnetic properties of the material under test obtained through the measurement of the reflection (S11) and transmission (S21) parameters at the same time. The SMM operates between 1 GHz and 20 GHz, current limit for the microwave matching of the cantilever, and the high frequency signal is recorded by means of a two-port Vector Network Analyzer, using both contact and no-contact modes of operation, the latter, especially minded for a fully nondestructive and topography-free characterization. This tool is an upgrade of the already established setup for the reflection mode S11 measurement. Actually, the proposed setup is able to give richer information in terms of scattering parameters, including amplitude and phase measurements, by means of the two-port arrangement.

  17. OSTEOBLAST ADHESION OF BREAST CANCER CELLS WITH SCANNING ACOUSTIC MICROSCOPY

    Energy Technology Data Exchange (ETDEWEB)

    Chiaki Miyasaka; Robyn R. Mercer; Andrea M. Mastro; Ken L. Telschow

    2005-03-01

    Breast cancer frequently metastasizes to the bone. Upon colonizing bone tissue, the cancer cells stimulate osteoclasts (cells that break bone down), resulting in large lesions in the bone. The breast cancer cells also affect osteoblasts (cells that build new bone). Conditioned medium was collected from a bone-metastatic breast cancer cell line, MDA-MB-231, and cultured with an immature osteoblast cell line, MC3T3-E1. Under these conditions the osteoblasts acquired a changed morphology and appeared to adherer in a different way to the substrate and to each other. To characterize cell adhesion, MC3T3-E1 osteoblasts were cultured with or without MDA-MB-231 conditioned medium for two days, and then assayed with a mechanical scanning acoustic reflection microscope (SAM). The SAM indicated that in normal medium the MC3T3-E1 osteoblasts were firmly attached to their plastic substrate. However, MC3T3-E1 cells cultured with MDA-MB-231 conditioned medium displayed both an abnormal shape and poor adhesion at the substrate interface. The cells were fixed and stained to visualize cytoskeletal components using optical microscopic techniques. We were not able to observe these differences until the cells were quite confluent after 7 days of culture. However, using the SAM, we were able to detect these changes within 2 days of culture with MDA-MB-231 conditioned medium

  18. Nanomaterial datasets to advance tomography in scanning transmission electron microscopy.

    Science.gov (United States)

    Levin, Barnaby D A; Padgett, Elliot; Chen, Chien-Chun; Scott, M C; Xu, Rui; Theis, Wolfgang; Jiang, Yi; Yang, Yongsoo; Ophus, Colin; Zhang, Haitao; Ha, Don-Hyung; Wang, Deli; Yu, Yingchao; Abruña, Hector D; Robinson, Richard D; Ercius, Peter; Kourkoutis, Lena F; Miao, Jianwei; Muller, David A; Hovden, Robert

    2016-06-07

    Electron tomography in materials science has flourished with the demand to characterize nanoscale materials in three dimensions (3D). Access to experimental data is vital for developing and validating reconstruction methods that improve resolution and reduce radiation dose requirements. This work presents five high-quality scanning transmission electron microscope (STEM) tomography datasets in order to address the critical need for open access data in this field. The datasets represent the current limits of experimental technique, are of high quality, and contain materials with structural complexity. Included are tomographic series of a hyperbranched Co2P nanocrystal, platinum nanoparticles on a carbon nanofibre imaged over the complete 180° tilt range, a platinum nanoparticle and a tungsten needle both imaged at atomic resolution by equal slope tomography, and a through-focal tilt series of PtCu nanoparticles. A volumetric reconstruction from every dataset is provided for comparison and development of post-processing and visualization techniques. Researchers interested in creating novel data processing and reconstruction algorithms will now have access to state of the art experimental test data.

  19. Scanning Electron Microscopy with Samples in an Electric Field

    Science.gov (United States)

    Frank, Ludĕk; Hovorka, Miloš; Mikmeková, Šárka; Mikmeková, Eliška; Müllerová, Ilona; Pokorná, Zuzana

    2012-01-01

    The high negative bias of a sample in a scanning electron microscope constitutes the “cathode lens” with a strong electric field just above the sample surface. This mode offers a convenient tool for controlling the landing energy of electrons down to units or even fractions of electronvolts with only slight readjustments of the column. Moreover, the field accelerates and collimates the signal electrons to earthed detectors above and below the sample, thereby assuring high collection efficiency and high amplification of the image signal. One important feature is the ability to acquire the complete emission of the backscattered electrons, including those emitted at high angles with respect to the surface normal. The cathode lens aberrations are proportional to the landing energy of electrons so the spot size becomes nearly constant throughout the full energy scale. At low energies and with their complete angular distribution acquired, the backscattered electron images offer enhanced information about crystalline and electronic structures thanks to contrast mechanisms that are otherwise unavailable. Examples from various areas of materials science are presented.

  20. Two-photon physics at LEP2

    Energy Technology Data Exchange (ETDEWEB)

    Cartwright, Susan; Lehto, Mark [University of Sheffield Department of Physics, Sheffield S3 7RH (United Kingdom); Seymour, Michael H.; Close, Frank; Wright, Alison [Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11 0QX (United Kingdom); Affholderbach, Klaus; Cowan, Glen [Universitaet Siegen, Fachbereich Physik, D-57068 Siegen (Germany); Finch, Alex [University of Lancaster, Lancaster LA1 4YB (United Kingdom); Lauber, Jan [University College London, Gower Street, London WC1E 6BT (United Kingdom)

    1998-02-01

    The working group on two-photon physics concentrated on three main subtopics: modelling the hadronic final state of deep inelastic scattering on a photon; unfolding the deep inelastic scattering data to obtain the photon structure function; and resonant production of exclusive final states, particularly of glueball candidates. In all three areas, new results were presented. (author)

  1. Environmental scanning electron microscopy of hydrated conditioned/etched dentine.

    Science.gov (United States)

    de Wet, F A; van der Vyver, P J; Eick, J D; Dusevich, V M

    2000-11-01

    Various etchants/conditioners are used during dental treatment to affect or remove the smear layer. The purpose of this study was to evaluate the effect of different treatments on moist dentine, using a field emission environmental scanning electron microscope (FE-ESEM). Twenty freshly extracted, human molar teeth were utilised. The roots and pulps were removed, and the crowns horizontally sectioned with a low speed diamond saw (Isomet) (with cooling in a saline solution) in order to expose superficial dentine. A smear layer was created on these surfaces by using 600 grit silicone carbide paper. Test surfaces were then treated in one of the following ways: 1. 37% phosphoric acid liquid 2. 37% phosphoric acid gel 3. NRC (non-rinse conditioner) without rinsing 4. NRC with rinsing. Shallow grooves were cut on the untreated sides, using a thin diamond bur. This enabled the samples to be split in half when pressure was applied in the grooves. Samples were maintained moist throughout specimen preparation. Samples were examined in the FE-ESEM (Philips XL 30) in such a way that the effect of the treatment could be viewed occlusally, as well as perpendicular to the treated interface. Phosphoric acid liquid and gel removed the smear layer, and demineralised the dentine for approximately 5-10 micrometers. NRC penetrated the smear layer and modified it to a lesser degree. However, washing of the NRC treated surface removed part of the smear layer, and opened up some dentinal tubules. Excellent resolution was possible with the FE-ESEM in both the wet and dry modes.

  2. Scanning electron microscopy of hair treated in hard water.

    Science.gov (United States)

    Srinivasan, Gautham; Chakravarthy Rangachari, Srinivas

    2016-06-01

    Hardness of water is determined by the amount of calcium carbonate (CaCO3 ) and magnesium sulfate (MgSO4 ) dissolved in it. Hardness of water used for washing hair may damage the hair. The objective of this study is to observe the surface changes due to hard water usage and compare the thickness of hair between hard and soft water treated samples. Ten to 15 hair strands of length 15-20 cm, which were lost during combing, were obtained from 15 healthy female volunteers. Each hair sample was cut into two equal halves to obtain two sets per volunteer. Each hair sample was wrapped around a glass rod. One set of 15 samples was washed with hard water, and the other set was washed with distilled water for 10 minutes on alternate days and air-dried. This procedure was carried out for 30 days. The surface of hair treated in hard and soft water was examined under a scanning electron microscope. The CaCO3 and MgSO4 content of hard and distilled water samples were determined as 212.5 ppm of CaCO3 and 10 ppm of CaCO3 respectively. The mean calcium deposition in hard and distilled water treated hair was determined as 0.804% and 0.26%, respectively. The mean magnesium deposition in hard and distilled water treated hair was determined as 0.34% and 0.078%, respectively. The mean thickness of hair treated in hard water and distilled water were 72.78 and 78.14 μm, respectively. The surface of hard water treated hair has a ruffled appearance with higher mineral deposition and decreased thickness when compared with the surface of distilled water treated hair. © 2015 The International Society of Dermatology.

  3. Scanning Capacitance Force Microscopy and Kelvin Probe Force Microscopy of Nanostructures Embedded in SiO2

    Science.gov (United States)

    Tallarida, G.; Spiga, S.; Fanciulli, M.

    Scanning capacitance force microscopy and Kelvin probe force microscopy are used to image Sn nanometer sized structures embedded in silicon oxide thin films. The capacitance variation occurring between probe and sample in presence of a metallic cluster modifies the oscillation amplitude of the AFM probe at twice the frequency of the applied voltage. The extreme localisation of the interaction due to the small geometries involved allows a lateral resolution of few nm. Issues related to the contrast mechanism and the spatial resolution are discussed with the support 2D finite element calculation of the electrostatic field distribution between probe and sample.

  4. Characterization of gold nanoparticle films: Rutherford backscattering spectroscopy, scanning electron microscopy with image analysis, and atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Pia C. Lansåker

    2014-10-01

    Full Text Available Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness dg—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM combined with image analysis as well as by atomic force microscopy (AFM. The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for dg were obtained by SEM with image analysis and by AFM.

  5. Transmission and scanning electron microscopy confirm that bone microstructure is similar in osteopenic and osteoporotic patients.

    Science.gov (United States)

    Gül, Orkun; Atik, O Sahap; Erdoğan, Deniz; Göktaş, Güleser; Elmas, Ciğdem

    2013-01-01

    The objective was to confirm the finding of "Bone microstructure is similar in osteopenic and osteoporotic patients with femoral neck fracture." obtained in previous "light microscopy study", which was new and important data. Fourteen patients (5 males, 9 females) who were admitted with proximal femoral fracture following low energy trauma (patients who participated in the light microscopy study) were included. The patients were divided into two groups based on the bone mineral density (BMD) measurement, including osteopenic group (n=7, mean age 69 years; range 63 to 74 years) and osteoporotic group (n=7, mean age 74.1 years; range 67 to 78 years). Cortical and trabecular bone samples were taken from the patients who underwent endoprosthesis during partial hip arthroplasty and these samples were analyzed using transmission electron microscopy and scanning electron microscopy evaluations which are more sophisticated higher resolution techniques. The mean cortical bone thickness was 3622.14 mm in osteopenic group and 2323.14 mm in osteoporotic group (pelectron microscopy and scanning electron microscopy evaluations revealed similar findings for both groups. Although a significant difference in cortical thickness was found between the groups, transmission and scanning electron microscopy confirmed that bone microstructure shared similar characteristics in osteopenic and osteoporotic patients with low-energy femoral neck fracture, as it was in previous light microscopy study.

  6. Fast and reliable method of conductive carbon nanotube-probe fabrication for scanning probe microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Dremov, Vyacheslav, E-mail: dremov@issp.ac.ru; Fedorov, Pavel; Grebenko, Artem [Institute of Solid State Physics, RAS, 142432 Chernogolovka (Russian Federation); Interdisciplinary Center for Basic Research, Moscow Institute of Physics and Technology, 141700 Dolgoprudniy (Russian Federation); Fedoseev, Vitaly [Institute of Solid State Physics, RAS, 142432 Chernogolovka (Russian Federation)

    2015-05-15

    We demonstrate the procedure of scanning probe microscopy (SPM) conductive probe fabrication with a single multi-walled carbon nanotube (MWNT) on a silicon cantilever pyramid. The nanotube bundle reliably attached to the metal-covered pyramid is formed using dielectrophoresis technique from the MWNT suspension. It is shown that the dimpled aluminum sample can be used both for shortening/modification of the nanotube bundle by applying pulse voltage between the probe and the sample and for controlling the probe shape via atomic force microscopy imaging the sample. Carbon nanotube attached to cantilever covered with noble metal is suitable for SPM imaging in such modulation regimes as capacitance contrast microscopy, Kelvin probe microscopy, and scanning gate microscopy. The majority of such probes are conductive with conductivity not degrading within hours of SPM imaging.

  7. Clinical applications of in vivo fluorescence confocal laser scanning microscopy

    Science.gov (United States)

    Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

    2008-02-01

    Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In

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

    Science.gov (United States)

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

    2014-03-01

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

  9. A custom CMOS imager for multi-beam laser scanning microscopy and an improvement of scanning speed

    Science.gov (United States)

    Seo, Min-Woong; Kagawa, Keiichiro; Yasutomi, Keita; Kawahito, Shoji

    2013-02-01

    Multi-beam laser scanning confocal microscopy with a 256 × 256-pixel custom CMOS imager performing focal-plane pinhole effect, in which any rotating disk is not required, is demonstrated. A specimen is illuminated by 32 × 32 diffraction limited light spots whose wavelength and pitch are 532nm and 8.4 μm, respectively. The spot array is generated by a microlens array, which is scanned by two-dimensional piezo actuator according to the scanning of the image sensor. The frame rate of the prototype is 0.17 Hz, which is limited by the actuator. The confocal effect has been confirmed by comparing the axial resolution in the confocal imaging mode with that of the normal imaging mode. The axial resolution in the confocal mode measured by the full width at half maximum (FWHM) for a planar mirror was 8.9 μm, which is showed that the confocality has been achieved with the proposed CMOS image sensor. The focal-plane pinhole effect in the confocal microscopy with the proposed CMOS imager has been demonstrated at low frame rate. An improvement of the scanning speed and a CMOS imager with photo-sensitivity modulation pixels suitable for high-speed scanning are also discussed.

  10. Artifact mitigation of ptychography integrated with on-the-fly scanning probe microscopy

    Science.gov (United States)

    Huang, Xiaojing; Yan, Hanfei; Ge, Mingyuan; Öztürk, Hande; Nazaretski, Evgeny; Robinson, Ian K.; Chu, Yong S.

    2017-07-01

    We report our experiences with conducting ptychography simultaneously with the X-ray fluorescence measurement using the on-the-fly mode for efficient multi-modality imaging. We demonstrate that the periodic artifact inherent to the raster scan pattern can be mitigated using a sufficiently fine scan step size to provide an overlap ratio of >70%. This allows us to obtain transmitted phase contrast images with enhanced spatial resolution from ptychography while maintaining the fluorescence imaging with continuous-motion scans on pixelated grids. This capability will greatly improve the competence and throughput of scanning probe X-ray microscopy.

  11. Dimensional comparison between amplitude-modulation atomic force microscopy and scanning ion conductance microscopy of biological samples

    Science.gov (United States)

    Kim, Joonhui; Choi, MyungHoon; Jung, Goo-Eun; Rahim Ferhan, Abdul; Cho, Nam-Joon; Cho, Sang-Joon

    2016-08-01

    The range of scanning probe microscopy (SPM) applications for atomic force microscopy (AFM) is expanding in the biological sciences field, reflecting an increasing demand for tools that can improve our fundamental understanding of the physics behind biological systems. However, the complexity associated with applying SPM techniques in biomedical research hampers the full exploitation of its capabilities. Recently, the development of scanning ion conductance microscopy (SICM) has overcome these limitations and enabled contact-free, high resolution imaging of live biological specimens. In this work, we demonstrate the limitation of AFM for imaging biological samples in liquid due to artifacts arising from AFM tip-sample interaction, and how SICM imaging is able to overcome those limitations with contact-free scanning. We also demonstrate that SICM measurements, when compared to AFM, show better fit to the actual dimensions of the biological samples. Our results highlight the superiority of SICM imaging, enabling it to be widely adopted as a general and versatile research tool for biological studies in the nanoscale.

  12. Transparency induced by two photon interference in a beam splitter

    Institute of Scientific and Technical Information of China (English)

    Wang Kai-Ge; Yang Guo-Jian

    2004-01-01

    We propose a special two-photon state which is completely transparent in a 50/50 beam splitter. This effect is caused by the destructive two-photon interference and shows the signature of photon entanglement. We find that the symmetry of the two-photon spectrum plays the key role for the properties of two-photon interference.

  13. Scanning electron microscopy and transmission electron microscopy study of hot-deformed gamma-TiAl-based alloy microstructure.

    Science.gov (United States)

    Chrapoński, J; Rodak, K

    2006-09-01

    The aim of this work was to assess the changes in the microstructure of hot-deformed specimens made of alloys containing 46-50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 degrees C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.

  14. STED microscopy--super-resolution bio-imaging utilizing a stimulated emission depletion.

    Science.gov (United States)

    Otomo, Kohei; Hibi, Terumasa; Kozawa, Yuichi; Nemoto, Tomomi

    2015-08-01

    One of the most popular super-resolution microscopies that breaks the diffraction barrier is stimulated emission depletion (STED) microscopy. As the optical set-up of STED microscopy is based on a laser scanning microscopy (LSM) system, it potentially has several merits of LSM like confocal or two-photon excitation LSM. In this article, we first describe the principles of STED microscopy and then describe the features of our newly developed two-photon excitation STED microscopy. On the basis of our recent results and those of other researchers, we conclude by discussing future research and new technologies in this field.

  15. Single-molecule chemistry and physics explored by low-temperature scanning probe microscopy.

    Science.gov (United States)

    Swart, Ingmar; Gross, Leo; Liljeroth, Peter

    2011-08-28

    It is well known that scanning probe techniques such as scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) routinely offer atomic scale information on the geometric and the electronic structure of solids. Recent developments in STM and especially in non-contact AFM have allowed imaging and spectroscopy of individual molecules on surfaces with unprecedented spatial resolution, which makes it possible to study chemistry and physics at the single molecule level. In this feature article, we first review the physical concepts underlying image contrast in STM and AFM. We then focus on the key experimental considerations and use selected examples to demonstrate the capabilities of modern day low-temperature scanning probe microscopy in providing chemical insight at the single molecule level.

  16. Thermal Properties of Materials Characterized by Scanning Electron-Acoustic Microscopy

    Institute of Scientific and Technical Information of China (English)

    GAO Chun-Ming; ZHANG Shu-Yi; ZHANG Zhong-Ning; SHUI Xiu-Ji; JIANG Tao

    2005-01-01

    @@ A modified technique of scanning electron-acoustic microscopy is employed to determine thermal diffusivity of materials. Using the dependence of the electron-acoustic signal on modulation frequency of the electron beam,the thermal diffusivity of materials is characterized based on a simplified thermoelastic theory. The thermal diffusivities of several metals characterized by the modified scanning electron-acoustic microscopy are in good agreement with the referential values of the corresponding materials, which proves that the scanning electronacoustic microscopy can be used to characterize the thermal diffusivity of materials effectively. In addition, for micro-inhomogeneous materials, such as biological tissues, the macro-effective (average) thermal diffusivities are characterized by the technique.

  17. Emulation and design of terahertz reflection-mode confocal scanning microscopy based on virtual pinhole

    Science.gov (United States)

    Yang, Yong-fa; Li, Qi

    2014-12-01

    In the practical application of terahertz reflection-mode confocal scanning microscopy, the size of detector pinhole is an important factor that determines the performance of spatial resolution characteristic of the microscopic system. However, the use of physical pinhole brings some inconvenience to the experiment and the adjustment error has a great influence on the experiment result. Through reasonably selecting the parameter of matrix detector virtual pinhole (VPH), it can efficiently approximate the physical pinhole. By using this approach, the difficulty of experimental calibration is reduced significantly. In this article, an imaging scheme of terahertz reflection-mode confocal scanning microscopy that is based on the matrix detector VPH is put forward. The influence of detector pinhole size on the axial resolution of confocal scanning microscopy is emulated and analyzed. Then, the parameter of VPH is emulated when the best axial imaging performance is reached.

  18. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking.

    Science.gov (United States)

    Dragoi, Ioan-Catalin; Stanciu, Stefan G; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E; Popescu, Marius; Coltuc, Dinu

    2016-04-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail.

  19. Two-photon cooling of magnesium atoms

    DEFF Research Database (Denmark)

    Malossi, N.; Damkjær, S.; Hansen, P. L.

    2005-01-01

    A two-photon mechanism for cooling atoms below the Doppler temperature is analyzed. We consider the magnesium ladder system (3s2)S01¿(3s3p)P11 at 285.2nm followed by the (3s3p)P11¿(3s3d)D21 transition at 880.7nm . For the ladder system quantum coherence effects may become important. Combined...... with the basic two-level Doppler cooling process this allows for reduction of the atomic sample temperature by more than a factor of 10 over a broad frequency range. First experimental evidence for the two-photon cooling process is presented and compared to model calculations. Agreement between theory...... and experiment is excellent. In addition, by properly choosing the Rabi frequencies of the two optical transitions a velocity independent atomic dark state is observed....

  20. Magnetic two-photon scattering and two-photon emission - Cross sections and redistribution functions

    Science.gov (United States)

    Alexander, S. G.; Meszaros, P.

    1991-01-01

    The magnetic two-photon scattering cross section is discussed within the framework of QED, and the corresponding scattering redistribution function for this process and its inverse, as well as the scattering source function are calculated explicitly. In a similar way, the magnetic two-photon emission process which follows the radiative excitation of Landau levels above ground is calculated. The two-photon scattering and two-photon emission are of the same order as the single-photon magnetic scattering. All three of these processes, and in optically thick cases also their inverses, are included in radiative transport calculations modeling accreting pulsars and gamma-ray bursters. These processes play a prominent role in determining the relative strength of the first two cyclotron harmonics, and their effects extend also to the higher harmonics.

  1. Two-photon ionization of colliding atoms

    Energy Technology Data Exchange (ETDEWEB)

    Nayfeh, M.H.

    1977-09-01

    Semiclassical expressions of two-photon ionization of two colliding atoms are derived for a wide range of electromagnetic field intensity and detunings from the isolated atom line. The dependence of the ionization yield on the details of the interaction potential of the system is derived. This process promises an extremely sensitive method for studying line broadening on the far wing, especially when absorption or fluorescence becomes very weak.

  2. Two-photon cooling of magnesium atoms

    DEFF Research Database (Denmark)

    Malossi, N.; Damkjær, S.; Hansen, P. L.;

    2005-01-01

    A two-photon mechanism for cooling atoms below the Doppler temperature is analyzed. We consider the magnesium ladder system (3s2)S01¿(3s3p)P11 at 285.2nm followed by the (3s3p)P11¿(3s3d)D21 transition at 880.7nm . For the ladder system quantum coherence effects may become important. Combined...

  3. Two-Photon Collective Atomic Recoil Lasing

    Directory of Open Access Journals (Sweden)

    James A. McKelvie

    2015-11-01

    Full Text Available We present a theoretical study of the interaction between light and a cold gasof three-level, ladder configuration atoms close to two-photon resonance. In particular, weinvestigate the existence of collective atomic recoil lasing (CARL instabilities in differentregimes of internal atomic excitation and compare to previous studies of the CARL instabilityinvolving two-level atoms. In the case of two-level atoms, the CARL instability is quenchedat high pump rates with significant atomic excitation by saturation of the (one-photoncoherence, which produces the optical forces responsible for the instability and rapid heatingdue to high spontaneous emission rates. We show that in the two-photon CARL schemestudied here involving three-level atoms, CARL instabilities can survive at high pump rateswhen the atoms have significant excitation, due to the contributions to the optical forces frommultiple coherences and the reduction of spontaneous emission due to transitions betweenthe populated states being dipole forbidden. This two-photon CARL scheme may form thebasis of methods to increase the effective nonlinear optical response of cold atomic gases.

  4. Applications of confocal laser scanning microscopy in research into organic semiconductor thin films

    DEFF Research Database (Denmark)

    Schiek, Manuela; Balzer, Frank

    2014-01-01

    At the center of opto-electronic devices are thin layers of organic semiconductors, which need to be sandwiched between planar electrodes. With the growing demand for opto-electronic devices now and in the future, new electrode materials are needed to meet the requirements of organic semiconductors...... laser scanning microscopy has emerged as a versatile tool for optical metrology while atomic force microscopy adds detailed structural information....

  5. Correlation of live-cell imaging with volume scanning electron microscopy.

    Science.gov (United States)

    Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

    2017-01-01

    Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Apedo, K.L., E-mail: apedo@unistra.fr [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Munzer, C.; He, H. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Montgomery, P. [ICube, Université de Strasbourg, CNRS, 23 rue du Loess, 67037 Strasbourg (France); Serres, N. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France); Fond, C. [ICube, Université de Strasbourg, CNRS, 2 rue Boussingault, 67000 Strasbourg (France); Feugeas, F. [ICube, INSA de Strasbourg, CNRS, 24 Bld de la Victoire, 67084 Strasbourg (France)

    2015-02-15

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

  7. A reverse pendulum bath cryostat design suitable for low temperature scanning probe microscopy

    Science.gov (United States)

    Heyde, M.; Thielsch, G.; Rust, H.-P.; Freund, H.-J.

    2005-03-01

    A new low temperature, ultrahigh vacuum cryostat design has been developed for atomic force and scanning tunnelling microscopy measurements. A microscope can be operated at 5 K in ultrahigh vacuum. The microscope body is thermally connected to a reverse pendulum and completely surrounded by a radiation shield. The design allows in situ dosing and irradiation of the sample as well as for easy access of tip and sample. The temperature performance and the vibrational properties of the reverse pendulum design are demonstrated in detail. A brief overview of low temperature instrumentation in scanning probe microscopy is given.

  8. Thin films of metal oxides on metal single crystals: Structure and growth by scanning tunneling microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Galloway, Heather Claire [Univ. of California, Berkeley, CA (United States)

    1995-12-01

    Detailed studies of the growth and structure of thin films of metal oxides grown on metal single crystal surfaces using Scanning Tunneling Microscopy (STM) are presented. The oxide overlayer systems studied are iron oxide and titanium oxide on the Pt(III) surface. The complexity of the metal oxides and large lattice mismatches often lead to surface structures with large unit cells. These are particularly suited to a local real space technique such as scanning tunneling microscopy. In particular, the symmetry that is directly observed with the STM elucidates the relationship of the oxide overlayers to the substrate as well as distinguishing, the structures of different oxides.

  9. Scanning electron microscopy of the human endolymphatic sac: a preliminary report.

    Science.gov (United States)

    Galey, F R; House, W F

    1980-04-01

    Scanning electron microscopy has been used to examine and compare one normal endolymphatic sac with one endolymphatic sac from a patient with Meniere's disease. The surgical procedure for obtaining these specimens and their preparation for scanning electron microscopy are described. The luminal surface of the rugose portion of both specimens was lined with two populations of epithelial cells: one with a dome-shaped apical surface, the other with a flattened polygonal surface. The surface of dome-shaped cells in both specimens was covered with microvilli. Neither specimen had observable loss of epithelial integrity or fibrosis.

  10. Scanning tunneling microscopy I general principles and applications to clean and adsorbate-covered surfaces

    CERN Document Server

    Wiesendanger, Roland

    1992-01-01

    Scanning Tunneling Microscopy I provides a unique introduction to a novel and fascinating technique that produces beautiful images of nature on an atomic scale. It is the first of three volumes that together offer a comprehensive treatment of scanning tunneling microscopy, its diverse applications, and its theoretical treatment. In this volume the reader will find a detailed description of the technique itself and of its applications to metals, semiconductors, layered materials, adsorbed molecules and superconductors. In addition to the many representative results reviewed, extensive references to original work will help to make accessible the vast body of knowledge already accumulated in this field.

  11. Sub-micron imaging of buried integrated circuit structures using scanning confocal electron microscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Frigo, S. P.; Levine, Z.; Zaluzec, N. J.; Materials Science Division; Northern Arizona Univ.; NIST

    2002-09-09

    Two-dimensional images of model integrated circuit components were collected using the technique of scanning confocal electron microscopy. For structures embedded about 5 {mu}m below the surface of a silicon oxide dielectric, a lateral resolution of 76{+-}9 nm was measured. Elemental mapping via x-ray emission spectrometry is demonstrated. A parallax analysis of images taken for various tilt angles to the electron beam allowed determination of the spacing between two wiring planes. The results show that scanning confocal electron microscopy is capable of probing buried structures at resolutions that will be necessary for the inspection of next-generation integrated circuit technology.

  12. Heterodyne method of apertureless near-field scanning optical microscopy on periodic gold nanowells.

    Energy Technology Data Exchange (ETDEWEB)

    Hall, J. E.; Wiederrecht, G. P.; Gray, S. K.; Chang, S.-H.; Jeon, S.; Rogers, J. A.; Bachelot, R.; Royer, P.; Univ. of Illinois; Univ. of Technology at Troyes; Inst. of Electro-Optical Science and Engineering

    2007-04-02

    Heterodyne detection for apertureless near-field scanning optical microscopy was used to study periodic gold nanowell arrays. Optical near-field amplitude and phase signals were obtained simultaneously with the topography of the gold nanowells and with different polarizations. Theoretical calculations of the near-fields were consistent with the experiments; in particular, the calculated amplitudes were in especially good agreement. The heterodyne method is shown to be particularly effective for these types of periodic photonic structures and other highly scattering media, which can overwhelm the near-field scattered signal when conventional apertureless near-field scanning optical microscopy is used.

  13. Imaging by Electrochemical Scanning Tunneling Microscopy and Deconvolution Resolving More Details of Surfaces Nanomorphology

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    to crystallographic-surface structures. Within the wide range of new technologies, those images surface features, the electrochemical scanning tunneling microscope (ESTM) provides means of atomic resolution where the tip participates actively in the process of imaging. Two metallic surfaces influence ions trapped...... of the characteristic details of the images. A large proportion of the observed noise may be explained by the scanning actions of the feedback circuitry while a minor fraction of the image details may be explained by surface drift phenomena. As opposed to the method of deconvolution, conventional methods of filtering......Upon imaging, electrochemical scanning tunneling microscopy (ESTM), scanning electrochemical micro-scopy (SECM) and in situ STM resolve information on electronic structures and on surface topography. At very high resolution, imaging processing is required, as to obtain information that relates...

  14. Local analysis of semiconductor nanoobjects by scanning tunneling atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Natalia A. Lashkova

    2015-03-01

    Full Text Available The features of the current–voltage (I–V measurements in local regions of semiconductor nanostructures by conductive atomic force microscopy (AFM are discussed. The standard procedure of I–V measurements in conductive AFM leads not infrequently to the thermomechanical stresses in the sample and, as a consequence, nonreproducibility and unreliability of measurements. The technique of obtaining reproducible current–voltage characteristics is proposed. According to the technique, a series of measurements of the selected scanning area in the mode of conducting AFM should be taken, each at the certain value of the potential. According to a series of scans I–V curve at a particular point (for any point of the scan was plotted. The program is realized in the LabVIEW software. The proposed method extends the capabilities of scanning probe microscopy in the diagnosis of nanostructured semiconductor materials.

  15. Two-photon super bunching of thermal light via multiple two-photon-path interference

    CERN Document Server

    Hong, Peilong; Zhang, Guoquan

    2012-01-01

    We propose a novel scheme to achieve two-photon super bunching of thermal light through multiple two-photon-path interference, in which two mutually first-order incoherent optical channels are introduced by inserting a modified Michelson interferometer into a traditional two-photon HBT interferometer, and the bunching peak-to-background ratio can reach 3 theoretically. Experimentally, the super bunching peak-to-background ratio was measured to be 2.4, much larger than the ratio 1.7 measured with the same thermal source in a traditional HBT interferometer. The peak-to-background ratio of two-photon super bunching of thermal light can be increased up to $2\\times1.5^n$ by inserting cascadingly $n$ pairs of mutually first-order incoherent optical channels into the traditional two-photon HBT interferometer. The two-photon super bunching of thermal light should be of great significance in improving the visibility of classical ghost imaging.

  16. Aggregation induced enhanced emission of conjugated dendrimers with a large intrinsic two-photon absorption cross-section

    NARCIS (Netherlands)

    Xu, Bin; Zhang, Jibo; Fang, Honghua; Ma, Suqian; Chen, Qidai; Sun, Hongbo; Im, Chan; Tian, Wenjing

    2014-01-01

    Organic nonlinear optical materials combining high luminescence quantum yields and large two-photon absorption cross-sections are attractive for both fundamental research and practical applications, such as up-converted lasers and two-photon fluorescence microscopy. Herein, we reported a series of

  17. Morphological aspects of Angiostrongylus costaricensis by light and scanning electron microscopy.

    Science.gov (United States)

    Rebello, Karina M; Menna-Barreto, Rubem F S; Chagas-Moutinho, Vanessa A; Mota, Ester M; Perales, Jonas; Neves-Ferreira, Ana Gisele C; Oliveira-Menezes, Aleksandra; Lenzi, Henrique

    2013-09-01

    Angiostrongylus costaricensis is a parasitic nematode that can cause severe gastrointestinal disease, known as abdominal angiostrongiliasis, in humans. This paper presents the characterization of first- and third-stage larvae and male and female adult worms of A. costaricensis by scanning electron and light microscopy. Several novel anatomical structures were identified by scanning electron microscopy, including details of the cuticular striations of the spicules in male worms and a protective flap of the cuticle covering the vulvar aperture in female worms. Other taxonomic features revealed by light microscopy include the gubernaculum and the esophageal-intestinal valve. The use of two microscopy techniques allowed a detailed characterization of the morphology of this nematode. A number of previously identified taxonomic features, such as the striated nature of the spicules and the lateral alae were confirmed; however, the use of scanning electron microscopy resulted in a reassessment of the correct number of papillae distributed around the oral opening and behind the cloacal opening. These observations, in combination with light microscopy-based characterization of the gubernaculum and esophageal valves, have allowed a more detailed description of this nematode taxonomy.

  18. Correlative Light and Scanning X-Ray Scattering Microscopy of Healthy and Pathologic Human Bone Sections

    OpenAIRE

    Giannini, C.; D. Siliqi; Bunk, O.; Beraudi, A.; Ladisa, M.; Altamura, D.; Stea, S.; Baruffaldi, F.

    2012-01-01

    Scanning small and wide angle X-ray scattering (scanning SWAXS) experiments were performed on healthy and pathologic human bone sections. Via crystallographic tools the data were transformed into quantitative images and as such compared with circularly polarized light (CPL) microscopy images. SWAXS and CPL images allowed extracting information of the mineral nanocrystalline phase embedded, with and without preferred orientation, in the collagen fibrils, mapping local changes at sub-osteon res...

  19. Gene sequencing by scanning molecular excitation microscopy. Progress report, June 1990--June 1993

    Energy Technology Data Exchange (ETDEWEB)

    Kopelman, R.

    1993-07-01

    We propose to complete the development of scanning molecular excitation microscopy to rapidly sequence DNA. This new type of scanned-tip microscopy is specifically designed to map individual DNA bases in a non-destructive fashion. Base recognition is based on the external heavy atom effect between a heavy atom label on a specific DNA base and a fluorescent sensor molecule at the end of a scanned optical tip. As the tip is scanned very close to the heavy atom its emissions will change intensity and wavelength. For sequencing, heavy-atom labeled single-stranded DNA molecules will be synthesized and oriented on a flat substrate such as mica. An optical tag an one end of each DNA molecule will be rapidly located at low resolution using conventional fluorescence or scanning near-field optical microscopy. Scanning with the same optical tip, the low-resolution path of the DNA will be mapped using long-range interactions such as attractive van der Waals forces or Forester energy transfer. Subsequently, the labeled bases will be mapped at better than 5{Angstrom} resolution with the same tip using the external heavy atom effect, and the coordinates stored and processed by computer. The proposed microscope could be automated to quickly sequence intact lambda clones, YACK, or genomic Not I fragments, making genomic sequencing much more rapid and economical.

  20. Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection.

    Science.gov (United States)

    Zhi, Yanan; Wang, Benquan; Yao, Xincheng

    2015-01-01

    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.

  1. A spirobifluorene-based two-photon fluorescence probe for mercury ions and its applications in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Haibo, E-mail: xiaohb@shnu.edu.cn; Zhang, Yanzhen; Zhang, Wu; Li, Shaozhi; Tan, Jingjing; Han, Zhongying

    2017-05-01

    A novel spirobifluorene derivative SPF-TMS, which containing dithioacetal groups and triphenylamine units, was synthesized. The probing behaviors toward various metal ions were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. The results indicated that SPF-TMS exhibits high sensitivity and selectivity for mercury ions. The detection limit was at least 8.6 × 10{sup −8}M, which is excellent comparing with other optical sensors for Hg{sup 2+}. When measured by two-photon excited fluorescence technique in THF at 800 nm, the two-photon cross-section of SPF-TMS is 272 GM. Especially, upon reaction with mercury species, SPF-TMS yielded another two-photon dye SPF-DA. Both SPF-TMS and SPF-DA emit strong two-photon induced fluorescence and can be applied in cell imaging by two-photon microscopy. - Highlights: • We report a spirobifluorene-based molecule as two-photon fluorescent probe with large two-photon cross-section. • The molecule has exclusive selectivity and sensitivity for mercury species. • The molecule has large two-photon emission changes before and after addition of Hg{sup 2+}. • Both the probe and the mercury ion-promoted reaction product can be applied in cell imaging by two-photon microscopy.

  2. Two-axis water-immersible microscanning mirror for scanning optics and acoustic microscopy

    Science.gov (United States)

    Xu, Song; Zou, Jun

    2016-10-01

    Fast multiaxis scanning is useful for not only optical but also acoustic microscopic imaging. Although they have been used for optical scanning, the application of (MEMS) scanning mirrors in acoustic microscopy is still very limited due to their small mirror plate size, and more importantly, inability to operate in liquids (as ultrasound coupling media). A microfabricated two-axis water-immersible scanning mirror for optical and acoustic microscopy is reported. It has an optical and acoustically reflective mirror plate (6 mm×4 mm) to provide numerical aperture for ultrasound beam steering. Electromagnetic and mechanical analysis and simulation were conducted to estimate the mechanical tilting angle and resonance frequency of both fast and slow axes, which matches well with the measurement results. The fast axis has a resonant frequency of 320 Hz in air and 220 Hz in water, which is more than 10 times higher than that of the slow axis (24 Hz in air and 14 Hz in water). Under a 100-mA driving current, the scanning angles of the fast axis reached ±9.5 deg in both air and water at the resonance frequency, respectively. The scanning angles of the slow axis reached ±15 deg in air and ±12.5 deg in water at resonant frequencies, respectively. Raster scanning of a collimated laser beam was achieved by driving both axes simultaneously close to their own resonance frequencies. The feasibility of using the two-axis water-immersible scanning mirror in scanning acoustic microscopy was also demonstrated.

  3. In situ surface reduction of a NiO-YSZ-alumina composite using scanning probe microscopy

    DEFF Research Database (Denmark)

    Hansen, Karin Vels; Jacobsen, Torben; Thydén, Karl Tor Sune

    2014-01-01

    In situ surface reductions of NiO-YSZ-Al2O3 composites into Ni-YSZ-Al2O3 cermets were carried out at 312–525 °C in a controlled atmosphere high-temperature scanning probe microscope (CAHT-SPM) in dry and humidified 9 % H2 in N2. The reduction of NiO was followed by contact mode scanning...... dependent and followed the Arrhenius equation. For samples reduced in dry hydrogen, the Arrhenius plot showed two regions with different activation energies. Scanning electron microscopy confirmed a difference in microstructure between these temperature regimes. A strong retarding effect of steam (H2O...

  4. Development of a fast electromagnetic shutter for compressive sensing imaging in scanning transmission electron microscopy

    CERN Document Server

    Béché, Armand; Freitag, Bert; Verbeeck, Jo

    2015-01-01

    The concept of compressive sensing was recently proposed to significantly reduce the electron dose in scanning transmission electron microscopy (STEM) while still maintaining the main features in the image. Here, an experimental setup based on an electromagnetic shutter placed in the condenser plane of a STEM is proposed. The shutter blanks the beam following a random pattern while the scanning coils are moving the beam in the usual scan pattern. Experimental images at both medium scale and high resolution are acquired and then reconstructed based on a discrete cosine algorithm. The obtained results confirm the predicted usefulness of compressive sensing in experimental STEM even though some remaining artifacts need to be resolved.

  5. Telocytes and putative stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy.

    Science.gov (United States)

    Popescu, Laurentiu M; Gherghiceanu, Mihaela; Suciu, Laura C; Manole, Catalin G; Hinescu, Mihail E

    2011-09-01

    This study describes a novel type of interstitial (stromal) cell - telocytes (TCs) - in the human and mouse respiratory tree (terminal and respiratory bronchioles, as well as alveolar ducts). TCs have recently been described in pleura, epicardium, myocardium, endocardium, intestine, uterus, pancreas, mammary gland, etc. (see www.telocytes.com ). TCs are cells with specific prolongations called telopodes (Tp), frequently two to three per cell. Tp are very long prolongations (tens up to hundreds of μm) built of alternating thin segments known as podomers (≤ 200 nm, below the resolving power of light microscope) and dilated segments called podoms, which accommodate mitochondria, rough endoplasmic reticulum and caveolae. Tp ramify dichotomously, making a 3-dimensional network with complex homo- and heterocellular junctions. Confocal microscopy reveals that TCs are c-kit- and CD34-positive. Tp release shed vesicles or exosomes, sending macromolecular signals to neighboring cells and eventually modifying their transcriptional activity. At bronchoalveolar junctions, TCs have been observed in close association with putative stem cells (SCs) in the subepithelial stroma. SCs are recognized by their ultrastructure and Sca-1 positivity. Tp surround SCs, forming complex TC-SC niches (TC-SCNs). Electron tomography allows the identification of bridging nanostructures, which connect Tp with SCs. In conclusion, this study shows the presence of TCs in lungs and identifies a TC-SC tandem in subepithelial niches of the bronchiolar tree. In TC-SCNs, the synergy of TCs and SCs may be based on nanocontacts and shed vesicles.

  6. Morphologic differences observed by scanning electron microscopy according to the reason for pseudophakic IOL explantation

    DEFF Research Database (Denmark)

    Fernandez-Buenaga, Roberto; Alio, Jorge L.; Ramirez, Jose M.

    2015-01-01

    Purpose To compare variations in surface morphology, as studied by scanning electron microscopy (SEM), of explanted intraocular lenses (IOLs) concerning the cause leading to the explantation surgery. Methods In this prospective multicenter study, explanted IOLs were analyzed by SEM and energy-dis...

  7. Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

    1997-01-01

    In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms a...

  8. Calibration-free quantitative surface topography reconstruction in scanning electron microscopy

    NARCIS (Netherlands)

    Faber, E.T.; Martinez-Martinez, D.; Mansilla, C.; Ocelik, V.; De Hosson, J. Th. M.

    2015-01-01

    This work presents a new approach to obtain reliable surface topography reconstructions from 2D Scanning Electron Microscopy (SEM) images. In this method a set of images taken at different tilt angles are compared by means of digital image correlation (DlC). It is argued that the strength of the met

  9. Dental wax impressions of plant tissues for viewing with scanning electron microscopy (SEM).

    Science.gov (United States)

    Beermann, Anke; Hülskamp, Martin

    2010-09-01

    Scanning electron microscopy (SEM) is a valuable method for examining surface structures. Taking wax impressions of plant structures, such as leaves, is a nondestructive procedure that makes it possible to view changes in surface structures over time, such as during development. This protocol describes a method for making dental wax impressions of plant tissues.

  10. Chemical Silver Coating of Fiber Tips in Near-Field Scanning Optical Microscopy

    Science.gov (United States)

    Vikram, Chandra S.; Witherow, William K.

    1998-01-01

    We report what is believed to be the first experimental demonstration of silver coating by a wet chemical process on tapered fiber tips used in near-field scanning optical microscopy. The process is at room temperature and pressure and takes only a few minutes to complete. Many tips can be simultaneously coated.

  11. Stochastic and photochromic switching of diarylethenes studied by scanning tunnelling microscopy

    NARCIS (Netherlands)

    Molen, S.J. van der; Vegte, H. van der; Kudernac, T.; Amin, I.; Feringa, B.L.; Wees, B.J. van

    2006-01-01

    We investigate switching of photochromic, thiophene-substituted diarylethene switches on gold. For this, the conjugated isomer is inserted in a dodecanethiol self-assembled monolayer. This layer is subsequently scanned by tunnelling microscopy. First, we study the statistics of stochastic switching

  12. Visualisation of biopolymer mixtures using confocal scanning laser microscopy (CSLM) and covalent labelling techniques

    NARCIS (Netherlands)

    Velde, van de F.; Weinbreck, F.; Edelman, M.W.; Linden, van der E.; Tromp, R.H.

    2003-01-01

    Confocal scanning laser microscopy (CSLM) has been used to study the behaviour of mixtures of proteins, gelatine, whey proteins and ß-lactoglobulin, and polysaccharides, dextran, gellan gum, carrageenan, gum Arabic, and starch. CSLM proved to be a suitable technique to visualise the microstructure o

  13. Adsorption of Cu phthalocyanine on Pt modified Ge(001): A scanning tunneling microscopy study

    NARCIS (Netherlands)

    Saedi, Amirmehdi; Berkelaar, Robin P.; Kumar, Avijit; Poelsema, Bene; Zandvliet, Harold J.W.

    2010-01-01

    The adsorption configurations of copper phthalocyanine (CuPc) molecules on platinum-modified Ge(001) have been studied using scanning tunneling microscopy. After deposition at room temperature and cooling down to 77 K the CuPc molecules are still dynamic. However, after annealing at 550±50 K, the ad

  14. Developments in application of light and scanning electron microscopy techniques for cell wall degradation studies.

    NARCIS (Netherlands)

    Engels, F.M.

    1996-01-01

    The results of recent technological developments in light and scanning electron microscopy closely used for research on forage cell wall degradation in ruminants, are reviewed. The indigestibility of forages by rumen microorganisms used to be ascribed mainly to an overall presence of lignin in the p

  15. Second-Harmonic Generation Scanning Microscopy on Domains in Al Surfaces

    DEFF Research Database (Denmark)

    Pedersen, Kjeld; Bozhevolnyi, Sergey I.

    1999-01-01

    Scanning optical second-harmonic generation microscopy has been used to investigate domains in the surface of polycrystaline Al. Strong contrast among the crystalline grains is obtained due to variations in their crystallographic orientations and thus also nonlinear response. The origin of the co...

  16. CONFOCAL SCANNING LASER MICROSCOPY OF MITOCHONDRIA - A POSSIBLE TOOL IN THE DIAGNOSIS OF MITOCHONDRIAL DISORDERS

    NARCIS (Netherlands)

    RUITERS, MHJ; VANSPRONSEN, EA; SKJELDAL, OH; STROMME, P; SCHOLTE, HR; PZYREMBEL, H; SMIT, GPA; RUITENBEEK, W; AGSTERIBBE, E

    1991-01-01

    This paper describes a non-invasive method for the study of mitochondrial morphology in cultured human skin fibroblasts by confocal scanning laser microscopy after staining the mitochondria with 2-[4-(dimethyl-aminostyryl]-1-methylpyridinium iodide. This method is applied to compare mitochondria in

  17. Elastic Changes of Capsule in a Rat Knee Contracture Model Assessed by Scanning Acoustic Microscopy

    Science.gov (United States)

    Hagiwara, Y.; Chimoto, E.; Ando, A.; Saijo, Y.; Itoi, E.

    Sound speed of a capsule in a rat knee contracture model was measured by scanning acoustic microscopy. There was no statistical significant difference in the anterior capsule compared with the control group. However, the sound speed of the posterior capsule was significantly greater compared with the control group after prolonged immobilization.

  18. Batch fabrication of scanning microscopy probes for thermal and magnetic imaging using standard micromachining

    NARCIS (Netherlands)

    Sarajlic, Edin; Vermeer, Rolf; Delalande, M.Y.; Siekman, Martin Herman; Huijink, R.; Fujita, H.; Abelmann, Leon

    2010-01-01

    We present a process for batch fabrication of a novel scanning microscopy probe for thermal and magnetic imaging using standard micromachining and conventional optical contact lithography. The probe features an AFM-type cantilever with a sharp pyramidal tip composed of four freestanding silicon

  19. Imaging inclusion complex formation in starch granules using confocal laser scanning microscopy

    NARCIS (Netherlands)

    Manca, Marianna; Woortman, Albert J. J.; Loos, Katja; Loi, Maria A.

    2015-01-01

    The tendency of amylose to form inclusion complexes with guest molecules has been an object of wide interest due to its fundamental role in food processing. Here we investigated the features of starch granules from several botanical sources using confocal laser scanning microscopy (CLSM) and uncover

  20. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis

    DEFF Research Database (Denmark)

    Skytte, Jacob Lercke; Ghita, Ovidiu; Whelan, Paul F.

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermente...