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Sample records for 4-dimensional intravital microscopy

  1. Understanding liver immunology using intravital microscopy.

    PubMed

    Marques, Pedro Elias; Oliveira, André Gustavo; Chang, Lynne; Paula-Neto, Heitor Affonso; Menezes, Gustavo Batista

    2015-09-01

    The liver has come a long way since it was considered only a metabolic organ attached to the gastrointestinal tract. The simultaneous ascension of immunology and intravital microscopy evidenced the liver as a central axis in the immune system, controlling immune responses to local and systemic agents as well as disease tolerance. The multiple hepatic cell populations are organized in a vascular environment that promotes intimate cellular interactions, including initiation of innate and adaptive immune responses, rapid leukocyte recruitment, pathogen clearance and production of a variety of immune mediators. In this review, we focus on the advances in liver immunology supported by intravital microscopy in diseases such as isquemia/reperfusion, acute liver injury and infections.

  2. Intravital Microscopy for THz-Bio Analysis

    NASA Astrophysics Data System (ADS)

    Kim, Pilhan

    Intravital microscopy is a high-resolution imaging technique to observe biological phenomena in living organisms. It often also stated as in vivo microscopy. Literal meaning of in vivo is "within the living" and there is another term, ex vivo of which literal meaning is "out of the living". Both terms are commonly used to describe the status of sample at the moment of biological manipulations or investigations are done. In vivo study is a form of research using whole living organism in experiment to investigate a certain biological phenomenon in its natural environment, whereas ex vivo study uses non-living subjects such as tissues or organs dissected from dead animal. In addition, in vitro of which literal meaning is "within the glass" is another commonly used term. In vitro study is a form of research using small living subject such as cell in a controlled environment such as petri dish or test tube. Cell culture, the process of growing cells in a petri dish, is the most common form of in vitro study. Figure 1 summarizes the status of samples for biological study categorized by in vivo, in vitro and ex vivo.

  3. Multiphoton intravital microscopy setup to visualize the mouse mammary gland

    NASA Astrophysics Data System (ADS)

    Adur, Javier; Herrera Torres, Ana M.; Masedunskas, Andrius; Baratti, Mariana O.; de Thomaz, Andre A.; Pelegati, Vitor B.; Carvalho, Hernandes F.; Cesar, Carlos L.

    2013-06-01

    Recently, light microscopy-based techniques have been extended to live mammalian models leading to the development of a new imaging approach called intravital microscopy (IVM). Although IVM has been introduced at the beginning of the last century, its major advancements have occurred in the last twenty years with the development of non-linear microscopy that has enabled performing deep tissue imaging. IVM has been utilized to address many biological questions in basic research and is now a fundamental tool that provide information on tissues such as morphology, cellular architecture, and metabolic status. IVM has become an indispensable tool in numerous areas. This study presents and describes the practical aspects of IVM necessary to visualize epithelial cells of live mouse mammary gland with multiphoton techniques.

  4. In vivo Evaluation of Venular Glycocalyx during Hemorrhagic Shock in Rats using Intravital Microscopy

    DTIC Science & Technology

    2013-01-01

    Brief Communication In vivo evaluation of venular glycocalyx during hemorrhagic shock in rats using intravital microscopy☆,☆☆ Ivo Torres Filho...pathophysiology and tested the hypothesis that hemorrhage causes glycocalyx degrada- tion in cremaster muscle microvessels. We utilized intravital microscopy...bound (Reitsma et al., 2007; Weinbaum et al., 2007). Intravital microscopy has been an invaluable resource for in vivo measurements of critically

  5. Imaging Nanotherapeutics in Inflamed Vasculature by Intravital Microscopy

    PubMed Central

    Wang, Zhenjia

    2016-01-01

    Intravital microscopy (IVM) is the application of light microscopy to real time study biology of live animal tissues in intact and physiological conditions with the high spatial and temporal resolution. Advances in imaging systems, genetic animal models and imaging probes, IVM has offered quantitative and dynamic insight into cell biology, immunology, neurobiology and cancer. In this review, we will focus on the targeting of nanotherapeutics to inflamed vasculature. We will introduce the basic concept and principle of IVM and demonstrate that IVM is a powerful tool used to quantitatively determine the molecular mechanisms of interactions between nanotherapeutics and neutrophils or endothelium in living mice. In the future, it is needed to develop new imaging systems and novel imaging contrast agents to better understand molecular mechanisms of tissue processing of nanotherapeutics in vivo. PMID:27877245

  6. Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Turcotte, Raphaël; Rutledge, Danette J.; Bélanger, Erik; Dill, Dorothy; Macklin, Wendy B.; Côté, Daniel C.

    2016-08-01

    Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo.

  7. Intravital assessment of myelin molecular order with polarimetric multiphoton microscopy

    PubMed Central

    Turcotte, Raphaël; Rutledge, Danette J.; Bélanger, Erik; Dill, Dorothy; Macklin, Wendy B.; Côté, Daniel C.

    2016-01-01

    Myelin plays an essential role in the nervous system and its disruption in diseases such as multiple sclerosis may lead to neuronal death, thus causing irreversible functional impairments. Understanding myelin biology is therefore of fundamental and clinical importance, but no tools currently exist to describe the fine spatial organization of myelin sheaths in vivo. Here we demonstrate intravital quantification of the myelin molecular structure using a microscopy method based on polarization-resolved coherent Raman scattering. Developmental myelination was imaged noninvasively in live zebrafish. Longitudinal imaging of individual axons revealed changes in myelin organization beyond the diffraction limit. Applied to promyelination drug screening, the method uniquely enabled the identification of focal myelin regions with differential architectures. These observations indicate that the study of myelin biology and the identification of therapeutic compounds will largely benefit from a method to quantify the myelin molecular organization in vivo. PMID:27538357

  8. Advanced Motion Compensation Methods for Intravital Optical Microscopy

    PubMed Central

    Vinegoni, Claudio; Lee, Sungon; Feruglio, Paolo Fumene; Weissleder, Ralph

    2013-01-01

    Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions. PMID:24273405

  9. Improved intravital microscopy via synchronization of respiration and holder stabilization

    NASA Astrophysics Data System (ADS)

    Lee, Sungon; Vinegoni, Claudio; Feruglio, Paolo Fumene; Weissleder, Ralph

    2012-09-01

    A major challenge in high-resolution intravital confocal and multiphoton microscopy is physiologic tissue movement during image acquisition. Of the various physiological sources of movement, respiration has arguably the largest and most wide-ranging effect. We describe a technique for achieving stabilized microscopy imaging using a dual strategy. First, we designed a mechanical stabilizer for constraining physical motion; this served to simultaneously increase the in-focus range over which data can be acquired as well as increase the reproducibility of imaging a certain position within each confocal imaging plane. Second, by implementing a retrospective breathing-gated imaging modality, we performed selective image extraction gated to a particular phase of the respiratory cycle. Thanks to the high reproducibility in position, all gated images presented a high degree of correlation over time. The images obtained using this technique not only showed significant improvements over images acquired without the stabilizer, but also demonstrated accurate in vivo imaging during longitudinal studies. The described methodology is easy to implement with any commercial imaging system, as are used by most biological imaging laboratories, and can be used for both confocal and multiphoton laser scanning microscopy.

  10. Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

    PubMed Central

    Andresen, Volker; Sporbert, Anje

    2014-01-01

    Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells – on the level of a few protein molecules in germinal centers. PMID:24748007

  11. Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model.

    PubMed

    Pavlovic, Dragan; Frieling, Helge; Lauer, Kai-Stephan; Bac, Vo Hoai; Richter, Joern; Wendt, Michael; Lehmann, Christian; Usichenko, Taras; Meissner, Konrad; Gruendling, Matthias

    2006-11-01

    Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ-support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine-support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2-3 cm long) was gently exteriorized and placed on the 'hand' of the organ support. A small part of the intestine formed a small 'island' in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a 'hanging drop'. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The 'hanging drop' technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.

  12. Microbial pathogenesis revealed by intravital microscopy: pros, cons and cautions.

    PubMed

    Stolp, Bettina; Melican, Keira

    2016-07-01

    Intravital multiphoton imaging allows visualization of infections and pathogenic mechanisms within intact organs in their physiological context. Today, most organs of mice and rats are applicable to in vivo or ex vivo imaging, opening completely new avenues for many researchers. Advances in fluorescent labeling of pathogens and infected cells, as well as improved small animal models for human pathogens, led to the increased application of in vivo imaging in infectious diseases research in recent years. Here, we review the latest literature on intravital or ex vivo imaging of viral and bacterial infections and critically discuss requirements, benefits and drawbacks of applied animal models, labeling strategies, and imaged organs.

  13. Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation.

    PubMed

    Burkovskiy, I; Lehmann, C; Jiang, C; Zhou, J

    2016-11-01

    Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single-step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.

  14. Intravital microscopy as a tool to study drug delivery in preclinical studies

    PubMed Central

    Amornphimoltham, Panomwat; Masedunskas, Andrius; Weigert, Roberto

    2010-01-01

    The technical developments in the field of non-linear microscopy have made intravital microscopy one of the most successful techniques for studying physiological and pathological processes in live animals. Intravital microscopy has been utilized to address many biological questions in basic research and is now a fundamental tool for preclinical studies, with an enormous potential for clinical applications. The ability to dynamically image cellular and subcellular structures combined with the possibility to perform longitudinal studies have empowered investigators to use this discipline to study the mechanisms of action of therapeutic agents and assess the efficacy on their targets in vivo. The goal of this review is to provide a general overview of the recent advances in intravital microscopy and to discuss some of its applications in preclinical studies. PMID:20933026

  15. Early development of cutaneous cancer revealed by intravital nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Chin; Li, Feng-Chieh; Lin, Wei-Chou; Chen, Yang-Fang; Chen, Shean-Jen; Lin, Sung-Jan; Dong, Chen-Yuan

    2010-09-01

    We performed intravital multiphoton microscopy to image and analyze normal and carcinogen treated skin tissues of nude mice in vivo. Using intravital images and the quantitative pixel to pixel ratiometric processing of multiphoton autofluorescence to second harmonic generation index (MAFSI), we can visualize the interaction between epithelial cells and extracellular matrix. We found that as the imaging depth increases, MAFSI has different distribution in normal and treated cutaneous specimens. Since the treated skin eventually became squamous cell carcinoma, our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by multiphoton microscopy.

  16. Two-photon microscopy of deep intravital tissues and its merits in clinical research.

    PubMed

    Wang, B-G; König, K; Halbhuber, K-J

    2010-04-01

    Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

  17. Automated motion artifact removal for intravital microscopy, without a priori information

    NASA Astrophysics Data System (ADS)

    Lee, Sungon; Vinegoni, Claudio; Sebas, Matthew; Weissleder, Ralph

    2014-03-01

    Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.

  18. Imaging the beating heart in the mouse using intravital microscopy techniques

    PubMed Central

    Vinegoni, Claudio; Aguirre, Aaron D; Lee, Sungon; Weissleder, Ralph

    2017-01-01

    Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies. PMID:26492138

  19. Intravital Microscopy in the Cremaster Muscle Microcirculation for Endothelial Dysfunction Studies.

    PubMed

    Rius, Cristina; Sanz, María J

    2015-01-01

    The intravital microscopy in the mouse cremaster muscle microcirculation is a method widely used to visualize in vivo blood cells interacting with the endothelium and within the vessels. Therefore, it is a suitable technique to study leukocyte-endothelial cell interactions along every stage of the canonical leukocyte recruitment cascade: rolling, adhesion, intravascular crawling, and migration both in postcapillary venules and arterioles of the mouse cremasteric microcirculation. This technique also enables to assess vessel functionality, since hemodynamic parameters such as shear stress, flow rate, and vasodilatation/vasoconstriction, among other vascular events, can be additionally determined. Furthermore, response to multiple drugs and mechanisms underlying blood cells interactions within the vascular system can be studied in a real scenario. This chapter describes a protocol for intravital microscopy in the mouse cremaster muscle microcirculation.

  20. Intravital microscopy: a novel tool to study cell biology in living animals

    PubMed Central

    Weigert, Roberto; Sramkova, Monika; Parente, Laura; Masedunskas, Andrius

    2011-01-01

    Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criteria. Indeed, first we will focus on those studies in which organs where imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures. PMID:20372919

  1. Intravital microscopy: a novel tool to study cell biology in living animals.

    PubMed

    Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

    2010-05-01

    Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

  2. Expanding two-photon intravital microscopy to the infrared by means of optical parametric oscillator.

    PubMed

    Herz, Josephine; Siffrin, Volker; Hauser, Anja E; Brandt, Alexander U; Leuenberger, Tina; Radbruch, Helena; Zipp, Frauke; Niesner, Raluca A

    2010-02-17

    Chronic inflammation in various organs, such as the brain, implies that different subpopulations of immune cells interact with the cells of the target organ. To monitor this cellular communication both morphologically and functionally, the ability to visualize more than two colors in deep tissue is indispensable. Here, we demonstrate the pronounced power of optical parametric oscillator (OPO)-based two-photon laser scanning microscopy for dynamic intravital imaging in hardly accessible organs of the central nervous and of the immune system, with particular relevance for long-term investigations of pathological mechanisms (e.g., chronic neuroinflammation) necessitating the use of fluorescent proteins. Expanding the wavelength excitation farther to the infrared overcomes the current limitations of standard Titanium:Sapphire laser excitation, leading to 1), simultaneous imaging of fluorophores with largely different excitation and emission spectra (e.g., GFP-derivatives and RFP-derivatives); and 2), higher penetration depths in tissue (up to 80%) at higher resolution and with reduced photobleaching and phototoxicity. This tool opens up new opportunities for deep-tissue imaging and will have a tremendous impact on the choice of protein fluorophores for intravital applications in bioscience and biomedicine, as we demonstrate in this work.

  3. Integrated intravital microscopy and mathematical modeling to optimize nanotherapeutics delivery to tumors

    NASA Astrophysics Data System (ADS)

    van de Ven, Anne L.; Wu, Min; Lowengrub, John; McDougall, Steven R.; Chaplain, Mark A. J.; Cristini, Vittorio; Ferrari, Mauro; Frieboes, Hermann B.

    2012-03-01

    Inefficient vascularization hinders the optimal transport of cell nutrients, oxygen, and drugs to cancer cells in solid tumors. Gradients of these substances maintain a heterogeneous cell-scale microenvironment through which drugs and their carriers must travel, significantly limiting optimal drug exposure. In this study, we integrate intravital microscopy with a mathematical model of cancer to evaluate the behavior of nanoparticle-based drug delivery systems designed to circumvent biophysical barriers. We simulate the effect of doxorubicin delivered via porous 1000 x 400 nm plateloid silicon particles to a solid tumor characterized by a realistic vasculature, and vary the parameters to determine how much drug per particle and how many particles need to be released within the vasculature in order to achieve remission of the tumor. We envision that this work will contribute to the development of quantitative measures of nanoparticle design and drug loading in order to optimize cancer treatment via nanotherapeutics.

  4. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography

    SciTech Connect

    Umetani, K.; Fukushima, K.

    2013-03-15

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 {mu}m, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 {mu}m diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 {mu}m was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  5. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography

    NASA Astrophysics Data System (ADS)

    Umetani, K.; Fukushima, K.

    2013-03-01

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 μm, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 μm diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 μm was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  6. X-ray intravital microscopy for functional imaging in rat hearts using synchrotron radiation coronary microangiography.

    PubMed

    Umetani, K; Fukushima, K

    2013-03-01

    An X-ray intravital microscopy technique was developed to enable in vivo visualization of the coronary, cerebral, and pulmonary arteries in rats without exposure of organs and with spatial resolution in the micrometer range and temporal resolution in the millisecond range. We have refined the system continually in terms of the spatial resolution and exposure time. X-rays transmitted through an object are detected by an X-ray direct-conversion type detector, which incorporates an X-ray SATICON pickup tube. The spatial resolution has been improved to 6 μm, yielding sharp images of small arteries. The exposure time has been shortened to around 2 ms using a new rotating-disk X-ray shutter, enabling imaging of beating rat hearts. Quantitative evaluations of the X-ray intravital microscopy technique were extracted from measurements of the smallest-detectable vessel size and detection of the vessel function. The smallest-diameter vessel viewed for measurements is determined primarily by the concentration of iodinated contrast material. The iodine concentration depends on the injection technique. We used ex vivo rat hearts under Langendorff perfusion for accurate evaluation. After the contrast agent is injected into the origin of the aorta in an isolated perfused rat heart, the contrast agent is delivered directly into the coronary arteries with minimum dilution. The vascular internal diameter response of coronary arterial circulation is analyzed to evaluate the vessel function. Small blood vessels of more than about 50 μm diameters were visualized clearly at heart rates of around 300 beats/min. Vasodilation compared to the control was observed quantitatively using drug manipulation. Furthermore, the apparent increase in the number of small vessels with diameters of less than about 50 μm was observed after the vasoactive agents increased the diameters of invisible small blood vessels to visible sizes. This technique is expected to offer the potential for direct

  7. Tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy.

    PubMed

    Xu, Najia; Lei, Xi; Liu, Lixin

    2011-09-24

    The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation(1,2). In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling(3). Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)(4). Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment(2,5). In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm(3) size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these

  8. Video-rate resonant scanning multiphoton microscopy: An emerging technique for intravital imaging of the tumor microenvironment.

    PubMed

    Kirkpatrick, Nathaniel D; Chung, Euiheon; Cook, Daniel C; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L; Padera, Timothy P; Fukumura, Dai; Jain, Rakesh K

    2012-01-01

    The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.

  9. Fluorescent imaging of endothelial glycocalyx layer with wheat germ agglutinin using intravital microscopy.

    PubMed

    Kataoka, Hanae; Ushiyama, Akira; Kawakami, Hayato; Akimoto, Yoshihiro; Matsubara, Sachie; Iijima, Takehiko

    2016-01-01

    Endothelial glycocalyx (GCX) is located on the apical surface of vascular endothelial cells and is composed of a negatively-charged network of proteoglycans and glycoproteins. The GCX plays an important role in maintaining the integrity of vascular walls and preventing leakage of plasma. Therefore, degradation of the GCX is believed to lead to pathological leakage of plasma. Because the GCX is a very thin layer, its ultrastructural image has been demonstrated on electron microscope. To explore the function of the GCX, it should be visualized by a microscope in vivo. Thus, we developed in vivo visualization technique of the GCX under fluorescence microscopy using a mouse dorsal skinfold chamber (DSC) model. To label and visualize the GCX, we used fluorescein isothiocyanate (FITC)-labeled lectin, which has a high specificity for sugar moieties. We examined the affinity of the different lectins to epivascular regions under an intravital fluorescent microscope. Among seven different lectins we examined, FITC labeled Triticum vulgaris (wheat germ) agglutinin (WGA) delineated the GCX most clearly. Binding of WGA to the GCX was inhibited by chitin hydrolysate, which contained WGA-binding polysaccharide chains. Furthermore, the septic condition attenuated this structure, suggesting structural degradation of endothelial GCX layer. In conclusion, FITC-labeled WGA lectin enabled visualization of endothelial GCX under in vivo fluorescence microscopy.

  10. Just Look! Intravital Microscopy as the Best Means to Study Kidney Cell Death Dynamics.

    PubMed

    Schießl, Ina Maria; Hammer, Anna; Riquier-Brison, Anne; Peti-Peterdi, Janos

    2016-05-01

    Kidney cell death plays a key role in the progression of life-threatening renal diseases, such as acute kidney injury and chronic kidney disease. Injured and dying epithelial and endothelial cells take part in complex communication with the innate immune system, which drives the progression of cell death and the decrease in renal function. To improve our understanding of kidney cell death dynamics and its impact on renal disease, a study approach is needed that facilitates the visualization of renal function and morphology in real time. Intravital multiphoton microscopy of the kidney has been used for more than a decade and made substantial contributions to our understanding of kidney physiology and pathophysiology. It is a unique tool that relates renal structure and function in a time- and spatial-dependent manner. Basic renal function, such as microvascular blood flow regulation and glomerular filtration, can be determined in real time and homeostatic alterations, which are linked inevitably to cell death and can be depicted down to the subcellular level. This review provides an overview of the available techniques to study kidney dysfunction and inflammation in terms of cell death in vivo, and addresses how this novel approach can be used to improve our understanding of cell death dynamics in renal disease.

  11. Treadmill exercise induces neutrophil recruitment into muscle tissue in a reactive oxygen species-dependent manner. An intravital microscopy study.

    PubMed

    Nunes-Silva, Albená; Bernardes, Priscila T T; Rezende, Bárbara M; Lopes, Fernando; Gomes, Elisa C; Marques, Pedro E; Lima, Paulo M A; Coimbra, Cândido C; Menezes, Gustavo B; Teixeira, Mauro M; Pinho, Vanessa

    2014-01-01

    Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process.

  12. Treadmill Exercise Induces Neutrophil Recruitment into Muscle Tissue in a Reactive Oxygen Species-Dependent Manner. An Intravital Microscopy Study

    PubMed Central

    Nunes-Silva, Albená; Bernardes, Priscila T. T.; Rezende, Bárbara M.; Lopes, Fernando; Gomes, Elisa C.; Marques, Pedro E.; Lima, Paulo M. A.; Coimbra, Cândido C.; Menezes, Gustavo B.; Teixeira, Mauro M.; Pinho, Vanessa

    2014-01-01

    Intense exercise is a physiological stress capable of inducing the interaction of neutrophils with muscle endothelial cells and their transmigration into tissue. Mechanisms driving this physiological inflammatory response are not known. Here, we investigate whether production of reactive oxygen species is relevant for neutrophil interaction with endothelial cells and recruitment into the quadriceps muscle in mice subjected to the treadmill fatiguing exercise protocol. Mice exercised until fatigue by running for 56.3±6.8 min on an electric treadmill. Skeletal muscle was evaluated by intravital microscopy at different time points after exercise, and then removed to assess local oxidative stress and histopathological analysis. We observed an increase in plasma lactate and creatine kinase (CK) concentrations after exercise. The numbers of monocytes, neutrophils, and lymphocytes in blood increased 12 and 24 hours after the exercise. Numbers of rolling and adherent leukocytes increased 3, 6, 12, and 24 hours post-exercise, as assessed by intravital microscopy. Using LysM-eGFP mice and confocal intravital microscopy technology, we show that the number of transmigrating neutrophils increased 12 hours post-exercise. Mutant gp91phox-/- (non-functional NADPH oxidase) mice and mice treated with apocynin showed diminished neutrophil recruitment. SOD treatment promoted further adhesion and transmigration of leukocytes 12 hours after the exercise. These findings confirm our hypothesis that treadmill exercise increases the recruitment of leukocytes to the postcapillary venules, and NADPH oxidase-induced ROS plays an important role in this process. PMID:24798414

  13. Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

    PubMed Central

    Peusquens, Julia; Ergen, Can; Kohlhepp, Marlene; Mossanen, Jana C.; Schneider, Carlo; Vogt, Michael; Tolba, Rene H.; Trautwein, Christian; Martin, Christian; Tacke, Frank

    2015-01-01

    Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring. The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange. To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4). CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a

  14. Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

    PubMed Central

    Chan, KY; Gupta, S; de Vries, R; Danser, AHJ; Villalón, CM; Muñoz-Islas, E; Maassen Van Den Brink, A

    2010-01-01

    Background and purpose: During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by α-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy. Experimental approach: Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. α-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists. Key results: α-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to α-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous α-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP. Conclusions and implications: Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects. PMID:20590623

  15. Automatic detection of motion blur in intravital video microscopy image sequences via directional statistics of log-Gabor energy maps.

    PubMed

    Ferrari, Ricardo J; Pinto, Carlos H Villa; da Silva, Bruno C Gregório; Bernardes, Danielle; Carvalho-Tavares, Juliana

    2015-02-01

    Intravital microscopy is an important experimental tool for the study of cellular and molecular mechanisms of the leukocyte-endothelial interactions in the microcirculation of various tissues and in different inflammatory conditions of in vivo specimens. However, due to the limited control over the conditions of the image acquisition, motion blur and artifacts, resulting mainly from the heartbeat and respiratory movements of the in vivo specimen, will very often be present. This problem can significantly undermine the results of either visual or computerized analysis of the acquired video images. Since only a fraction of the total number of images are usually corrupted by severe motion blur, it is necessary to have a procedure to automatically identify such images in the video for either further restoration or removal. This paper proposes a new technique for the detection of motion blur in intravital video microscopy based on directional statistics of local energy maps computed using a bank of 2D log-Gabor filters. Quantitative assessment using both artificially corrupted images and real microscopy data were conducted to test the effectiveness of the proposed method. Results showed an area under the receiver operating characteristic curve (AUC) of 0.95 (AUC = 0.95; 95 % CI 0.93-0.97) when tested on 329 video images visually ranked by four observers.

  16. Direct observation of liposome uptake by leukocytes in vivo in skin blood vessels using intravital fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Devoisselle, Jean-Marie; Mordon, Serge R.; Begu, Sylvie; Desmettre, Thomas

    2000-04-01

    This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5,6-CF-encapsulated PEGylated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope fitted with a Xenon light source and an epi-fluorescence assembly. An ultra-high sensitivity video-camera mounted on the microscope projected the image onto a monitor, and the images were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using these model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 hours. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up liposomes. This observation is in accordance with previous in vitro studies.

  17. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  18. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  19. Combined application of dynamic light scattering imaging and fluorescence intravital microscopy in vascular biology

    NASA Astrophysics Data System (ADS)

    Kalchenko, V.; Ziv, K.; Addadi, Y.; Madar-Balakirski, N.; Meglinski, I.; Neeman, M.; Harmelin, A.

    2010-08-01

    The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels.

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

    PubMed

    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.

  1. Quantification of Cy-5 siRNA signal in the intra-vital multi-photon microscopy images.

    PubMed

    Chen, Antong; Dogdas, Belma; Mehta, Saurin; Haskell, Kathleen; Ng, Bruce; Keough, Ed; Howell, Bonnie; Meacham, D Adam; Aslamkhan, Amy G; Davide, Joseph; Stanton, Matthew; Bagchi, Ansuman; Sepp-Lorenzino, Laura; Tao, Weikang

    2012-01-01

    Transgenic mice with Tie2- green fluorescent protein (GFP) are used as a model to study the kinetic distribution of the Cy5-siRNA delivered by lipid nanoparticles (LNP) into the liver. After the mouse is injected with the LNP, it undergoes a procedure of intra-vital multi-photon microscopy imaging over a period of two hours, during which the process for the nanoparticle to diffuse into the hepatocytes from the vasculature system is monitored. Since the images are obtained in-vivo, the quantification of Cy5 kinetics suffers from the moving field of view (FOV). A method is proposed to register the sequence of images through template matching. Based on the semi-automatic segmentations of the vessels in the common FOV, the registered images are segmented into three regions of interest (ROI) in which the Cy5 signals are quantified. Computation of the percentage signal strength in the ROIs over time allows for the analysis of the diffusion of Cy5-siRNA into the hepatocytes, and helps demonstrate the effectiveness of the Cy5-siRNA delivery vehicle.

  2. Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy.

    PubMed

    Huang, Alex Y; Myers, Jay T; Barkauskas, Deborah; Howell, Scott J; Oleinick, Nancy L; McCormick, Thomas S; Cooper, Kevin D; Baron, Elma D; Lam, Minh

    2012-09-01

    The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT.

  3. Improving signal levels in intravital multiphoton microscopy using an objective correction collar

    NASA Astrophysics Data System (ADS)

    Muriello, Pamela A.; Dunn, Kenneth W.

    2008-04-01

    Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.

  4. Two photon microscopy intravital study of DC-mediated anti-tumor response of NK cells

    NASA Astrophysics Data System (ADS)

    Caccia, Michele; Gorletta, Tatiana; Sironi, Laura; Zanoni, Ivan; Salvetti, Cristina; Collini, Maddalena; Granucci, Francesca; Chirico, Giuseppe

    2010-02-01

    Recent studies have demonstrated that dendritic cells (DCs) play a crucial role in the activation of Natural Killer cells (NKs) that are responsible for anti-tumor innate immune responses. The focus of this report is on the role of pathogen associated molecular pattern (PAMP) activated-DCs in inducing NK cell-mediated anti-tumor responses. Mice transplanted sub-cute (s.c.) with AK7 cells, a mesothelioma cell line sensitive to NK cell responses, are injected with fluorescent NK cells and DC activation is then induced by s.c. injection of Lipopolysaccharide (LPS). Using 4 dimensional tracking we follow the kinetic behavior of NK cells at the Draining Lymph-Node (DLN). As control, noninflammatory conditions are also evaluated. Our data suggest that NK cells are recruited to the DLN where they can interact with activated-DCs with a peculiar kinetic behavior: short lived interactions interleaved by rarer longer ones. We also found that the changes in the NK dynamic behavior in inflammatory conditions clearly affect relevant motility parameters such as the instantaneous and average velocity and the effective diffusion coefficient. This observation suggests that NK cells and activated-DCs might efficiently interact in the DLN, where cells could be activated. Therefore the interaction between activated-DCs and NK cells in DLN is not only a reality but it may be also crucial for the start of the immune response of the NKs.

  5. Stabilizing 3D in vivo intravital microscopy images with an iteratively refined soft-tissue model for immunology experiments.

    PubMed

    Gómez-Conde, Iván; Caetano, Susana S; Tadokoro, Carlos E; Olivieri, David N

    2015-09-01

    We describe a set of new algorithms and a software tool, StabiTissue, for stabilizing in vivo intravital microscopy images that suffer from soft-tissue background movement. Because these images lack predetermined anchors and are dominated by noise, we use a pixel weighted image alignment together with a correction for nonlinear tissue deformations. We call this correction a poor man׳s diffeomorphic map since it ascertains the nonlinear regions of the image without resorting to a full integral equation method. To determine the quality of the image stabilization, we developed an ensemble sampling method that quantifies the coincidence between image pairs from randomly distributed image regions. We obtain global stabilization alignment through an iterative constrained simulated annealing optimization procedure. To show the accuracy of our algorithm with existing software, we measured the misalignment error rate in datasets taken from two different organs and compared the results to a similar and popular open-source solution. Present open-source stabilization software tools perform poorly because they do not treat the specific needs of the IV-2pM datasets with soft-tissue deformation, speckle noise, full 5D inter- and intra-stack motion error correction, and undefined anchors. In contrast, the results of our tests demonstrate that our method is more immune to noise and provides better performance for datasets' possessing nonlinear tissue deformations. As a practical application of our software, we show how our stabilization improves cell tracking, where the presence of background movement would degrade track information. We also provide a qualitative comparison of our software with other open-source libraries/applications. Our software is freely available at the open source repository http://sourceforge.net/projects/stabitissue/.

  6. From optical bench to cageside: intravital microscopy on the long road to rational vaccine design

    PubMed Central

    Hickman, Heather D.; Bennink, Jack R.; Yewdell, Jonathan W.

    2012-01-01

    Summary No anti-viral vaccine is perfect. For some important pathogens, there are no effective vaccines. Many current vaccines are based on the working principles of Jenner and Pasteur, i.e. empiric administration of attenuated or inactivated forms of the pathogen. Tapping the full potential of vaccination requires a thorough understanding of the mechanism of immune activation by pathogens and their individual components. Though the rate of discovery continues to accelerate, the complexity of the immune system is daunting, particularly when integrated into the overall physiology of the host. Here, we review the application of multiphoton microscopy to examine host-pathogen interactions, focusing on our recent efforts to understand mouse CD8+ T-cell responses to viruses at the level of cellular interactions in lymph nodes draining the infection site. We also discuss our recent efforts to understand the influence of the sympathetic nervous system on antiviral immunity, with the ultimate goal of appreciating the traditional elements of immunity as just one facet of the total organismal response to infection and immunization. PMID:21198674

  7. 4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

    PubMed Central

    Lee, Juhyun; Fei, Peng; Packard, René R. Sevag; Kang, Hanul; Xu, Hao; Baek, Kyung In; Jen, Nelson; Chen, Junjie; Yen, Hilary; Chi, Neil C.; Ho, Chih-Ming; Hsiai, Tzung K.

    2016-01-01

    Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress–mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress–activated Notch signaling to modulate trabeculation. PMID:27018592

  8. 4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation.

    PubMed

    Lee, Juhyun; Fei, Peng; Packard, René R Sevag; Kang, Hanul; Xu, Hao; Baek, Kyung In; Jen, Nelson; Chen, Junjie; Yen, Hilary; Kuo, C-C Jay; Chi, Neil C; Ho, Chih-Ming; Li, Rongsong; Hsiai, Tzung K

    2016-05-02

    Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

  9. Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two-photon microscopy.

    PubMed

    Ricard, Clément; Lamasse, Lisa; Jaouen, Alexandre; Rougon, Geneviève; Debarbieux, Franck

    2016-06-01

    Simultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord.

  10. Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

    PubMed Central

    Rodriguez-Tirado, Carolina; Kitamura, Takanori; Kato, Yu; Pollard, Jeffery W.; Condeelis, John S.; Entenberg, David

    2017-01-01

    Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the

  11. Visualizing the Acute Effects of Vascular-Targeted Therapy In Vivo Using Intravital Microscopy and Magnetic Resonance Imaging: Correlation with Endothelial Apoptosis, Cytokine Induction, and Treatment Outcome1

    PubMed Central

    Seshadri, Mukund; Spernyak, Joseph A; Maiery, Patricia G; Cheney, Richard T; Mazurchuk, Richard; Bellnier, David A

    2007-01-01

    Abstract The acute effects of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were investigated in vivo using intravital microscopy (IVM) and magnetic resonance imaging (MRI). Changes in vascular permeability and blood flow of syngeneic CT-26 murine colon adenocarcinomas were assessed at 4 and 24 hours after DMXAA treatment (30 mg/kg, i.p.) and correlated with induction of tumor necrosis factor-α (TNF-α), endothelial damage [CD31/terminal deoxynucleotidyl transferase (TdT)], and treatment outcome. Intravital imaging revealed a marked increase in vascular permeability 4 hours after treatment, consistent with increases in intratumoral mRNA and protein levels of TNF-α. Parallel contrast-enhanced MRI studies showed a ∼ 4-fold increase in longitudinal relaxation rates (ΔR1), indicative of increased contrast agent accumulation within the tumor. Dual immunostained tumor sections (CD31/TdT) revealed evidence of endothelial apoptosis at this time point. Twenty-four hours after treatment, extensive hemorrhage and complete disruption of vascular architecture were observed with IVM, along with a significant reduction in ΔR1; and virtual absence of CD31 immunostaining. DMXAA-induced tumor vascular damage resulted in significant long-term (60-day) cures compared to untreated controls. Multimodality imaging approaches are useful in visualizing the effects of antivascular therapy in vivo. Such approaches allow cross validation and correlation of findings with underlying molecular changes contributing to treatment outcome. PMID:17356709

  12. Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy

    PubMed Central

    Nemoto, Tomomi

    2016-01-01

    Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions. PMID:27657513

  13. Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing

    PubMed Central

    Pang, Yonggang; Tsigkou, Olga; Spencer, Joel A.; Lin, Charles P.; Neville, Craig

    2015-01-01

    Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (d,l-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad

  14. The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

    PubMed Central

    Jenne, Craig N.; Wong, Connie H. Y.; Petri, Björn; Kubes, Paul

    2011-01-01

    Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation. PMID:21949865

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

    PubMed

    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. Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches.

    PubMed

    Wang, Lin; Kamocka, Malgorzata M; Zollman, Amy; Carlesso, Nadia

    2017-03-21

    Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions(1)(,)(2). Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny(3)(,)(4)(,)(5). New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function(6)(,)(7)(,)(8). Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress. Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific "niche" cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)(9) and RBPJ knock-out mice (lacking canonical Notch signaling)(10) are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

  17. Fluorescein Derivatives in Intravital Fluorescence Imaging

    PubMed Central

    Robertson, Thomas A.; Bunel, Florestan; Roberts, Michael S.

    2013-01-01

    Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM) enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT)] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples. PMID:24709799

  18. Recent advances in intravital imaging of dynamic biological systems.

    PubMed

    Kikuta, Junichi; Ishii, Masaru

    2012-01-01

    Intravital multiphoton microscopy has opened a new era in the field of biological imaging. Focal excitation of fluorophores by simultaneous attack of multiple (normally "two") photons generates images with high spatial resolution, and use of near-infrared lasers for multiphoton excitation allows penetration of thicker specimens, enabling biologists to visualize living cellular dynamics deep inside tissues and organs without thin sectioning. Moreover, the minimized photo-bleaching and toxicity associated with multiphoton techniques is beneficial for imaging of live specimens for extended observation periods. Here we focus on recent findings using intravital multiphoton imaging of dynamic biological systems such as the immune system and bone homeostasis. The immune system comprises highly dynamic networks, in which many cell types actively travel throughout the body and interact with each other in specific areas. Therefore, real-time intravital imaging represents a powerful tool for understanding the mechanisms underlying this dynamic system.

  19. Intravital Microscopic Interrogation of Peripheral Taste Sensation

    PubMed Central

    Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

    2015-01-01

    Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo. PMID:25726964

  20. Intravital Microscopic Interrogation of Peripheral Taste Sensation

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

    2015-03-01

    Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

  1. Intravital microscopic interrogation of peripheral taste sensation.

    PubMed

    Choi, Myunghwan; Lee, Woei Ming; Yun, Seok Hyun

    2015-03-02

    Intravital microscopy is a powerful tool in neuroscience but has not been adapted to the taste sensory organ due to anatomical constraint. Here we developed an imaging window to facilitate microscopic access to the murine tongue in vivo. Real-time two-photon microscopy allowed the visualization of three-dimensional microanatomy of the intact tongue mucosa and functional activity of taste cells in response to topically administered tastants in live mice. Video microscopy also showed the calcium activity of taste cells elicited by small-sized tastants in the blood circulation. Molecular kinetic analysis suggested that intravascular taste sensation takes place at the microvilli on the apical side of taste cells after diffusion of the molecules through the pericellular capillaries and tight junctions in the taste bud. Our results demonstrate the capabilities and utilities of the new tool for taste research in vivo.

  2. Intravital autofluorescence 2-photon microscopy of murine intestinal mucosa with ultra-broadband femtosecond laser pulse excitation: image quality, photodamage, and inflammation

    NASA Astrophysics Data System (ADS)

    Klinger, Antje; Krapf, Lisa; Orzekowsky-Schroeder, Regina; Koop, Norbert; Vogel, Alfred; Hüttmann, Gereon

    2015-11-01

    Ultra-broadband excitation with ultrashort pulses may enable simultaneous excitation of multiple endogenous fluorophores in vital tissue. Imaging living gut mucosa by autofluorescence 2-photon microscopy with more than 150 nm broad excitation at an 800-nm central wavelength from a sub-10 fs titanium-sapphire (Ti:sapphire) laser with a dielectric mirror based prechirp was compared to the excitation with 220 fs pulses of a tunable Ti:sapphire laser at 730 and 800 nm wavelengths. Excitation efficiency, image quality, and photochemical damage were evaluated. At similar excitation fluxes, the same image brightness was achieved with both lasers. As expected, with ultra-broadband pulses, fluorescence from NAD(P)H, flavines, and lipoproteins was observed simultaneously. However, nonlinear photodamage apparent as hyperfluorescence with functional and structural alterations of the tissue occurred earlier when the laser power was adjusted to the same image brightness. After only a few minutes, the immigration of polymorphonuclear leucocytes into the epithelium and degranulation of these cells, a sign of inflammation, was observed. Photodamage is promoted by the higher peak irradiances and/or by nonoptimal excitation of autofluorescence at the longer wavelength. We conclude that excitation with a tunable narrow bandwidth laser is preferable to ultra-broadband excitation for autofluorescence-based 2-photon microscopy, unless the spectral phase can be controlled to optimize excitation conditions.

  3. Intravital Multiphoton Imaging of the Kidney: Tubular Structure and Metabolism.

    PubMed

    Small, David M; Sanchez, Washington Y; Gobe, Glenda C

    2016-01-01

    Multiphoton microscopy (MPM) allows the visualization of dynamic pathophysiological events in real time in live animals. Intravital imaging can be applied to investigate novel mechanisms and treatments of different forms of kidney disease as well as improve our understanding of normal kidney physiology. Using rodent models, in conjunction with endogenous fluorescence and infused exogenous fluorescent dyes, measurement can be made of renal processes such as glomerular permeability, juxtaglomerular apparatus function, interactions of the tubulointerstitium, tubulovascular interactions, vascular flow rate, and the renin-angiotensin-aldosterone system. Subcellular processes including mitochondrial dynamics, reactive oxygen species production, cytosolic ion concentrations, and death processes of apoptosis and necrosis can also be seen and measured by MPM. The current methods chapter presents an overview of MPM with a focus on techniques for intravital kidney imaging and gives examples of instances where intravital MPM has been utilized to study renal pathophysiology. Suggestions are provided for MPM methods within the confines of intravital microscopy and selected kidney structure. MPM is undoubtedly a powerful new technique for application in experimental nephrology, and we believe it will continue to create new paradigms for understanding and treating kidney disease.

  4. [Bone and Stem Cells. Intravital imaging of bone marrow microenvironment].

    PubMed

    Mizuno, Hiroki; Kikuta, Junichi; Ishii, Masaru

    2014-04-01

    Various kinds of cell types, such as osteoclasts, osteoblasts, hematopoietic cells, and mesenchymal cells, have been reported to exist in the bone marrow and communicate with each other. Although there have been many previous studies about bone marrow microenvironment, most of them were analyzed by conventional methods such as histological analysis and flow cytometry. These methods could not observe the dynamic cell movement in living bone marrow. Recently rapid development of fluorescent imaging techniques enables us to understand the cellular dynamics in vivo . That's why we have originally established an advanced imaging system for visualizing living bone tissues with intravital two-photon microscopy. Here we show the latest data and the detailed methodology of intravital imaging of bone marrow microenvironment, and also discuss its further application.

  5. Intravital characterization of tumor cell migration in pancreatic cancer

    PubMed Central

    Beerling, Evelyne; Oosterom, Ilse; Voest, Emile; Lolkema, Martijn; van Rheenen, Jacco

    2016-01-01

    ABSTRACT Curing pancreatic cancer is difficult as metastases often determine the poor clinical outcome. To gain more insight into the metastatic behavior of pancreatic cancer cells, we characterized migratory cells in primary pancreatic tumors using intravital microscopy. We visualized the migratory behavior of primary tumor cells of a genetically engineered pancreatic cancer mouse model and found that pancreatic tumor cells migrate with a mesenchymal morphology as single individual cells or collectively as a stream of non-cohesive single motile cells. These findings may improve our ability to conceive treatments that block metastatic behavior. PMID:28243522

  6. Intravital multiphoton imaging of mouse tibialis anterior muscle

    PubMed Central

    Lau, Jasmine; Goh, Chi Ching; Devi, Sapna; Keeble, Jo; See, Peter; Ginhoux, Florent; Ng, Lai Guan

    2016-01-01

    ABSTRACT Intravital imaging by multiphoton microscopy is a powerful tool to gain invaluable insight into tissue biology and function. Here, we provide a step-by-step tissue preparation protocol for imaging the mouse tibialis anterior skeletal muscle. Additionally, we include steps for jugular vein catheterization that allow for well-controlled intravenous reagent delivery. Preparation of the tibialis anterior muscle is minimally invasive, reducing the chances of inducing damage and inflammation prior to imaging. The tibialis anterior muscle is useful for imaging leukocyte interaction with vascular endothelium, and to understand muscle contraction biology. Importantly, this model can be easily adapted to study neuromuscular diseases and myopathies. PMID:28243520

  7. Imaging windows for long-term intravital imaging

    PubMed Central

    Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

    2014-01-01

    Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure. PMID:28243510

  8. Elucidation of monocyte/macrophage dynamics and function by intravital imaging

    PubMed Central

    Rua, Rejane; McGavern, Dorian B.

    2015-01-01

    Monocytes and macrophages are a diverse population of innate immune cells that play a critical role in homeostasis and inflammation. These cells are surveillant by nature and closely monitor the vasculature and surrounding tissue during states of health and disease. Given their abundance and strategic positioning throughout the body, myeloid cells are among the first responders to any inflammatory challenge and are active participants in most immune-mediated diseases. Recent studies have shed new light on myeloid cell dynamics and function by use of an imaging technique referred to as intravital microscopy (IVM). This powerful approach allows researchers to gain real-time insights into monocytes and macrophages performing homeostatic and inflammatory tasks in living tissues. In this review, we will present a contemporary synopsis of how intravital microscopy has revolutionized our understanding of myeloid cell contributions to vascular maintenance, microbial defense, autoimmunity, tumorigenesis, and acute/chronic inflammatory diseases. PMID:26162402

  9. Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures.

    PubMed

    Hato, Takashi; Winfree, Seth; Day, Richard; Sandoval, Ruben M; Molitoris, Bruce A; Yoder, Mervin C; Wiggins, Roger C; Zheng, Yi; Dunn, Kenneth W; Dagher, Pierre C

    2017-03-01

    In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.

  10. Intravital imaging of the mouse popliteal lymph node.

    PubMed

    Liou, H L Rachel; Myers, Jay T; Barkauskas, Deborah S; Huang, Alex Y

    2012-02-08

    Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network. Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow

  11. Fluorescent Tobacco mosaic virus-Derived Bio-Nanoparticles for Intravital Two-Photon Imaging

    PubMed Central

    Niehl, Annette; Appaix, Florence; Boscá, Sonia; van der Sanden, Boudewijn; Nicoud, Jean-François; Bolze, Frédéric; Heinlein, Manfred

    2016-01-01

    Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature. PMID:26793221

  12. Intravital Microscopic Methods to Evaluate Anti-inflammatory Effects and Signaling Mechanisms Evoked by Hydrogen Sulfide

    PubMed Central

    Zuidema, Mozow Y.; Korthuis, Ronald J.

    2016-01-01

    Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule with potent anti-inflammatory properties. Exogenous application of H2S donors, administered either acutely during an inflammatory response or as an antecedent preconditioning intervention that invokes the activation of anti-inflammatory cell survival programs, effectively limits leukocyte rolling, adhesion and emigration, generation of reactive oxygen species, chemokine and cell adhesion molecule expression, endothelial barrier disruption,capillary perfusion deficits, and parenchymal cell dysfunction and injury. This chapter focuses on intravital microscopic methods that can be used to assess the anti-inflammatory effects exerted by H2S, as well as to explore the cellular signaling mechanisms by which this gaseous molecule limits the aforementioned inflammatory responses. Recent advances include use of intravital multiphoton microscopy and optical biosensor technology to explore signaling mechanisms in vivo. PMID:25747477

  13. Intravital imaging of dendritic spine plasticity

    PubMed Central

    Sau Wan Lai, Cora

    2014-01-01

    Abstract Dendritic spines are the postsynaptic part of most excitatory synapses in the mammalian brain. Recent works have suggested that the structural and functional plasticity of dendritic spines have been associated with information coding and memories. Advances in imaging and labeling techniques enable the study of dendritic spine dynamics in vivo. This perspective focuses on intravital imaging studies of dendritic spine plasticity in the neocortex. I will introduce imaging tools for studying spine dynamics and will further review current findings on spine structure and function under various physiological and pathological conditions. PMID:28243511

  14. Methodological advances in imaging intravital axonal transport

    PubMed Central

    Sleigh, James N.; Vagnoni, Alessio; Twelvetrees, Alison E.; Schiavo, Giampietro

    2017-01-01

    Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer’s disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions. PMID:28344778

  15. Methodological advances in imaging intravital axonal transport.

    PubMed

    Sleigh, James N; Vagnoni, Alessio; Twelvetrees, Alison E; Schiavo, Giampietro

    2017-01-01

    Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer's disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions.

  16. Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

    PubMed Central

    Harney, Allison S.; Wang, Yarong; Condeelis, John S.; Entenberg, David

    2016-01-01

    In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment. PMID:27341448

  17. Imaging windows for long-term intravital imaging: General overview and technical insights.

    PubMed

    Alieva, Maria; Ritsma, Laila; Giedt, Randy J; Weissleder, Ralph; van Rheenen, Jacco

    2014-01-01

    Intravital microscopy is increasingly used to visualize and quantitate dynamic biological processes at the (sub)cellular level in live animals. By visualizing tissues through imaging windows, individual cells (e.g., cancer, host, or stem cells) can be tracked and studied over a time-span of days to months. Several imaging windows have been developed to access tissues including the brain, superficial fascia, mammary glands, liver, kidney, pancreas, and small intestine among others. Here, we review the development of imaging windows and compare the most commonly used long-term imaging windows for cancer biology: the cranial imaging window, the dorsal skin fold chamber, the mammary imaging window, and the abdominal imaging window. Moreover, we provide technical details, considerations, and trouble-shooting tips on the surgical procedures and microscopy setups for each imaging window and explain different strategies to assure imaging of the same area over multiple imaging sessions. This review aims to be a useful resource for establishing the long-term intravital imaging procedure.

  18. Intravital insights in skin wound healing using the mouse dorsal skin fold chamber.

    PubMed

    Sorg, Heiko; Krueger, Christian; Vollmar, Brigitte

    2007-12-01

    The skin fold chamber is one of the most accepted animal models for studying the microcirculation both in health and disease. Here we describe for the first time the alternative use of the skin fold chamber in mice for intravital microscopic investigation of skin regeneration after creating a full dermal thickness wound. The dorsal skin fold chamber was implanted in hairless SKH1-hr mice and a full dermal thickness wound (area approximately 4 mm2) was created. By means of intravital fluorescence microscopy, the kinetics of wound healing were analyzed for 12 days post wounding with assessment of epithelialization and nutritive perfusion. The morphology of the regenerating skin was characterized by hematoxylin-eosin histology and immunohistochemistry for proliferation and microvessel density. The model allows the continuous visualization of wound closure with complete epithelialization at day 12. Furthermore, a sola cutis se reficientis could be described by an inner circular ring of vessels at the wound margin surrounded by outer radial passing vessels. Inner circular vessels presented initially with large diameters and matured towards diameters of less than 15 microm for conversion into radial spreading outer vessels. Furthermore, wound healing showed all diverse core issues of skin repair. In summary, we were able to establish a model for the analysis of microcirculation in the healing skin of the mouse. This versatile model allows distinct analysis of new vessel formation and maturation in regenerating skin as well as evaluation of skin healing under different pathologic conditions.

  19. Fungal Infection in the Brain: What We Learned from Intravital Imaging

    PubMed Central

    Shi, Meiqing; Mody, Christopher H.

    2016-01-01

    Approximately 1.2 billion people suffer from fungal diseases worldwide. Arguably, the most serious manifestation occurs when pathogenic fungi infect the brain, often causing fatal meningoencephalitis. For most fungi, infection occurs via the vascular route. The organism must first be arrested in the brain microvasculature and transmigrate into the brain parenchyma across the blood–brain barrier. As a result, host immune cells are recruited into the brain to contain the fungi. However, it remains poorly understood how fungi traffic to, and migrate into the brain and how immune cells interact with invading fungi in the brain. A new era of intravital fluorescence microscopy has begun to provide insights. We are able to employ this powerful approach to study dynamic interactions of disseminating fungi with brain endothelial cells as well as resident and recruited immune cells during the brain infection. In this review, with a focus on Cryptococcus neoformans, we will provide an overview of the application of intravital imaging in fungal infections in the brain, discuss recent findings and speculate on possible future research directions. PMID:27532000

  20. Neutrophil Extravasation Cascade: What Can We Learn from Two-photon Intravital Imaging?

    PubMed Central

    Park, Sang A

    2016-01-01

    Immune cells (leukocytes or white blood cells) move actively and sensitively based on body conditions. Despite their important role as protectors inside the body, it is difficult to directly observe the spatiotemporal momentum of leukocytes. With advances in imaging technology, the introduction of two-photon microscopy has enabled researchers to look deeper inside tissues in a three-dimensional manner. In observations of immune cell movement along the blood vessel, vascular permeability and innate immune cell movements remain unclear. Here, we describe the neutrophil extravasation cascade, which were observed using a two-photon intravital imaging technique. We also provide evidence for novel mechanisms such as neutrophil body extension and microparticle formation as well as their biological roles during migration. PMID:28035206

  1. Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

    PubMed Central

    Evans, Teresa A.; Barkauskas, Deborah S.; Myers, Jay T.; Huang, Alex Y.

    2014-01-01

    Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury. PMID:25489963

  2. The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging.

    PubMed

    Coles, Jonathan A; Stewart-Hutchinson, Phillip J; Myburgh, Elmarie; Brewer, James M

    2017-03-27

    A wide range of viral and microbial infections are known to cause meningitis, and there is evidence that the meninges are the gateway to pathogenic invasion of the brain parenchyma. Hence observation of these regions has wide application to understanding host-pathogen interactions. Interactions between pathogens and cells of the immune response can be modified by changes in their environment, such as suppression of the flow of blood and lymph, and, particularly in the case of the meninges, with their unsupported membranes, invasive dissection can alter the tissue architecture. For these reasons, intravital imaging through the unperforated skull is the method of choice. We give a protocol for a simple method of two-photon microscopy through the thinned cortical skull of the anesthetized mouse to enable real-time imaging with sub-micron resolution through the meninges and into the superficial brain parenchyma. In reporter mice in which selected cell types express fluorescent proteins, imaging after infection with fluorescent pathogens (lymphocytic choriomeningitis virus, Trypanosoma brucei or Plasmodium berghei) has shown strong recruitment to the cortical meninges of immune cells, including neutrophils, T cells, and putative dendritic cells and macrophages. Without special labeling, the boundaries between the dura mater, the leptomeninx, and the parenchyma are not directly visualized in intravital two-photon microscopy, but other landmarks and characteristics, which we illustrate, allow the researcher to identify the compartment being imaged. While most infectious meningitides are localized mainly in the dura mater, others involve recruitment of immune cells to the leptomeninx.

  3. A novel model for ectopic, chronic, intravital multiphoton imaging of bone marrow vasculature and architecture in split femurs

    PubMed Central

    Bălan, Mirela; Kiefer, Friedemann

    2015-01-01

    Creating a model for intravital visualization of femoral bone marrow, a major site of hematopoiesis in adult mammalian organisms, poses a serious challenge, in that it needs to overcome bone opacity and the inaccessibility of marrow. Furthermore, meaningful analysis of bone marrow developmental and differentiation processes requires the repetitive observation of the same site over long periods of time, which we refer to as chronic imaging. To surmount these issues, we developed a chronic intravital imaging model that allows the observation of split femurs, ectopically transplanted into a dorsal skinfold chamber of a host mouse. Repeated, long term observations are facilitated by multiphoton microscopy, an imaging technique that combines superior imaging capacity at greater tissue depth with low phototoxicity. The transplanted, ectopic femur was stabilized by its sterile environment and rapidly connected to the host vasculature, allowing further development and observation of extended processes. After optimizing transplant age and grafting procedure, we observed the development of new woven bone and maturation of secondary ossification centers in the transplanted femurs, preceded by the sprouting of a sinusoidal-like vascular network, which was almost entirely composed of femoral endothelial cells. After two weeks, the transplant was still populated with stromal and haematopoietic cells belonging both to donor and host. Over this time frame, the transplant partially retained myeloid progenitor cells with single and multi-lineage differentiation capacity. In summary, our model allowed repeated intravital imaging of bone marrow angiogenesis and hematopoiesis. It represents a promising starting point for the development of improved chronic optical imaging models for femoral bone marrow. PMID:28243515

  4. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles

    PubMed Central

    Leong, Hon Sing; Steinmetz, Nicole F; Ablack, Amber; Destito, Giuseppe; Zijlstra, Andries; Stuhlmann, Heidi; Manchester, Marianne; Lewis, John D

    2011-01-01

    Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of cpMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application. PMID:20671724

  5. Intravital fluorescence microscopic study of the behavior of long-circulating liposomes during microvascular thrombosis

    NASA Astrophysics Data System (ADS)

    Dvoisselle, Jean-Marie; Begu, Sylvie; Tourne-Peteilh, Corine; Buys, Bruno; Mordon, Serge R.

    2002-06-01

    Treatment of thrombosis depends on the selectivity of thrombolytic agents to the clot. It has been already demonstrated that liposomes can provide a better selectivity of such agents to the clot site. We have recently shown that intravital fluorescence microscopy is a powerful tool to image in situ and in real time the labeling of leukocytes by long circulating liposomes. The aim of this study was to monitor the in vivo behavior of such liposomes in a clot site. Carboxyfluorescein-loaded long circulating liposomes were prepared and characterized in term of size and permeability. The liposomes suspension was injected intravenously to golden hamsters. The skin microcirculation was observed using a dorsal skin-fold chamber by fluorescence microscopy. Thrombosis were obtained as the consequence of the inflammatory response due to the surgery. Using this model, fluorescent dots were observed at the site of the clot. Liposomes accumulate at the clot site whatever the mechanism (passive deposition or uptake). There is a period of latency and 30 seconds after the blood flow stop, fluorescence increases very rapidly and a bright fluorescent spot is observed at the site of the clot. Further studies are needed to determine the exact localization of liposomes in the clot and the mechanism of interaction.

  6. Intravital imaging reveals new ancillary mechanisms co-opted by cancer cells to drive tumor progression

    PubMed Central

    Lucas, Morghan C.; Timpson, Paul

    2016-01-01

    Intravital imaging is providing new insights into the dynamics of tumor progression in native tissues and has started to reveal the layers of complexity found in cancer. Recent advances in intravital imaging have allowed us to look deeper into cancer behavior and to dissect the interactions between tumor cells and the ancillary host niche that promote cancer development. In this review, we provide an insight into the latest advances in cancer biology achieved by intravital imaging, focusing on recently discovered mechanisms by which tumor cells manipulate normal tissue to facilitate disease progression. PMID:27239290

  7. Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

    PubMed Central

    Radbruch, Helena; Andresen, Volker; Mossakowski, Agata; Siffrin, Volker; Seelemann, Thomas; Spiecker, Heinrich; Moll, Ingrid; Herz, Josephine; Hauser, Anja E.; Zipp, Frauke; Behne, Martin J.; Niesner, Raluca

    2013-01-01

    Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm2) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm2) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal

  8. Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging

    PubMed Central

    Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K.; Wells, Sam; Wikswo, John P.; Zijlstra, Andries; Richmond, Ann

    2016-01-01

    ABSTRACT We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment. PMID:28243517

  9. Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.

    PubMed

    Sobolik, Tammy; Su, Ying-Jun; Ashby, Will; Schaffer, David K; Wells, Sam; Wikswo, John P; Zijlstra, Andries; Richmond, Ann

    2016-01-01

    We developed mammary imaging windows (MIWs) to evaluate leukocyte infiltration and cancer cell dissemination in mouse mammary tumors imaged by confocal microscopy. Previous techniques relied on surgical resection of a skin flap to image the tumor microenvironment restricting imaging time to a few hours. Utilization of mammary imaging windows offers extension of intravital imaging of the tumor microenvironment. We have characterized strengths and identified some previously undescribed potential weaknesses of MIW techniques. Through iterative enhancements of a transdermal portal we defined conditions for improved quality and extended confocal imaging time for imaging key cell-cell interactions in the tumor microenvironment.

  10. What Ticks Do Under Your Skin: Two-Photon Intravital Imaging of Ixodes Scapularis Feeding in the Presence of the Lyme Disease Spirochete

    PubMed Central

    Bockenstedt, Linda K.; Gonzalez, David; Mao, Jialing; Li, Ming; Belperron, Alexia A.; Haberman, Ann

    2014-01-01

    Lyme disease, due to infection with the Ixodes-tick transmitted spirochete Borrelia burgdorferi, is the most common tick-transmitted disease in the northern hemisphere. Our understanding of the tick-pathogen-vertebrate host interactions that sustain an enzootic cycle for B. burgdorferi is incomplete. In this article, we describe a method for imaging the feeding of Ixodes scapularis nymphs in real-time using two-photon intravital microscopy and show how this technology can be applied to view the response of Lyme borrelia in the skin of an infected host to tick feeding. PMID:24600332

  11. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

    PubMed Central

    Bixler, Joel N.; Kong, Ying; Cirillo, Jeffrey D.; Maitland, Kristen C.

    2016-01-01

    Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU. PMID:26901051

  12. Intravital Fluorescence Excitation in Whole-Animal Optical Imaging.

    PubMed

    Nooshabadi, Fatemeh; Yang, Hee-Jeong; Bixler, Joel N; Kong, Ying; Cirillo, Jeffrey D; Maitland, Kristen C

    2016-01-01

    Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 103 colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 106 CFU.

  13. In vivo microscopy.

    PubMed

    Peti-Peterdi, János

    2016-04-01

    This article summarizes the past, present, and future promise of multiphoton excitation fluorescence microscopy for intravital kidney imaging. During the past 15years, several high-power visual research approaches have been developed using multiphoton imaging to study the normal functions of the healthy, intact, living kidney, and the various molecular and cellular mechanisms of the development of kidney diseases. In this review, the main focus will be on intravital multiphoton imaging of the glomerulus, the structure and function of the glomerular filtration barrier, especially the podocyte. Examples will be given for the combination of two powerful research tools, in vivo multiphoton imaging and mouse genetics using commercially available whole animal models for the detailed characterization of glomerular cell types, their function and fate, and for the better understanding of the molecular mechanisms of glomerular pathologies. One of the new modalities of multiphoton imaging, serial imaging of the same glomerulus in the same animal over several days will be emphasized for its potential for further advancing the field of nephrology research.

  14. Intravital Imaging Reveals Angiotensin II-Induced Transcytosis of Albumin by Podocytes.

    PubMed

    Schießl, Ina Maria; Hammer, Anna; Kattler, Veronika; Gess, Bernhard; Theilig, Franziska; Witzgall, Ralph; Castrop, Hayo

    2016-03-01

    Albuminuria is a hallmark of kidney disease of various etiologies and usually caused by deterioration of glomerular filtration barrier integrity. We recently showed that angiotensin II (Ang II) acutely increases albumin filtration in the healthy kidney. Here, we used intravital microscopy to assess the effects of Ang II on podocyte function in rats. Acute infusion of 30, 60, or 80 ng/kg per minute Ang II enhanced the endocytosis of albumin by activation of the type 1 Ang II receptor and resulted in an average (±SEM) of 3.7±2.2, 72.3±18.6 (P<0.001), and 239.4±34.6 µm(3) (P<0.001) albumin-containing vesicles per glomerulus, respectively, compared with none at baseline or 10 ng/kg per minute Ang II. Immunostaining of Ang II-infused kidneys confirmed the presence of albumin-containing vesicles, which colocalized with megalin, in podocin-positive cells. Furthermore, podocyte endocytosis of albumin was markedly reduced in the presence of gentamicin, a competitive inhibitor of megalin-dependent endocytosis. Ang II infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and gentamicin further increased this concentration. Some endocytic vesicles were acidified and colocalized with LysoTracker. Most vesicles migrated from the capillary to the apical aspect of the podocyte and were eventually released into the urinary space. This transcytosis accounted for approximately 10% of total albumin filtration. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. Ang II enhances the endocytosis and transcytosis of plasma albumin by podocytes, which may eventually impair podocyte function.

  15. Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow.

    PubMed

    Shi, Xiaorui; Zhang, Fei; Urdang, Zachary; Dai, Min; Neng, Lingling; Zhang, Jinhui; Chen, Songlin; Ramamoorthy, Sripriya; Nuttall, Alfred L

    2014-07-01

    Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions.

  16. Immunohistochemical expression of fibronectin in the lungs of fire victims proves intravital reaction in fatal burns.

    PubMed

    Bohnert, Michael; Anderson, Jürina; Rothschild, Markus A; Böhm, Joachim

    2010-11-01

    Immunohistochemical studies about the presence of fibronectin in the lungs were performed in a group of 73 fire victims (63 cases of intravital and 10 cases of postmortem burn) as well as in an unselected control group of 55 individuals not exposed to fire before death. The cases of intravital burn showed a significantly stronger fibronectin expression than the control cases and the cases of postmortem burn. Fibronectin was mainly present in macrophages of the peribronchial lung parenchyma and, not associated with cells, in the matrix of peribronchial tissue. Our findings suggest that higher levels of fibronectin expression in the lung tissue of burn victims compared to fire-unrelated deaths may serve as an indicator of an early intravital inflammatory response to fire damage.

  17. Live-Animal Imaging of Renal Function by Multiphoton Microscopy

    PubMed Central

    Dunn, Kenneth W.; Sutton, Timothy A.; Sandoval, Ruben M.

    2015-01-01

    Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. PMID:23042524

  18. In vivo cell cycle profiling in xenograft tumors by quantitative intravital microscopy

    PubMed Central

    Chittajallu, Deepak R; Florian, Stefan; Kohler, Rainer H; Iwamoto, Yoshiko; Orth, James D; Weissleder, Ralph; Danuser, Gaudenz; Mitchison, Timothy J

    2015-01-01

    Quantification of cell-cycle state at a single-cell level is essential to understand fundamental three-dimensional biological processes such as tissue development and cancer. Analysis of 3D in vivo images, however, is very challenging. Today’s best practice, manual annotation of select image events, generates arbitrarily sampled data distributions, unsuitable for reliable mechanistic inferences. Here, we present an integrated workflow for quantitative in vivo cell-cycle profiling. It combines image analysis and machine learning methods for automated 3D segmentation and cell-cycle state identification of individual cell-nuclei with widely varying morphologies embedded in complex tumor environments. We applied our workflow to quantify cell-cycle effects of three antimitotic cancer drugs over 8 days in HT-1080 fibrosarcoma xenografts in living mice using a dataset of 38,000 cells and compared the induced phenotypes. In contrast to 2D culture, observed mitotic arrest was relatively low, suggesting involvement of additional mechanisms in their antitumor effect in vivo. PMID:25867850

  19. Quantum error correcting codes and 4-dimensional arithmetic hyperbolic manifolds

    SciTech Connect

    Guth, Larry; Lubotzky, Alexander

    2014-08-15

    Using 4-dimensional arithmetic hyperbolic manifolds, we construct some new homological quantum error correcting codes. They are low density parity check codes with linear rate and distance n{sup ε}. Their rate is evaluated via Euler characteristic arguments and their distance using Z{sub 2}-systolic geometry. This construction answers a question of Zémor [“On Cayley graphs, surface codes, and the limits of homological coding for quantum error correction,” in Proceedings of Second International Workshop on Coding and Cryptology (IWCC), Lecture Notes in Computer Science Vol. 5557 (2009), pp. 259–273], who asked whether homological codes with such parameters could exist at all.

  20. Tumor Microvasculature and Microenvironment: Novel Insights Through Intravital Imaging in Pre-Clinical Models

    PubMed Central

    Fukumura, Dai; Duda, Dan G.; Munn, Lance L.; Jain, Rakesh K.

    2010-01-01

    Intravital imaging techniques have provided unprecedented insight into tumor microcirculation and microenvironment. For example, these techniques allowed quantitative evaluations of tumor blood vasculature to uncover its abnormal organization, structure and function (e.g., hyper-permeability, heterogeneous and compromised blood flow). Similarly, imaging of functional lymphatics has documented their absence inside tumors. These abnormalities result in elevated interstitial fluid pressure and hinder the delivery of therapeutic agents to tumors. In addition, they induce a hostile microenvironment characterized by hypoxia and acidosis, as documented by intravital imaging. The abnormal microenvironment further lowers the effectiveness of anti-tumor treatments such as radiation therapy and chemotherapy. In addition to these mechanistic insights, intravital imaging may also offer new opportunities to improve therapy. For example, tumor angiogenesis results in immature, dysfunctional vessels—primarily caused by an imbalance in production of pro- and anti-angiogenic factors by the tumors. Restoring the balance of pro- and anti-angiogenic signaling in tumors can “normalize” tumor vasculature and thus, improve its function, as demonstrated by intravital imaging studies in preclinical models and in cancer patients. Administration of cytotoxic therapy during periods of vascular normalization has the potential to enhance treatment efficacy. PMID:20374484

  1. Time dependent electromagnetic fields and 4-dimensional Stokes' theorem

    NASA Astrophysics Data System (ADS)

    Andosca, Ryan; Singleton, Douglas

    2016-11-01

    Stokes' theorem is central to many aspects of physics—electromagnetism, the Aharonov-Bohm effect, and Wilson loops to name a few. However, the pedagogical examples and research work almost exclusively focus on situations where the fields are time-independent so that one need only deal with purely spatial line integrals (e.g., ∮ A . d x ) and purely spatial area integrals (e.g., ∫ ( ∇ × A ) . d a = ∫ B . d a ). Here, we address this gap by giving some explicit examples of how Stokes' theorem plays out with time-dependent fields in a full 4-dimensional spacetime context. We also discuss some unusual features of Stokes' theorem with time-dependent fields related to gauge transformations and non-simply connected topology.

  2. REAL-TIME INTRAVITAL IMAGING ESTABLISHES TUMOUR-ASSOCIATED MACROPHAGES AS THE EXTRASKELETAL TARGET OF BISPHOSPHONATE ACTION IN CANCER

    PubMed Central

    Junankar, Simon; Shay, Gemma; Jurczyluk, Julie; Ali, Naveid; Down, Jenny; Pocock, Nicholas; Parker, Andrew; Nguyen, Akira; Sun, Shuting; Kashemirov, Boris; McKenna, Charles E.; Croucher, Peter I.; Swarbrick, Alexander; Weilbaecher, Katherine; Phan, Tri Giang; Rogers, Michael J.

    2014-01-01

    Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their anti-resorptive effects on the skeleton. However, since bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their anti-tumour action, particularly on cells outside of bone, remain unknown. Here we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumours, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumour-associated macrophages (TAMs), but not tumour cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumour of a patient and showed the resected tumour to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumours outside the skeleton and that their anti-tumour activity is likely to be mediated via TAMs. PMID:25312016

  3. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    PubMed Central

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-01-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. PMID:27004454

  4. From morphology to biochemical state – intravital multiphoton fluorescence lifetime imaging of inflamed human skin

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Getova, Valentina; Niemeyer, Verena; Zens, Katharina; Unnerstall, Tim R.; Feger, Julia S.; Fallah, Mohammad A.; Metze, Dieter; Ständer, Sonja; Luger, Thomas A.; Koenig, Karsten; Mess, Christian; Schneider, Stefan W.

    2016-03-01

    The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPT-FLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

  5. Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging

    PubMed Central

    Eng, Diana G.; Rusiniak, Michael E.; Sequeira-Lopez, Maria Luisa S.; Gomez, R. Ariel; Pippin, Jeffrey W.; Gross, Kenneth W.; Peti-Peterdi, Janos; Shankland, Stuart J.

    2017-01-01

    Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman’s capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS. PMID:28329012

  6. Tracking the stochastic fate of cells of the renin lineage after podocyte depletion using multicolor reporters and intravital imaging.

    PubMed

    Kaverina, Natalya V; Kadoya, Hiroyuki; Eng, Diana G; Rusiniak, Michael E; Sequeira-Lopez, Maria Luisa S; Gomez, R Ariel; Pippin, Jeffrey W; Gross, Kenneth W; Peti-Peterdi, Janos; Shankland, Stuart J

    2017-01-01

    Podocyte depletion plays a major role in focal segmental glomerular sclerosis (FSGS). Because cells of the renin lineage (CoRL) serve as adult podocyte and parietal epithelial cell (PEC) progenitor candidates, we generated Ren1cCre/R26R-ConfettiTG/WT and Ren1dCre/R26R-ConfettiTG/WT mice to determine CoRL clonality during podocyte replacement. Four CoRL reporters (GFP, YFP, RFP, CFP) were restricted to cells in the juxtaglomerular compartment (JGC) at baseline. Following abrupt podocyte depletion in experimental FSGS, all four CoRL reporters were detected in a subset of glomeruli at day 28, where they co-expressed de novo four podocyte proteins (podocin, nephrin, WT-1 and p57) and two glomerular parietal epithelial cell (PEC) proteins (claudin-1, PAX8). To monitor the precise migration of a subset of CoRL over a 2w period following podocyte depletion, intravital multiphoton microscopy was used. Our findings demonstrate direct visual support for the migration of single CoRL from the JGC to the parietal Bowman's capsule, early proximal tubule, mesangium and glomerular tuft. In summary, these results suggest that following podocyte depletion, multi-clonal CoRL migrate to the glomerulus and replace podocyte and PECs in experimental FSGS.

  7. Dynamic phase imaging utilizing a 4-dimensional microscope system

    PubMed Central

    Creath, Katherine

    2011-01-01

    This paper describes a new, novel interference Linnik microscope system and presents images and data of live biological samples. The specially designed optical system enables instantaneous 4-dimensional video measurements of dynamic motions within and among live cells without the need for contrast agents. This "label-free", vibration insensitive imaging system enables measurement of biological objects in reflection using harmless light levels with a variety of magnifications and wavelengths with fields of view from several hundred microns up to a millimeter. At the core of the instrument is a phase measurement camera (PMC) enabling simultaneous measurement of multiple interference patterns utilizing a pixelated phase mask taking advantage of the polarization properties of light. Utilizing this technology enables the creation of phase image movies in real time at video rates so that dynamic motions and volumetric changes can be tracked. Objects are placed on a reflective surface in liquid under a coverslip. Phase values are converted to optical thickness data enabling volumetric, motion and morphological studies. Data from a number of different organisms such as flagellates and rotifers will be presented, as will measurements of human breast cancer cells with the addition of various agents that break down the cells. These data highlight examples of monitoring different biological processes and motions. PMID:24357901

  8. Respiratory Amplitude Guided 4-Dimensional Magnetic Resonance Imaging

    SciTech Connect

    Hu, Yanle; Caruthers, Shelton D.; Low, Daniel A.; Parikh, Parag J.; Mutic, Sasa

    2013-05-01

    Purpose: To evaluate the feasibility of prospectively guiding 4-dimensional (4D) magnetic resonance imaging (MRI) image acquisition using triggers at preselected respiratory amplitudes to achieve T{sub 2} weighting for abdominal motion tracking. Methods and Materials: A respiratory amplitude-based triggering system was developed and integrated into a commercial turbo spin echo MRI sequence. Initial feasibility tests were performed on healthy human study participants. Four respiratory states, the middle and the end of inhalation and exhalation, were used to trigger 4D MRI image acquisition of the liver. To achieve T{sub 2} weighting, the echo time and repetition time were set to 75 milliseconds and 4108 milliseconds, respectively. Single-shot acquisition, together with parallel imaging and partial k-space imaging techniques, was used to improve image acquisition efficiency. 4D MRI image sets composed of axial or sagittal slices were acquired. Results: Respiratory data measured and logged by the MRI scanner showed that the triggers occurred at the appropriate respiratory levels. Liver motion could be easily observed on both 4D MRI image datasets by sensing either the change of liver in size and shape (axial) or diaphragm motion (sagittal). Both 4D MRI image datasets were T{sub 2}-weighted as expected. Conclusions: This study demonstrated the feasibility of achieving T{sub 2}-weighted 4D MRI images using amplitude-based respiratory triggers. With the aid of the respiratory amplitude-based triggering system, the proposed method is compatible with most MRI sequences and therefore has the potential to improve tumor-tissue contrast in abdominal tumor motion imaging.

  9. Intravital imaging of podocyte calcium in glomerular injury and disease

    PubMed Central

    Burford, James L.; Villanueva, Karie; Lam, Lisa; Riquier-Brison, Anne; Hackl, Matthias J.; Pippin, Jeffrey; Shankland, Stuart J.; Peti-Peterdi, János

    2014-01-01

    Intracellular calcium ([Ca2+]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca2+]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca2+]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca2+]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca2+]i around the injury site and promoted cell-to-cell propagating podocyte [Ca2+]i waves along capillary loops. [Ca2+]i wave propagation was ameliorated by inhibitors of purinergic [Ca2+]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca2+]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca2+]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca2+]i in glomerular pathology and suggest that purinergic [Ca2+]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney. PMID:24713653

  10. Intravital imaging of podocyte calcium in glomerular injury and disease.

    PubMed

    Burford, James L; Villanueva, Karie; Lam, Lisa; Riquier-Brison, Anne; Hackl, Matthias J; Pippin, Jeffrey; Shankland, Stuart J; Peti-Peterdi, János

    2014-05-01

    Intracellular calcium ([Ca²⁺]i) signaling mediates physiological and pathological processes in multiple organs, including the renal podocyte; however, in vivo podocyte [Ca²⁺]i dynamics are not fully understood. Here we developed an imaging approach that uses multiphoton microscopy (MPM) to directly visualize podocyte [Ca²⁺]i dynamics within the intact kidneys of live mice expressing a fluorescent calcium indicator only in these cells. [Ca²⁺]i was at a low steady-state level in control podocytes, while Ang II infusion caused a minor elevation. Experimental focal podocyte injury triggered a robust and sustained elevation of podocyte [Ca²⁺]i around the injury site and promoted cell-to-cell propagating podocyte [Ca²⁺]i waves along capillary loops. [Ca²⁺]i wave propagation was ameliorated by inhibitors of purinergic [Ca²⁺]i signaling as well as in animals lacking the P2Y2 purinergic receptor. Increased podocyte [Ca²⁺]i resulted in contraction of the glomerular tuft and increased capillary albumin permeability. In preclinical models of renal fibrosis and glomerulosclerosis, high podocyte [Ca²⁺]i correlated with increased cell motility. Our findings provide a visual demonstration of the in vivo importance of podocyte [Ca²⁺]i in glomerular pathology and suggest that purinergic [Ca²⁺]i signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

  11. Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury.

    PubMed

    Honda, Masaki; Takeichi, Takayuki; Hashimoto, Shintaro; Yoshii, Daiki; Isono, Kaori; Hayashida, Shintaro; Ohya, Yuki; Yamamoto, Hidekazu; Sugawara, Yasuhiko; Inomata, Yukihiro

    2017-02-15

    Neutrophils are considered responsible for the pathophysiological changes resulting from hepatic ischemia-reperfusion (I/R) injury, which is a complication of trauma, shock, liver resection, and transplantation. Recently, evidence is accumulating that formyl-peptide receptor (FPR) signaling constitutes an important danger signal that guides neutrophils to sites of inflammation. This study aimed to investigate dynamic neutrophil recruitment using two-photon laser-scanning microscopy (TPLSM) in response to FPR1 blockade during hepatic I/R. LysM-eGFP mice were subjected to partial warm hepatic I/R. They were pretreated with an FPR1 antagonist, cyclosporine H (CsH), or formyl peptide, fMLF. Liver was imaged after hepatic laser irradiation or I/R using the TPLSM technique. CsH treatment alleviated hepatic I/R injury, as evidenced by decreased serum transaminase levels, reduced hepatocyte necrosis/apoptosis, and diminished inflammatory cytokine, chemokine, and oxidative stress. In contrast, systemic administration of fMLF showed few effects. Time-lapse TPLSM showed that FPR1 blockade inhibited the accumulation of neutrophils in the necrotic area induced by laser irradiation in vivo. In the CsH-treated I/R group, the number and crawling velocity of neutrophils in the nonperfused area were lower than those in the control group. Meanwhile, FPR1 blockade did not affect monocyte/macrophage recruitment. Hepatic I/R promoted the retention of neutrophils and their active behavior in the spleen, whereas CsH treatment prevented their changes. Intravital TPLSM revealed that formyl-peptide-FPR1 signaling is responsible for regulating neutrophil chemotaxis to allow migration into the necrotic area in hepatic I/R. Our findings suggest effective approaches for elucidating the mechanisms of immune cell responses in hepatic I/R.

  12. Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

    NASA Astrophysics Data System (ADS)

    McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

    2015-02-01

    Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

  13. Intravital lectin perfusion analysis of vascular permeability in human micro- and macro- blood vessels.

    PubMed

    Debbage, P L; Sölder, E; Seidl, S; Hutzler, P; Hugl, B; Ofner, D; Kreczy, A

    2001-10-01

    We previously applied intravital lectin perfusion in mouse models to elucidate mechanisms underlying vascular permeability. The present work transfers this technique to human models, analysing vascular permeability in macro- and microvessels. Human vascular endothelial surface carbohydrate biochemistry differs significantly from its murine counterpart, lacking alpha-galactosyl epitopes and expressing the L-fucose moiety in the glycocalyx; the poly-N-lactosamine glycan backbone is common to all mammals. We examined extensively lectin binding specificities in sections and in vivo, and then applied the poly-N-lactosamine-specific lectin LEA and the L-fucose-specific lectin UEA-I in human intravital perfusions. Transendothelial transport differed in macrovessels and microvessels. In microvessels of adult human fat tissue, rectal wall and rectal carcinomas, slow transendothelial transport by vesicles was followed by significant retention at the subendothelial basement membrane; paracellular passage was not observed. Passage time exceeded 1 h. Thus we found barrier mechanisms resembling those we described previously in murine tissues. In both adult and fetal macrovessels, the vena saphena magna and the umbilical vein, respectively, rapid passage across the endothelial lining was observed, the tracer localising completely in the subendothelial tissues within 15 min; vesicular transport was more rapid than in microvessels, and retention at the subendothelial basement membrane briefer.

  14. A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

    PubMed Central

    Tao, Wen; Rubart, Michael; Ryan, Jennifer; Xiao, Xiao; Qiao, Chunping; Hato, Takashi; Davidson, Michael W.; Dunn, Kenneth W.

    2015-01-01

    The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. PMID:26333599

  15. Intravital imaging of multicolor-labeled tumor immune microenvironment through skin-fold window chamber

    NASA Astrophysics Data System (ADS)

    Qi, Shuhong; Zhang, Zhihong

    2015-03-01

    Tumor immune microenvironment became very important for the tumor immunotherapy. There were several kinds of immune cells in tumor stromal, and they played very different roles in tumor growth. In order to observe the behaviors of multiple immune cells in tumor microenvironment and the interaction between immune cells and tumor cells at the same time, we generated a multicolor-labeled tumor immune microenvironment model. The tumor cells and immune cells were labeled by different fluorescent proteins. By using of skin-fold window chamber implanted into mice and intravital imaging technology, we could dynamically observe the different immune cells in tumor microenvironment. After data analysis from the video, we could know the behavior of TILs, DCs and Tregs in tumor immune microenvironment; furthermore, we could know these immune cells play different roles in the tumor microenvironment.

  16. Impact of rapamycin on phenotype and tolerogenic function of dendritic cells via intravital optical imaging

    NASA Astrophysics Data System (ADS)

    Luo, Meijie; Zhang, Zhihong

    2014-03-01

    Rapamycin (RAPA) as a unique tolerance-promoting therapeutic drug is crucial to successful clinical organ transplantation. DC (Dendritic cells) play a critical role in antigen presentation to T cells to initiate immune responses involved in tissue rejection. Although the influence of RAPA on DC differentiation and maturation had been reported by some research groups, it is still controversial and unclear right now. In addition, it is also lack of study on investigating the role of DC in DTH reaction via intravital optical imaging. Herein, we investigated the effect of rapamycin on phenotype and function of bone marrow monocyte-derived DC both in vitro and in vivo. In vitro experiments by flow cytometry (FACS) showed that DC displayed decreased cell size and lower expression levels of surface molecule CD80 induced by RAPA; Furthermore, the phagocytic ability to OVA of DC was inhibited by RAPA started from 1 h to 2 h post co-incubation, but recovered after 4 h; In addition, the capacity of DC to activate naïve OT-II T cell proliferation was also inhibited at 3 day post co-incubation, but had no effect at 5 day, the data indicated this effect was reversible when removing the drug. More importantly, the DC-T interaction was monitored both in vitro and in intravital lymph node explant, and showed that RAPA-DC had a significant lower proportion of long-lived (>15min) contacts. Thus, RAPA displayed immunosuppressive to phenotypic and functional maturation of DC, and this phenomenon induced by RAPA may favorable in the clinical organ transplantation in future.

  17. Intravital Microscopy for Identifying Tumor Vessels in Patients With Stage IA-IV Melanoma That is Being Removed by Surgery

    ClinicalTrials.gov

    2016-01-13

    Recurrent Melanoma; Stage IA Skin Melanoma; Stage IB Skin Melanoma; Stage IIA Skin Melanoma; Stage IIB Skin Melanoma; Stage IIC Skin Melanoma; Stage IIIA Skin Melanoma; Stage IIIB Skin Melanoma; Stage IIIC Skin Melanoma; Stage IV Skin Melanoma

  18. Intravital and whole-organ imaging reveals capture of melanoma-derived antigen by lymph node subcapsular macrophages leading to widespread deposition on follicular dendritic cells.

    PubMed

    Moalli, Federica; Proulx, Steven T; Schwendener, Reto; Detmar, Michael; Schlapbach, Christoph; Stein, Jens V

    2015-01-01

    Aberrant antigens expressed by tumor cells, such as in melanoma, are often associated with humoral immune responses, which may in turn influence tumor progression. Despite recent data showing the central role of adaptive immune responses on cancer spread or control, it remains poorly understood where and how tumor-derived antigen (TDA) induces a humoral immune response in tumor-bearing hosts. Based on our observation of TDA accumulation in B cell areas of lymph nodes (LNs) from melanoma patients, we developed a pre-metastatic B16.F10 melanoma model expressing a fluorescent fusion protein, tandem dimer tomato, as a surrogate TDA. Using intravital two-photon microscopy (2PM) and whole-mount 3D LN imaging of tumor-draining LNs in immunocompetent mice, we report an unexpectedly widespread accumulation of TDA on follicular dendritic cells (FDCs), which were dynamically scanned by circulating B cells. Furthermore, 2PM imaging identified macrophages located in the subcapsular sinus of tumor-draining LNs to capture subcellular TDA-containing particles arriving in afferent lymph. As a consequence, depletion of macrophages or genetic ablation of B cells and FDCs resulted in dramatically reduced TDA capture in tumor-draining LNs. In sum, we identified a major pathway for the induction of humoral responses in a melanoma model, which may be exploitable to manipulate anti-TDA antibody production during cancer immunotherapy.

  19. Red blood cells affect the margination of microparticles in synthetic microcapillaries and intravital microcirculation as a function of their size and shape.

    PubMed

    D'Apolito, Rosa; Tomaiuolo, Giovanna; Taraballi, Francesca; Minardi, Silvia; Kirui, Dickson; Liu, Xuewu; Cevenini, Armando; Palomba, Roberto; Ferrari, Mauro; Salvatore, Francesco; Tasciotti, Ennio; Guido, Stefano

    2015-11-10

    A key step in particle-based drug delivery throughmicrocirculation is particlemigration from blood flow to vesselwalls, also known as “margination”,which promotes particle contact and adhesion to the vesselwall. Margination and adhesion should be independently addressed as two distinct phenomena, considering that the former is a fundamental prerequisite to achieve particle adhesion and subsequent extravasation. Although margination has beenmodeled by numerical simulations and investigated inmodel systems in vitro, experimental studies including red blood cells (RBCs) are lacking. Here, we evaluate the effect of RBCs on margination through microfluidic studies in vitro and by intravital microscopy in vivo.We showthatmargination,which is almost absent when particles are suspended in a cell-free medium, is drastically enhanced by RBCs. This effect is size- and shape-dependent, larger spherical/discoid particles being more effectively marginated both in vitro and in vivo. Our findings can be explained by the collision of particles with RBCs that induces the drifting of the particles towards the vessel walls where they become trapped in the cell-free layer. These results are relevant for the design of drug delivery strategies based on systemically administered carriers.

  20. Implications of Intravital Imaging of Murine Germinal Centers on the Control of B Cell Selection and Division

    PubMed Central

    Binder, Sebastian C.; Meyer-Hermann, Michael

    2016-01-01

    Intravital imaging of antibody optimization in germinal center (GC) reactions has set a new dimension in the understanding of the humoral immune response during the last decade. The inclusion of spatio-temporal cellular dynamics in the research on GCs required analysis using the agent-based mathematical models. In this study, we integrate the available intravital imaging data from various research groups and incorporate these into a quantitative mathematical model of GC reactions and antibody affinity maturation. Interestingly, the integration of data concerning the spatial organization of GCs and B cell motility allows to draw conclusions on the strength of the selection pressure and the control of B cell division by T follicular helper cells. PMID:28066409

  1. Long-term intravital imaging of the multicolor-coded tumor microenvironment during combination immunotherapy

    PubMed Central

    Qi, Shuhong; Li, Hui; Lu, Lisen; Qi, Zhongyang; Liu, Lei; Chen, Lu; Shen, Guanxin; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    The combined-immunotherapy of adoptive cell therapy (ACT) and cyclophosphamide (CTX) is one of the most efficient treatments for melanoma patients. However, no synergistic effects of CTX and ACT on the spatio-temporal dynamics of immunocytes in vivo have been described. Here, we visualized key cell events in immunotherapy-elicited immunoreactions in a multicolor-coded tumor microenvironment, and then established an optimal strategy of metronomic combined-immunotherapy to enhance anti-tumor efficacy. Intravital imaging data indicated that regulatory T cells formed an 'immunosuppressive ring' around a solid tumor. The CTX-ACT combined-treatment elicited synergistic immunoreactions in tumor areas, which included relieving the immune suppression, triggering the transient activation of endogenous tumor-infiltrating immunocytes, increasing the accumulation of adoptive cytotoxic T lymphocytes, and accelerating the infiltration of dendritic cells. These insights into the spatio-temporal dynamics of immunocytes are beneficial for optimizing immunotherapy and provide new approaches for elucidating the mechanisms underlying the involvement of immunocytes in cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.14756.001 PMID:27855783

  2. Expanding Applications of the Nano Intravital Device as a Platform for Exploring Tumor Microenvironments

    NASA Astrophysics Data System (ADS)

    Padgen, Michael R.

    The tumor microenvironment has been demonstrated to be a key determinant in the progression of cancer. Unfortunately, the mechanisms behind the different microenvironments (cytokine gradients, hypoxia, hypoglycemia, etc) have not been fully elucidated. Identifying these mechanisms can lead to targeted, individualized therapy to prevent metastasis. The Nano Intravital Device (NANIVID) is a microfabricated, implantable device designed to initiate specific microenvironments in vivo so that the time course of the effects can be observed. With both spatial and temporal control over the induced environments, the affected regions of the tumor can be compared to the rest of the tumor. The NANIVID was first used to establish cytokine gradients to monitor the migration of invasive cancer cells. The three projects that comprise this work expand the applications of the NANIVID to establish the device as a robust platform for investigating tumor microenvironment interactions. The first project released chemical mimics from the device to induce the cellular hypoxic response in tumors to determine how hypoxia affects the fate of disseminated tumor cells. The second project used the NANIVID in combination with an atomic force microscope to investigate the altered mechanics of migrating invasive cancer cells. The final project was to develop a cell counter to monitor the isolation of the invasive subpopulation of cells that were drawn into the device using a chemoattractant. These three projects demonstrate the potential of the NANIVID as a platform for investigating the tumor microenvironment.

  3. Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes.

    PubMed

    Reiterová, K; Miterpáková, M; Turceková, L'; Antolová, D; Dubinský, P

    2005-03-10

    Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.

  4. A 4-Dimensional Representation of Antennal Lobe Output Based on an Ensemble of Characterized Projection Neurons

    PubMed Central

    Staudacher, Erich M.; Huetteroth, Wolf; Schachtner, Joachim; Daly, Kevin C.

    2009-01-01

    A central problem facing studies of neural encoding in sensory systems is how to accurately quantify the extent of spatial and temporal responses. In this study, we take advantage of the relatively simple and stereotypic neural architecture found in invertebrates. We combine standard electrophysiological techniques, recently developed population analysis techniques, and novel anatomical methods to form an innovative 4-dimensional view of odor output representations in the antennal lobe of the moth Manduca sexta. This novel approach allows quantification of olfactory responses of characterized neurons with spike time resolution. Additionally, arbitrary integration windows can be used for comparisons with other methods such as imaging. By assigning statistical significance to changes in neuronal firing, this method can visualize activity across the entire antennal lobe. The resulting 4-dimensional representation of antennal lobe output complements imaging and multi-unit experiments yet provides a more comprehensive and accurate view of glomerular activation patterns in spike time resolution. PMID:19464513

  5. Fluorescence Microscopy

    PubMed Central

    Sanderson, Michael J.; Smith, Ian; Parker, Ian; Bootman, Martin D.

    2016-01-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. PMID:25275114

  6. Intravital Imaging of the Kidney in a Rat Model of Salt-Sensitive Hypertension.

    PubMed

    Endres, Bradley T; Sandoval, Ruben M; Rhodes, George J; Campos-Bilderback, Silvia B; Kamocka, Malgorzata M; McDermott-Roe, Christopher; Staruschenko, Alexander; Molitoris, Bruce A; Geurts, Aron M; Palygin, Oleg

    2017-04-12

    Hypertension is one of the most prevalent diseases worldwide, and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb=0.0686), relative permeability to albumin (+∆16%) and impaired volume hemodynamics (-∆14%). Serum albumin, but not serum globulins or creatinine, concentration was decreased (-0.54g/dL), which was concomitant with increased filtration of albumin (3.7 vs 0.8 g per day normal diet). Pathologically, hypertensive animals had significant tubular damage as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+∆2.20score/image), progressive augmentation of red blood cell velocity (+∆269µm/s) and micro vessel diameter (+∆4.3µm), and increased vascular injury (+∆0.61leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.

  7. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

    PubMed Central

    Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

    2015-01-01

    Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

  8. The classification of the Weyl conformal tensor in 4-dimensional manifolds of neutral signature

    NASA Astrophysics Data System (ADS)

    Hall, Graham

    2017-01-01

    This paper presents a simple account of the algebraic classification of the Weyl conformal tensor on a 4-dimensional manifold with metric g of neutral signature (+ , + , - , -) . The classification is algebraically similar to the well-known Petrov classification in the Lorentz case and the various algebraic types and corresponding canonical forms are obtained. Criteria concerning principal, totally null 2-spaces are explored and which lead to principal null directions similar to those of L. Bel in the Lorentz case. The uniqueness, or otherwise, of the tetrads in which the canonical forms appear are investigated and some topological and differentiability properties of the algebraic types are also established.

  9. Analytical Microscopy

    SciTech Connect

    Not Available

    2006-06-01

    In the Analytical Microscopy group, within the National Center for Photovoltaic's Measurements and Characterization Division, we combine two complementary areas of analytical microscopy--electron microscopy and proximal-probe techniques--and use a variety of state-of-the-art imaging and analytical tools. We also design and build custom instrumentation and develop novel techniques that provide unique capabilities for studying materials and devices. In our work, we collaborate with you to solve materials- and device-related R&D problems. This sheet summarizes the uses and features of four major tools: transmission electron microscopy, scanning electron microscopy, the dual-beam focused-ion-beam workstation, and scanning probe microscopy.

  10. Intravital Computer Morphometry on Protozoa: A Method for Monitoring of the Morphofunctional Disorders in Cells Exposed in the Cell Phone Communication Electromagnetic Field.

    PubMed

    Uskalova, D V; Igolkina, Yu V; Sarapultseva, E I

    2016-08-01

    Morphofunctional disorders in unicellular aquatic protozoa - Spirostomum ambiguum infusorians after 30-, 60-, and 360-min exposure in electromagnetic field at a radiation frequency of 1 GHz and energy flow density of 50 μW/cm(2) were analyzed by intravital computer morphometry. Significant disorders in morphometric values correlated with low mobility of the protozoa. The results suggested the use of intravital computer morphometry on the protozoa for early diagnosis of radiation-induced effects of the mobile communication electromagnetic field, for example, low mobility of spermatozoa.

  11. Intravital excitation increases detection sensitivity for pulmonary tuberculosis by whole-body imaging with β-lactamase reporter enzyme fluorescence.

    PubMed

    Nooshabadi, Fatemeh; Yang, Hee-Jeong; Cheng, Yunfeng; Durkee, Madeleine S; Xie, Hexin; Rao, Jianghong; Cirillo, Jeffrey D; Maitland, Kristen C

    2016-10-18

    Tuberculosis is a pulmonary disease with an especially high mortality rate in immuno-compromised populations, specifically children and HIV positive individuals. The causative agent, Mycobacterium tuberculosis (Mtb), is a very slow growing and difficult organism to work with, making both diagnosis and development of effective treatments cumbersome. We utilize a fiber-optic fluorescence microendoscope integrated with a whole-body imaging system for in vivo Mtb detection. The system exploits an endogenous enzyme of Mtb (β-lactamase, or BlaC) using a BlaC-specific NIR fluorogenic substrate. In the presence of BlaC, this substrate is cleaved and becomes fluorescent. Using intravital illumination of the lung to excite this probe, sensitivity of the optical system increases over trans- and epi-illumination methods of whole-body fluorescence imaging. We demonstrate that integration of these imaging technologies with BlaC-specific fluorescent reporter probe improves the level of detection to ∼100 colony forming units, a 100× increase in sensitivity in comparison to epi-illumination and a 10× increase in sensitivity in comparison to previous work in intravital excitation of tdTomato-expressing Mtb. This lower detection threshold enables the study of early stage bacterial infections with clinical strains of Mtb and longitudinal studies of disease pathogenesis and therapeutic efficacy with multiple time points in a single animal.

  12. Evaluation of 4-dimensional Computed Tomography to 4-dimensional Cone-Beam Computed Tomography Deformable Image Registration for Lung Cancer Adaptive Radiation Therapy

    SciTech Connect

    Balik, Salim; Weiss, Elisabeth; Jan, Nuzhat; Roman, Nicholas; Sleeman, William C.; Fatyga, Mirek; Christensen, Gary E.; Zhang, Cheng; Murphy, Martin J.; Lu, Jun; Keall, Paul; Williamson, Jeffrey F.; Hugo, Geoffrey D.

    2013-06-01

    Purpose: To evaluate 2 deformable image registration (DIR) algorithms for the purpose of contour mapping to support image-guided adaptive radiation therapy with 4-dimensional cone-beam CT (4DCBCT). Methods and Materials: One planning 4D fan-beam CT (4DFBCT) and 7 weekly 4DCBCT scans were acquired for 10 locally advanced non-small cell lung cancer patients. The gross tumor volume was delineated by a physician in all 4D images. End-of-inspiration phase planning 4DFBCT was registered to the corresponding phase in weekly 4DCBCT images for day-to-day registrations. For phase-to-phase registration, the end-of-inspiration phase from each 4D image was registered to the end-of-expiration phase. Two DIR algorithms—small deformation inverse consistent linear elastic (SICLE) and Insight Toolkit diffeomorphic demons (DEMONS)—were evaluated. Physician-delineated contours were compared with the warped contours by using the Dice similarity coefficient (DSC), average symmetric distance, and false-positive and false-negative indices. The DIR results are compared with rigid registration of tumor. Results: For day-to-day registrations, the mean DSC was 0.75 ± 0.09 with SICLE, 0.70 ± 0.12 with DEMONS, 0.66 ± 0.12 with rigid-tumor registration, and 0.60 ± 0.14 with rigid-bone registration. Results were comparable to intraobserver variability calculated from phase-to-phase registrations as well as measured interobserver variation for 1 patient. SICLE and DEMONS, when compared with rigid-bone (4.1 mm) and rigid-tumor (3.6 mm) registration, respectively reduced the average symmetric distance to 2.6 and 3.3 mm. On average, SICLE and DEMONS increased the DSC to 0.80 and 0.79, respectively, compared with rigid-tumor (0.78) registrations for 4DCBCT phase-to-phase registrations. Conclusions: Deformable image registration achieved comparable accuracy to reported interobserver delineation variability and higher accuracy than rigid-tumor registration. Deformable image registration

  13. Synchrotron 4-dimensional imaging of two-phase flow through porous media

    PubMed Central

    Kim, F.H.; Penumadu, D.; Patel, P.; Xiao, X.; Garboczi, E.J.; Moylan, S.P.; Donmez, M.A.

    2016-01-01

    Near real-time visualization of complex two-phase flow in a porous medium was demonstrated with dynamic 4-dimensional (4D) (3D + time) imaging at the 2-BM beam line of the Advanced Photon Source (APS) at Argonne National Laboratory. Advancing fluid fronts through tortuous flow paths and their interactions with sand grains were clearly captured, and formations of air bubbles and capillary bridges were visualized. The intense X-ray photon flux of the synchrotron facility made 4D imaging possible, capturing the dynamic evolution of both solid and fluid phases. Computed Tomography (CT) scans were collected every 12 s with a pixel size of 3.25 µm. The experiment was carried out to improve understanding of the physics associated with two-phase flow. The results provide a source of validation data for numerical simulation codes such as Lattice-Boltzmann, which are used to model multi-phase flow through porous media. PMID:27891248

  14. Fast and precise targeting of single tumor cells in vivo by multimodal correlative microscopy

    PubMed Central

    Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Solecki, Gergely; Allio, Guillaume; Winkler, Frank; Ruthensteiner, Bernhard; Goetz, Jacky G.; Schwab, Yannick

    2016-01-01

    ABSTRACT Intravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and three-dimensional electron microscopy. It enables a rapid (c.a. 2 weeks) and accurate (<5 µm) correlation of functional imaging to ultrastructural analysis of single cells in a relevant context. We demonstrate the power of our approach by capturing single tumor cells in the vasculature of the cerebral cortex and in subcutaneous tumors, providing unique insights into metastatic events. Providing a significantly improved throughput, our workflow enables multiple sampling, a prerequisite for making correlative imaging a relevant tool to study cell biology in vivo. Owing to the versatility of this workflow, we envision broad applications in various fields of biological research, such as cancer or developmental biology. PMID:26659665

  15. Long-term time-lapse multimodal microscopy for tracking cell dynamics in live tissue

    NASA Astrophysics Data System (ADS)

    Graf, Benedikt W.; Valero, Maria C.; Chaney, Eric J.; Marjanovic, Marina; Boppart, Marni D.; Boppart, Stephen A.

    2011-02-01

    High speed intravital microscopy has emerged as an essential tool for studying cellular dynamics in live tissue. A limitation of this technique, however, is that the timescale that a sample can be continuously imaged is limited by practical considerations to several hours. Long term observation of live tissue is of great interest for a variety of research areas. We present methods for observing long term cellular dynamics in live tissue based on three-dimensional registration of time-lapse intravital microscopy images. For these experiments we utilized a custom multimodal microscope that allows simultaneous and co-registered acquisition of optical coherence (OCM) and multiphoton (MPM) microscopy images. OCM allows the structure of a sample to be visualized based on backscattered light while MPM excited fluorescence allows individual cells and cell function to be visualized. The OCM images of tissue structure are used to register data sets taken at different time points. The transformations of the OCM images are applied to MPM images to determine the migration of cell populations. This method of image registration is applied to in vivo tracking of bone-marrow derived GFP-labeled stem cells in mouse skin following bone marrow transplants from GFP donors into species-matched wildtype hosts. The use of three-dimensional image registration of time-lapse microscopy images enables tracking these cells after local cutaneous injury, and for investigating the role of skin stem cells in wound healing.

  16. Correlative Microscopy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microscopy and Imaging offers many opportunities to collaborate and cooperate with scientists in many different fields nationally and internationally. Images have proven to be very important components in basic research, product development and understanding structure/function relationships in addit...

  17. Correlative microscopy.

    PubMed

    Loussert Fonta, Céline; Humbel, Bruno M

    2015-09-01

    In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array

  18. Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue.

    PubMed

    Takehara, Hiroaki; Ohta, Yasumi; Motoyama, Mayumi; Haruta, Makito; Nagasaki, Mizuki; Takehara, Hironari; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Ohta, Jun

    2015-05-01

    The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 μm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation.

  19. Intravital fluorescence imaging of mouse brain using implantable semiconductor devices and epi-illumination of biological tissue

    PubMed Central

    Takehara, Hiroaki; Ohta, Yasumi; Motoyama, Mayumi; Haruta, Makito; Nagasaki, Mizuki; Takehara, Hironari; Noda, Toshihiko; Sasagawa, Kiyotaka; Tokuda, Takashi; Ohta, Jun

    2015-01-01

    The application of the fluorescence imaging method to living animals, together with the use of genetically engineered animals and synthesized photo-responsive compounds, is a powerful method for investigating brain functions. Here, we report a fluorescence imaging method for the brain surface and deep brain tissue that uses compact and mass-producible semiconductor imaging devices based on complementary metal-oxide semiconductor (CMOS) technology. An image sensor chip was designed to be inserted into brain tissue, and its size was 1500 × 450 μm. Sample illumination is also a key issue for intravital fluorescence imaging. Hence, for the uniform illumination of the imaging area, we propose a new method involving the epi-illumination of living biological tissues, and we performed investigations using optical simulations and experimental evaluation. PMID:26137364

  20. 5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin

    NASA Astrophysics Data System (ADS)

    König, Karsten; Weinigel, Martin; Breunig, Hans G.; Gregory, Axel; Fischer, Peter; Kellner-Höfer, Marcel; Bückle, Rainer; Schwarz, Martin; Riemann, Iris; Stracke, Frank; Huck, Volker; Gorzelanny, Christian; Schneider, Stefan W.

    2010-02-01

    Some years ago, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have been launched. These tomographs provide optical biopsies with submicron resolution based on two-photon excited autofluorescence (NAD(P)H, flavoproteins, keratin, elastin, melanin, porphyrins) and second harmonic generation by collagen. The 3D tomograph was now transferred into a 5D imaging system by the additional detection of the emission spectrum and the fluorescence lifetime based on spatially and spectrally resolved time-resolved single photon counting. The novel 5D intravital tomograph (5D-IVT) was employed for the early detection of atopic dermatitis and the analysis of treatment effects.

  1. Expansion Microscopy

    PubMed Central

    Chen, Fei; Tillberg, Paul W.; Boyden, Edward S.

    2014-01-01

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. Here we report the discovery that, by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable super-resolution microscopy with diffraction-limited microscopes. We demonstrate ExM with effective ~70 nm lateral resolution in both cultured cells and brain tissue, performing three-color super-resolution imaging of ~107 μm3 of the mouse hippocampus with a conventional confocal microscope. PMID:25592419

  2. Optimizing 4-Dimensional Magnetic Resonance Imaging Data Sampling for Respiratory Motion Analysis of Pancreatic Tumors

    SciTech Connect

    Stemkens, Bjorn; Tijssen, Rob H.N.; Senneville, Baudouin D. de

    2015-03-01

    Purpose: To determine the optimum sampling strategy for retrospective reconstruction of 4-dimensional (4D) MR data for nonrigid motion characterization of tumor and organs at risk for radiation therapy purposes. Methods and Materials: For optimization, we compared 2 surrogate signals (external respiratory bellows and internal MRI navigators) and 2 MR sampling strategies (Cartesian and radial) in terms of image quality and robustness. Using the optimized protocol, 6 pancreatic cancer patients were scanned to calculate the 4D motion. Region of interest analysis was performed to characterize the respiratory-induced motion of the tumor and organs at risk simultaneously. Results: The MRI navigator was found to be a more reliable surrogate for pancreatic motion than the respiratory bellows signal. Radial sampling is most benign for undersampling artifacts and intraview motion. Motion characterization revealed interorgan and interpatient variation, as well as heterogeneity within the tumor. Conclusions: A robust 4D-MRI method, based on clinically available protocols, is presented and successfully applied to characterize the abdominal motion in a small number of pancreatic cancer patients.

  3. Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system.

    PubMed

    Bénazet, Jean-Denis; Zeller, Rolf

    2009-10-01

    A wealth of classical embryological manipulation experiments taking mainly advantage of the chicken limb buds identified the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) as the respective ectodermal and mesenchymal key signaling centers coordinating proximodistal (PD) and anteroposterior (AP) limb axis development. These experiments inspired Wolpert's French flag model, which is a classic among morphogen gradient models. Subsequent molecular and genetic analysis in the mouse identified retinoic acid as proximal signal, and fibroblast growth factors (FGFs) and sonic hedgehog (SHH) as the essential instructive signals produced by AER and ZPA, respectively. Recent studies provide good evidence that progenitors are specified early with respect to their PD and AP fates and that morpho-regulatory signaling is also required for subsequent proliferative expansion of the specified progenitor pools. The determination of particular fates seems to occur rather late and depends on additional signals such as bone morphogenetic proteins (BMPs), which indicates that cells integrate signaling inputs over time and space. The coordinate regulation of PD and AP axis patterning is controlled by an epithelial-mesenchymal feedback signaling system, in which transcriptional regulation of the BMP antagonist Gremlin1 integrates inputs from the BMP, SHH, and FGF pathways. Vertebrate limb-bud development is controlled by a 4-dimensional (4D) patterning system integrating positive and negative regulatory feedback loops, rather than thresholds set by morphogen gradients.

  4. Cross-Sectional Shape of Rat Mesenteric Arterioles at Branching Studied by Confocal Laser Microscopy

    NASA Astrophysics Data System (ADS)

    Nakano, Atushi; Minamiyama, Motomu; Niimi, Hideyuki

    This study was aimed to investigate the cross-sectional shape of mesenteric arterioles at branching, using confocal laser microscopy. Wistar rats (8 weeks, male) were anesthetized with thiobutabarbital sodium. Blood flow and microvascular network in the mesentery were observed using video microscopy. The rat intestine with mesentery was extracted and the intestinal vasculature was perfused with Krebs-Ringer and then fixed with paraformaldehyde under a static pressure of 100mmHg. A section of mesentery was isolated from the intestine, and spread up to the in vivo geometry based on the intravital microscopic observation. The mesentery section was stained with tetramethyl rhodamine isothiocyanate (TRITC)-phalloidin. The samples were observed under a confocal laser microscope. The cross-sectional image was re-sliced to measure the cross-sectional area and major/minor axes of the best fitting ellipse. The aspect ratio was defined in terms of the minor/major diameter ratio. The extended focus image of mesenteric arterioles showed that the cross-sectional shape was not circular but elliptic-like. The cross-sectional area of the parent vessel decreased from proximal to distal positions. The mean aspect ratio of the parent vessel was approximately 0.5, while that of the branching vessel was approximately 0.8. The flattened shape and variation of the cross-sectional area of arterioles requires some correction of in vivo data of the two-dimensional mesenteric microvasculature obtained using intravital microscopy.

  5. Positron microscopy

    SciTech Connect

    Hulett, L.D. Jr.; Xu, J.

    1995-02-01

    The negative work function property that some materials have for positrons make possible the development of positron reemission microscopy (PRM). Because of the low energies with which the positrons are emitted, some unique applications, such as the imaging of defects, can be made. The history of the concept of PRM, and its present state of development will be reviewed. The potential of positron microprobe techniques will be discussed also.

  6. Substrate-Free Self-Assembled SiOx-Core Nanodots from Alkylalkoxysilane as a Multicolor Photoluminescence Source for Intravital Imaging

    PubMed Central

    Lin, Pei-Ying; Hsieh, Chiung-Wen; Kung, Mei-Lang; Hsieh, Shuchen

    2013-01-01

    Intravital fluorescence imaging has great potential in biological and biomedical research, as it provides the ability to directly observe biological structures and processes in their natural state. Contrast agents for intravital imaging applications should exhibit good biocompatibility, multiphoton fluorescence, and long emission. Carbon nanodots and semiconductor nanocrystals meet these requirements in most cases, with the added benefit that their properties can be ‘tuned' for specific applications by controlling the size and surface chemistry of the nanoparticles. Here, we report on a simple heat-assisted strategy to fabricate SiOx-core self-assembled nanodots using self-assembled monolayer (SAM) materials. Our results demonstrate that substrate-free self-assembled nanodots from alkylalkoxysilane exhibit controllable structural and chemical characteristics that are well suited for applications in biological, biomedical, and clinical research, and may find further use in optoelectronic and sensor devices. PMID:23609156

  7. Planning 4-Dimensional Computed Tomography (4DCT) Cannot Adequately Represent Daily Intrafractional Motion of Abdominal Tumors

    SciTech Connect

    Ge, Jiajia; Santanam, Lakshmi; Noel, Camille; Parikh, Parag J.

    2013-03-15

    Purpose: To evaluate whether planning 4-dimensional computed tomography (4DCT) can adequately represent daily motion of abdominal tumors in regularly fractionated and stereotactic body radiation therapy (SBRT) patients. Methods and Materials: Intrafractional tumor motion of 10 patients with abdominal tumors (4 pancreas-fractionated and 6 liver-stereotactic patients) with implanted fiducials was measured based on daily orthogonal fluoroscopic movies over 38 treatment fractions. The needed internal margin for at least 90% of tumor coverage was calculated based on a 95th and fifth percentile of daily 3-dimensional tumor motion. The planning internal margin was generated by fusing 4DCT motion from all phase bins. The disagreement between needed and planning internal margin was analyzed fraction by fraction in 3 motion axes (superior-inferior [SI], anterior-posterior [AP], and left-right [LR]). The 4DCT margin was considered as an overestimation/underestimation of daily motion when disagreement exceeded at least 3 mm in the SI axis and/or 1.2 mm in the AP and LR axes (4DCT image resolution). The underlying reasons for this disagreement were evaluated based on interfractional and intrafractional breathing variation. Results: The 4DCT overestimated daily 3-dimensional motion in 39% of the fractions in 7 of 10 patients and underestimated it in 53% of the fractions in 8 of 10 patients. Median underestimation was 3.9 mm, 3.0 mm, and 1.7 mm in the SI axis, AP axis, and LR axis, respectively. The 4DCT was found to capture irregular deep breaths in 3 of 10 patients, with 4DCT motion larger than mean daily amplitude by 18 to 21 mm. The breathing pattern varied from breath to breath and day to day. The intrafractional variation of amplitude was significantly larger than intrafractional variation (2.7 mm vs 1.3 mm) in the primary motion axis (ie, SI axis). The SBRT patients showed significantly larger intrafractional amplitude variation than fractionated patients (3.0 mm vs 2

  8. Management of Respiration-Induced Motion With 4-Dimensional Computed Tomography (4DCT) for Pancreas Irradiation

    SciTech Connect

    Tai, An; Liang, Zhiwen; Erickson, Beth; Li, X. Allen

    2013-08-01

    Purpose: The purposes of this study were to quantify respiration-induced organ motions for pancreatic cancer patients and to explore strategies to account for these motions. Methods and Materials: Both 3-dimensional computed tomography (3DCT) and 4-dimensional computed tomography (4DCT) scans were acquired sequentially for 15 pancreatic cancer patients, including 10 randomly selected patients and 5 patients selected from a subgroup of patients with large tumor respiratory motions. 3DCTs were fused with 2 sets of 4DCT data at the end of exhale phase (50%) and the end of inhale phase (0%). The target was delineated on the 50% and 0% phase CT sets, and the organs at risk were drawn on the 3DCT. These contours were populated to the CT sets at other respiratory phases based on deformable image registration. Internal target volumes (ITV) were generated by tracing the target contours of all phases (ITV{sub 10}), 3 phases of 0%, 20% and 50% (ITV{sub 3}), and 2 phases of 0% and 50% (ITV{sub 2}). ITVs generated from phase images were compared using percentage of volume overlap, Dice coefficient, geometric centers, and average surface distance. Results: Volume variations of pancreas, kidneys, and liver as a function of respiratory phases were small (<5%) during respiration. For the 10 randomly selected patients, peak-to-peak amplitudes of liver, left kidney, right kidney, and the target along the superior-inferior (SI) direction were 7.9 ± 3.2 mm, 7.1 ± 3.1 mm, 5.7 ± 3.2 mm, and 5.9 ± 2.8 mm, respectively. The percentage of volume overlap and Dice coefficient were 92% ± 1% and 96% ± 1% between ITV{sub 10} and ITV{sub 2} and 96% ± 1% and 98% ± 1% between ITV{sub 10} and ITV{sub 3}, respectively. The percentage of volume overlap between ITV{sub 10} and ITV{sub 3} was 93.6 ± 1.1 for patients with tumor motion >8 mm. Conclusions: Appropriate motion management strategies are proposed for radiation treatment planning of pancreatic tumors based on magnitudes of tumor

  9. Modeling Pancreatic Tumor Motion Using 4-Dimensional Computed Tomography and Surrogate Markers

    SciTech Connect

    Huguet, Florence; Yorke, Ellen D.; Davidson, Margaret; Zhang, Zhigang; Jackson, Andrew; Mageras, Gig S.; Wu, Abraham J.; Goodman, Karyn A.

    2015-03-01

    Purpose: To assess intrafractional positional variations of pancreatic tumors using 4-dimensional computed tomography (4D-CT), their impact on gross tumor volume (GTV) coverage, the reliability of biliary stent, fiducial seeds, and the real-time position management (RPM) external marker as tumor surrogates for setup of respiratory gated treatment, and to build a correlative model of tumor motion. Methods and Materials: We analyzed the respiration-correlated 4D-CT images acquired during simulation of 36 patients with either a biliary stent (n=16) or implanted fiducials (n=20) who were treated with RPM respiratory gated intensity modulated radiation therapy for locally advanced pancreatic cancer. Respiratory displacement relative to end-exhalation was measured for the GTV, the biliary stent, or fiducial seeds, and the RPM marker. The results were compared between the full respiratory cycle and the gating interval. Linear mixed model was used to assess the correlation of GTV motion with the potential surrogate markers. Results: The average ± SD GTV excursions were 0.3 ± 0.2 cm in the left-right direction, 0.6 ± 0.3 cm in the anterior-posterior direction, and 1.3 ± 0.7 cm in the superior-inferior direction. Gating around end-exhalation reduced GTV motion by 46% to 60%. D95% was at least the prescribed 56 Gy in 76% of patients. GTV displacement was associated with the RPM marker, the biliary stent, and the fiducial seeds. The correlation was better with fiducial seeds and with biliary stent. Conclusions: Respiratory gating reduced the margin necessary for radiation therapy for pancreatic tumors. GTV motion was well correlated with biliary stent or fiducial seed displacements, validating their use as surrogates for daily assessment of GTV position during treatment. A patient-specific internal target volume based on 4D-CT is recommended both for gated and not-gated treatment; otherwise, our model can be used to predict the degree of GTV motion.

  10. High-Quality T2-Weighted 4-Dimensional Magnetic Resonance Imaging for Radiation Therapy Applications

    SciTech Connect

    Du, Dongsu; Caruthers, Shelton D.; Glide-Hurst, Carri; Low, Daniel A.; Li, H. Harold; Mutic, Sasa; Hu, Yanle

    2015-06-01

    Purpose: The purpose of this study was to improve triggering efficiency of the prospective respiratory amplitude-triggered 4-dimensional magnetic resonance imaging (4DMRI) method and to develop a 4DMRI imaging protocol that could offer T2 weighting for better tumor visualization, good spatial coverage and spatial resolution, and respiratory motion sampling within a reasonable amount of time for radiation therapy applications. Methods and Materials: The respiratory state splitting (RSS) and multi-shot acquisition (MSA) methods were analytically compared and validated in a simulation study by using the respiratory signals from 10 healthy human subjects. The RSS method was more effective in improving triggering efficiency. It was implemented in prospective respiratory amplitude-triggered 4DMRI. 4DMRI image datasets were acquired from 5 healthy human subjects. Liver motion was estimated using the acquired 4DMRI image datasets. Results: The simulation study showed the RSS method was more effective for improving triggering efficiency than the MSA method. The average reductions in 4DMRI acquisition times were 36% and 10% for the RSS and MSA methods, respectively. The human subject study showed that T2-weighted 4DMRI with 10 respiratory states, 60 slices at a spatial resolution of 1.5 × 1.5 × 3.0 mm{sup 3} could be acquired in 9 to 18 minutes, depending on the individual's breath pattern. Based on the acquired 4DMRI image datasets, the ranges of peak-to-peak liver displacements among 5 human subjects were 9.0 to 12.9 mm, 2.5 to 3.9 mm, and 0.5 to 2.3 mm in superior-inferior, anterior-posterior, and left-right directions, respectively. Conclusions: We demonstrated that with the RSS method, it was feasible to acquire high-quality T2-weighted 4DMRI within a reasonable amount of time for radiation therapy applications.

  11. SU-E-T-267: Proton Pencil Beam Scanning for Mediastinal Lymphoma: 4-Dimensional Feasibility Study

    SciTech Connect

    Zeng, C; Plastaras, J; Tochner, Z; Hill-Kayser, C; Hahn, S; Both, S

    2014-06-01

    Purpose: To assess the feasibility of proton pencil beam scanning (PBS) for the treatment of mediastinal lymphoma. Methods: A group of 6 patients were planned using an anterior field with PBS. Spots with ∼5 mm σ were used for all patients, while large spots (∼10 mm σ) were employed for patients with motion perpendicular to the beam (≥5 mm). We considered volumetric repainting such that, in each fraction, the same field would be delivered twice. Four-dimensional dose was calculated on initial and verification 4-dimensional computed tomography (4D-CT) scans (2—3) based on respiratory trace and beam delivery sequence. This was implemented by binning the spots into separate plans on each 4D-CT phase respectively. Four starting phases were sampled for each painting and 4 energy switching times (0.5 s, 1 s, 3 s, and 5 s) were tested, resulting in 2560 dose distributions for the cohort. Plan robustness was measured for target and critical structures in terms of the percentage difference between delivered dose and planned dose. Results: For 5 of the 6 patients, the ITV (internal target volume) D98% was degraded by <3% (standard deviations ∼ 0.1%) when averaged over the whole course (up to 5% per fraction). Deviations of mean lung dose, heart maximum dose, and cord maximum dose were within 5% of prescribed dose. For one patient with motion perpendicular to the beam (up to 5 mm), the degradation of ITV D98% was 9% over the whole course (12% per fraction), which was mitigated to 1% (3% per fraction) by employing large spots and repainting. No significant difference in coverage was observed for different energy switching times. Conclusion: This feasibility study demonstrates that, for mediastinal lymphoma, the PBS plan robustness can be maintained during delivery when target motion is measured and volumetric repainting and/or large spots are employed. This work was supported by Ion Beam Application.

  12. The 4-Dimensional Plant: Effects of Wind-Induced Canopy Movement on Light Fluctuations and Photosynthesis

    PubMed Central

    Burgess, Alexandra J.; Retkute, Renata; Preston, Simon P.; Jensen, Oliver E.; Pound, Michael P.; Pridmore, Tony P.; Murchie, Erik H.

    2016-01-01

    accurate modeling of mechanical canopy excitation (here coined the 4-dimensional plant) and some associated biological and applied implications of such techniques. We hypothesize that biomechanical plant properties are a specific adaptation to achieve wind-induced photosynthetic enhancement and we outline how traits facilitating canopy excitation could be used as a route for improving crop yield. PMID:27708654

  13. The 4-Dimensional Plant: Effects of Wind-Induced Canopy Movement on Light Fluctuations and Photosynthesis.

    PubMed

    Burgess, Alexandra J; Retkute, Renata; Preston, Simon P; Jensen, Oliver E; Pound, Michael P; Pridmore, Tony P; Murchie, Erik H

    2016-01-01

    accurate modeling of mechanical canopy excitation (here coined the 4-dimensional plant) and some associated biological and applied implications of such techniques. We hypothesize that biomechanical plant properties are a specific adaptation to achieve wind-induced photosynthetic enhancement and we outline how traits facilitating canopy excitation could be used as a route for improving crop yield.

  14. Clinical Validation of 4-Dimensional Computed Tomography Ventilation With Pulmonary Function Test Data

    SciTech Connect

    Brennan, Douglas; Schubert, Leah; Diot, Quentin; Castillo, Richard; Castillo, Edward; Guerrero, Thomas; Martel, Mary K.; Linderman, Derek; Gaspar, Laurie E.; Miften, Moyed; Kavanagh, Brian D.; Vinogradskiy, Yevgeniy

    2015-06-01

    Purpose: A new form of functional imaging has been proposed in the form of 4-dimensional computed tomography (4DCT) ventilation. Because 4DCTs are acquired as part of routine care for lung cancer patients, calculating ventilation maps from 4DCTs provides spatial lung function information without added dosimetric or monetary cost to the patient. Before 4DCT-ventilation is implemented it needs to be clinically validated. Pulmonary function tests (PFTs) provide a clinically established way of evaluating lung function. The purpose of our work was to perform a clinical validation by comparing 4DCT-ventilation metrics with PFT data. Methods and Materials: Ninety-eight lung cancer patients with pretreatment 4DCT and PFT data were included in the study. Pulmonary function test metrics used to diagnose obstructive lung disease were recorded: forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity. Four-dimensional CT data sets and spatial registration were used to compute 4DCT-ventilation images using a density change–based and a Jacobian-based model. The ventilation maps were reduced to single metrics intended to reflect the degree of ventilation obstruction. Specifically, we computed the coefficient of variation (SD/mean), ventilation V20 (volume of lung ≤20% ventilation), and correlated the ventilation metrics with PFT data. Regression analysis was used to determine whether 4DCT ventilation data could predict for normal versus abnormal lung function using PFT thresholds. Results: Correlation coefficients comparing 4DCT-ventilation with PFT data ranged from 0.63 to 0.72, with the best agreement between FEV1 and coefficient of variation. Four-dimensional CT ventilation metrics were able to significantly delineate between clinically normal versus abnormal PFT results. Conclusions: Validation of 4DCT ventilation with clinically relevant metrics is essential. We demonstrate good global agreement between PFTs and 4DCT-ventilation, indicating that 4DCT

  15. Visualizing the podocyte with multiphoton microscopy

    PubMed Central

    Khoury, Charbel C.; Khayat, Mark F.; Yeo, Tet-Kin; Pyagay, Petr E.; Wang, Amy; Asuncion, Allan M.; Sharma, Kumar; Yu, Weiming; Chen, Sheldon

    2012-01-01

    The podocyte is a highly specialized kidney glomerular epithelial cell that plays an essential role in glomerular filtration and is believed to be the target of numerous glomerular diseases leading to proteinuria. Despite the leaps in our understanding of podocyte biology, new methodologies are needed to facilitate research into the cell. Multiphoton microscopy (MPM) was used to image the nephrin knockout/green fluorescent protein (GFP) knock-in heterozygote (Nphs1tm1Rkl/J) mouse. The nephrin promoter restricts GFP expression to the podocytes that fluoresce green under excitation. From the exterior of an intact kidney, MPM can peer into the renal parenchyma and visualize the podocytes that outline the globular shape of the glomeruli. Details as fine as the podocyte’s secondary processes can be resolved. In contrast, podocytes exhibit no fluorescence in the wildtype mouse and are invisible to MPM. Phenotypically, there are no significant differences between wildtype and Nphs1tm1Rkl/J mice in body weight, urinary albumin excretion, creatinine clearance, or glomerular depth. Interestingly, the glomeruli are closer to the kidney capsule in female mice, making the gender the preferred choice for MPM. For the first time, green fluorescent podocytes in a mouse model free of confounding phenotypes can be visualized unequivocally and in the “positive” by MPM, facilitating intravital studies of the podocyte. PMID:23022193

  16. New techniques for motion-artifact-free in vivo cardiac microscopy

    PubMed Central

    Vinegoni, Claudio; Lee, Sungon; Aguirre, Aaron D.; Weissleder, Ralph

    2015-01-01

    Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases. PMID:26029116

  17. Intravital imaging of mouse colonic adenoma using MMP-based molecular probes with multi-channel fluorescence endoscopy

    PubMed Central

    Oh, Gyungseok; Yoo, Su Woong; Jung, Yebin; Ryu, Yeon-Mi; Park, Youngrong; Kim, Sang-Yeob; Kim, Ki Hean; Kim, Sungjee; Myung, Seung-Jae; Chung, Euiheon

    2014-01-01

    Intravital imaging has provided molecular, cellular and anatomical insight into the study of tumor. Early detection and treatment of gastrointestinal (GI) diseases can be enhanced with specific molecular markers and endoscopic imaging modalities. We present a wide-field multi-channel fluorescence endoscope to screen GI tract for colon cancer using multiple molecular probes targeting matrix metalloproteinases (MMP) conjugated with quantum dots (QD) in AOM/DSS mouse model. MMP9 and MMP14 antibody (Ab)-QD conjugates demonstrate specific binding to colonic adenoma. The average target-to-background (T/B) ratios are 2.10 ± 0.28 and 1.78 ± 0.18 for MMP14 Ab-QD and MMP9 Ab-QD, respectively. The overlap between the two molecular probes is 67.7 ± 8.4%. The presence of false negative indicates that even more number of targeting could increase the sensitivity of overall detection given heterogeneous molecular expression in tumors. Our approach indicates potential for the screening of small or flat lesions that are precancerous. PMID:24877024

  18. Intravital imaging of mouse colonic adenoma using MMP-based molecular probes with multi-channel fluorescence endoscopy.

    PubMed

    Oh, Gyungseok; Yoo, Su Woong; Jung, Yebin; Ryu, Yeon-Mi; Park, Youngrong; Kim, Sang-Yeob; Kim, Ki Hean; Kim, Sungjee; Myung, Seung-Jae; Chung, Euiheon

    2014-05-01

    Intravital imaging has provided molecular, cellular and anatomical insight into the study of tumor. Early detection and treatment of gastrointestinal (GI) diseases can be enhanced with specific molecular markers and endoscopic imaging modalities. We present a wide-field multi-channel fluorescence endoscope to screen GI tract for colon cancer using multiple molecular probes targeting matrix metalloproteinases (MMP) conjugated with quantum dots (QD) in AOM/DSS mouse model. MMP9 and MMP14 antibody (Ab)-QD conjugates demonstrate specific binding to colonic adenoma. The average target-to-background (T/B) ratios are 2.10 ± 0.28 and 1.78 ± 0.18 for MMP14 Ab-QD and MMP9 Ab-QD, respectively. The overlap between the two molecular probes is 67.7 ± 8.4%. The presence of false negative indicates that even more number of targeting could increase the sensitivity of overall detection given heterogeneous molecular expression in tumors. Our approach indicates potential for the screening of small or flat lesions that are precancerous.

  19. Microcirculatory effects of a homeopathic preparation in patients with mild vertigo: an intravital microscopic study.

    PubMed

    Klopp, R; Niemer, W; Weiser, M

    2005-01-01

    The effects of the homeopathic preparation Vertigoheel on variables related to microcirculation were investigated using vital microscopy techniques in patients with vestibular vertigo. In a non-randomized, open study, 16 patients given Vertigoheel were compared with 16 untreated patients. Measurements were carried out in two areas (defined by selecting 60 blood-cell perfused nodal points of arterioles, venules, and capillaries with a mean diameter > or = 40 microm): the cuticulum/subcuticulum of the inside left lower arm and an area 5 mm behind the left earlobe. After 12 weeks of treatment, patients receiving the homeopathic preparation exhibited an increased number of nodal points, increased flow rates of erythrocytes in both arterioles and venules, increased vasomotion, and a slight reduction in hematocrit vs. baseline. None of these changes were observed in the control group and the differences between treatment groups were statistically significant. Partial oxygen pressure increased significantly in the Vertigoheel group compared with the control group. In addition, in Vertigoheel patients, significantly increased numbers of cell-wall adhering leucocytes were observed, accompanied by increased local concentrations of the adhesion molecules ICAM-1. The microcirculatory changes were associated with a reduction in the severity of vertigo in the actively treated patients, both as assessed by the treating physician and by the patients themselves. The data support a pharmacological effect on microcirculation from the treatment.

  20. Noninvasive intravital cellular diagnosis of atopic dermatitis by using harmonic optical virtual biopsy

    NASA Astrophysics Data System (ADS)

    Chen, Szu-Yu; Lee, Jyh-Hong; Chiang, Bor-Luen; Sun, Chi-Kuang

    2007-02-01

    Atopic dermatitis (AD) is now very common in people who live in cities, especially for babies and children. Since the cause of AD is still not completely understood and each person may have his own mixed symptoms that can change over time, diagnosis of AD can not be done precisely. Unlike some skin diseases, physical biopsy is rarely used in diagnosing AD on account of its low urgency. Thus, only indirect diagnoses, like asking for a medical history to learn about the symptoms and to rule out other diseases can be carried out. To gain insight into cellular details of AD for long-term diagnosing without physical biopsy, a noninvasive in vivo tool with a sub-micron subsurface resolution and high penetrability has to be used. In this presentation, we show that harmonic optical virtual biopsy can provide the required noninvasive cellular imaging, and is ideal for future clinical diagnosis of AD. Harmonic optical microscopy has been demonstrated to have the capability to reveal cellular morphology of human skin from epidermis to dermis layer. Third harmonic generation (THG), which is sensitive to inhomogeneous interfaces, can show the structures of skins, and can be used to reveal the morphological changes, for example, the thicken cuticle which is a common symptom of AD. Second harmonic generation (SHG), which occurs in non-centrosymmetric structures, has excellent contrast in collagen fibers and can show the pathological changes of dermis layer. Utilizing both THG and SHG, useful information may be given to facilitate the diagnosis of AD.

  1. Intravital Confocal and Two-photon Imaging of Dual-color Cells and Extracellular Matrix Mimics

    PubMed Central

    Bal, Ufuk; Andresen, Volker; Baggett, Brenda; Utzinger, Urs

    2013-01-01

    To optimize imaging of cells in three dimensional culture we studied confocal backscattering, Second Harmonic Generation (SHG) and autofluorescence as source of contrast in extracellular matrix (ECM) mimics and evaluated the attenuation as well as bleaching of endogenous cellular fluorescence signals. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence while still providing good reflectance to detect voids in the embedding medium. We labeled breast cancer cells’ outline with DsRed2 and nucleus with eGFP. DsRed2 can be excited with confocal imaging at 568nm, and with two photon excitation (TPE) in the red and longer NIR. eGFP was excited at 488nm for confocal and in the NIR for TPE. While there is small difference in the bleaching rate for eGFP between confocal and TPE we observed significant difference for DsRed2 where bleaching is strongest during TPE in the red wavelengths and smallest during confocal imaging. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence becomes twice as strong compared to confocal imaging. PMID:23380006

  2. Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging

    NASA Astrophysics Data System (ADS)

    Reitan, Nina Kristine; Thuen, Marte; Goa, Pa˚L. Erik; de Lange Davies, Catharina

    2010-05-01

    Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were used as molecular tracers for CLSM and DCE-MRI, respectively. A significant correlation was found between permeability indicators. The extravasation rate Ki as measured by CLSM correlated positively with DCE-MRI parameters, such as the volume transfer constant Ktrans and the initial slope of the contrast agent concentration-time curve. This demonstrates that these two techniques give complementary information. Extravasation was further related to microvascular structure and was found to correlate with the fractal dimension and vascular density. The structural parameter values that were obtained from CLSM images were higher for abnormal tumor vasculature than for normal vessels.

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

    PubMed Central

    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

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

    PubMed

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

    2017-03-09

    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.

  5. Defining the quantitative limits of intravital two-photon lymphocyte tracking

    PubMed Central

    Textor, Johannes; Peixoto, Antonio; Henrickson, Sarah E.; Sinn, Mathieu; von Andrian, Ulrich H.; Westermann, Jürgen

    2011-01-01

    Two-photon microscopy has substantially advanced our understanding of cellular dynamics in the immune system. Cell migration can now be imaged in real time in the living animal. Strikingly, the migration of naive lymphocytes in secondary lymphoid tissue appears predominantly random. It is unclear, however, whether directed migration may escape detection in this random background. Using a combination of mathematical modeling and experimental data, we investigate the extent to which modern two-photon imaging can rule out biologically relevant directed migration. For naive T cells migrating in uninfected lymph nodes (LNs) at average 3D speeds of around 18 μm/min, we rule out uniform directed migration of more than 1.7 μm/min at the 95% confidence level, confirming that T cell migration is indeed mostly random on a timescale of minutes. To investigate whether this finding still holds for longer timescales, we use a 3D simulation of the naive T cell LN transit. A pure random walk predicts a transit time of around 16 h, which is in good agreement with experimental results. A directional bias of only 0.5 μm/min—less than 3% of the cell speed—would already accelerate the transit twofold. These results jointly strengthen the random walk analogy for naive T cell migration in LNs, but they also emphasize that very small deviations from random migration can still be important. Our methods are applicable to cells of any type and can be used to reanalyze existing datasets. PMID:21734152

  6. Spatiotemporal Analyses of Osteogenesis and Angiogenesis via Intravital Imaging in Cranial Bone Defect Repair

    PubMed Central

    Huang, Chunlan; Ness, Vincent P.; Yang, Xiaochuan; Chen, Hongli; Luo, Jiebo; Brown, Edward B; Zhang, Xinping

    2015-01-01

    Osteogenesis and angiogenesis are two integrated components in bone repair and regeneration. A deeper understanding of osteogenesis and angiogenesis has been hampered by technical difficulties of analyzing bone and neovasculature simultaneously in spatiotemporal scales and in three-dimensional formats. To overcome these barriers, a cranial defect window chamber model was established that enabled high-resolution, longitudinal, and real-time tracking of angiogenesis and bone defect healing via Multiphoton Laser Scanning Microscopy (MPLSM). By simultaneously probing new bone matrix via second harmonic generation (SHG), neovascular networks via intravenous perfusion of fluorophore, and osteoblast differentiation via 2.3kb collagen type I promoter driven GFP (Col2.3GFP), we examined the morphogenetic sequence of cranial bone defect healing and further established the spatiotemporal analyses of osteogenesis and angiogenesis coupling in repair and regeneration. We demonstrated that bone defect closure was initiated in the residual bone around the edge of the defect. The expansion and migration of osteoprogenitors into the bone defect occurred during the first 3 weeks of healing, coupled with vigorous microvessel angiogenesis at the leading edge of the defect. Subsequent bone repair was marked by matrix deposition and active vascular network remodeling within new bone. Implantation of bone marrow stromal cells (BMSCs) isolated from Col2.3GFP mice further showed that donor-dependent bone formation occurred rapidly within the first 3 weeks of implantation, in concert with early angiogenesis. The subsequent bone wound closure was largely host-dependent, associated with localized modest induction of angiogenesis. The establishment of a live imaging platform via cranial window provides a unique tool to understand osteogenesis and angiogenesis in repair and regeneration, enabling further elucidation of the spatiotemporal regulatory mechanisms of osteoprogenitor cell interactions

  7. Intravital Förster resonance energy transfer imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli.

    PubMed

    Kamioka, Yuji; Takakura, Kanako; Sumiyama, Kenta; Matsuda, Michiyuki

    2017-02-01

    Myeloid-derived suppressor cells (MDSCs) cause paraneoplastic leukemoid reactions and facilitate tumor cell metastasis. However, the interaction of MDSCs with tumor cells in live tissue has not been adequately visualized. To accomplish this task, we developed an intravital imaging protocol to observe metastasized tumor cells in mouse lungs. For visualization of the activation of MDSCs, bone marrow cells derived from transgenic mice expressing a Förster resonance energy transfer biosensor for ERK were implanted into host mice. Under a two-photon excitation microscope, numerous polymorphonuclear cells (PMNs) were found to infiltrate the lungs of tumor-bearing mice in which 4T1 mammary tumor cells were implanted into the footpads. By Förster resonance energy transfer imaging, we found ERK activation in PMNs around the 4T1 tumor emboli in the lungs. Because antibody array analysis implied the involvement of osteopontin (OPN) in the metastasis of 4T1 cells, we further analyzed the effect of OPN knockdown. The OPN knockdown in 4T1 cells did not affect the cell growth, but markedly suppressed lung metastasis of 4T1 cells and ERK activation in PMNs in the lung. Intravenous injection of recombinant OPN restored the lung metastasis of OPN-deficient 4T1 cells, suggesting that OPN functioned in a paracrine manner. It has been reported that ERK activation of neutrophils causes NETosis and that PMNs promote metastasis of tumor cells by NETosis. In agreement with previous reports, the NETosis inhibitor DNase I inhibited lung metastasis of 4T1 cells. These observations suggest that OPN promotes metastasis of 4T1 cells by activating PMNs and inducing NETosis.

  8. In vivo imaging of the tumor and its associated microenvironment using combined CARS / 2-photon microscopy

    PubMed Central

    Lee, Martin; Downes, Andy; Chau, You-Ying; Serrels, Bryan; Hastie, Nick; Elfick, Alistair; Brunton, Valerie; Frame, Margaret; Serrels, Alan

    2015-01-01

    The use of confocal and multi-photon microscopy for intra-vital cancer imaging has impacted on our understanding of cancer cell behavior and interaction with the surrounding tumor microenvironment in vivo. However, many studies to-date rely on the use fluorescent dyes or genetically encoded probes that enable visualization of a structure or cell population of interest, but do not illuminate the complexity of the surrounding tumor microenvironment. Here, we show that multi-modal microscopy combining 2-photon fluorescence with CARS can begin to address this deficit, enabling detailed imaging of the tumor niche without the need for additional labeling. This can be performed on live tumor-bearing animals through optical observation windows, permitting real-time and longitudinal imaging of dynamic processes within the tumor niche. PMID:28243514

  9. All ASD complex and real 4-dimensional Einstein spaces with Λ≠0 admitting a nonnull Killing vector

    NASA Astrophysics Data System (ADS)

    Chudecki, Adam

    2016-12-01

    Anti-self-dual (ASD) 4-dimensional complex Einstein spaces with nonzero cosmological constant Λ equipped with a nonnull Killing vector are considered. It is shown that any conformally nonflat metric of such spaces can be always brought to a special form and the Einstein field equations can be reduced to the Boyer-Finley-Plebański equation (Toda field equation). Some alternative forms of the metric are discussed. All possible real slices (neutral, Euclidean and Lorentzian) of ASD complex Einstein spaces with Λ≠0 admitting a nonnull Killing vector are found.

  10. Intravital imaging of Ca2+ signals in lymphocytes of Ca2+ biosensor transgenic mice: indication of autoimmune diseases before the pathological onset

    PubMed Central

    Yoshikawa, Soichiro; Usami, Takako; Kikuta, Junichi; Ishii, Masaru; Sasano, Tetsuo; Sugiyama, Koji; Furukawa, Tetsushi; Nakasho, Eiji; Takayanagi, Hiroshi; Tedder, Thomas F.; Karasuyama, Hajime; Miyawaki, Atsushi; Adachi, Takahiro

    2016-01-01

    Calcium ion (Ca2+) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca2+ indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60flox). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca2+) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22−/− and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca2+ fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca2+ signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals. PMID:26732477

  11. Improved estimates of the dynamical state of the North Pacific Ocean from a 4 dimensional variational data assimilation

    NASA Astrophysics Data System (ADS)

    Masuda, Shuhei; Awaji, Toshiyuki; Sugiura, Nozomi; Ishikawa, Yoichi; Baba, Kinji; Horiuchi, Kazutoshi; Komori, Nobumasa

    2003-08-01

    A 4-dimensional variational data assimilation system has been used to better define the mean seasonal state of the North Pacific. The synthesis of available observational records and a sophisticated general circulation model produces a dynamically consistent time-varying dataset which exhibits realistic features of the global ocean circulation and requires no artificial sources or sinks for the temperature and salinity fields. The dataset enables us to clarify the water mass formation and movement processes. A sensitivity experiment using our system reveals that the origin of the North Pacific Intermediate Water can be traced back to the Okhotsk and Bering Seas in the subarctic region and to the subtropical Kuroshio region further south, consistent with recent observational findings. This result illustrates that the ocean state derived from our data assimilation has greater information and forecast potential than that obtained from earlier methods.

  12. Utilization of 4-Dimensional Data Visualization Modeling to Evaluate Burial Ground Contaminants at the Paducah Gaseous Diffusion Plant, Paducah, Kentucky

    SciTech Connect

    Brindley, T. L.; Tarantino, J. J.; Locke, A. L.; Dollins, D. W.

    2006-07-01

    This paper describes how 4-Dimensional (4D) Data Visualization Modeling was used to evaluate historical data and to help guide the decisions for the sampling necessary to complete a Remedial Investigation/Feasibility Study (RI/FS) for the burial ground sites at the Department of Energy (DOE) Paducah Gaseous Diffusion Plant (PGDP). DOE at the Paducah Site is primarily involved in environmental cleanup and landlord activities. The scope of this project was to prepare a work plan for identifying the data available and the data required to conduct an RI/FS for the Burial Ground Operable Unit (BGOU) located within and near PGDP. The work plan focuses on collecting existing information about contamination in and around the burial grounds and determining what additional data are required to support an assessment of risks to human health and the environment and to support future decisions regarding actions to reduce these risks. (authors)

  13. Investigating the effects of Panadol on mouse liver by in vivo multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Li, Feng Chieh; Liang, Huei, Jr.; Yang, Shu-Mei; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2008-02-01

    Conventional hepatic research relies heavily on histological images for obtaining morphological information of the liver. However, static histological images can not provide real-time dynamic information of in vivo physiological processes such as cellular motion or damage. For a long time, panadol has been used in pain relief. However, Panadol may have unwanted side effects and detailed information of the effects of Panadol on hepatic metabolism is unknown. In this work, we developed a high resolution intravital hepatic imaging chamber to study the effects of Panadol on liver. We expect this methodology to be useful in revealing the detailed metabolism of liver after using Panadol and this approach allows us to achieve a better understanding of hepatic processes. In our approach, we use multiphoton fluorescence (MPF) microscopy to observe the side effect of liver on using Panadol inside the in vivo mouse animal model.

  14. Longitudinal Intravital Imaging of the Retina Reveals Long-term Dynamics of Immune Infiltration and Its Effects on the Glial Network in Experimental Autoimmune Uveoretinitis, without Evident Signs of Neuronal Dysfunction in the Ganglion Cell Layer

    PubMed Central

    Bremer, Daniel; Pache, Florence; Günther, Robert; Hornow, Jürgen; Andresen, Volker; Leben, Ruth; Mothes, Ronja; Zimmermann, Hanna; Brandt, Alexander U.; Paul, Friedemann; Hauser, Anja E.; Radbruch, Helena; Niesner, Raluca

    2016-01-01

    A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4+ T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4+ T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile CX3CR1+ cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent

  15. Dictionary of Microscopy

    NASA Astrophysics Data System (ADS)

    Heath, Julian

    2005-10-01

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

  16. Cell-based and in vivo spectral analysis of fluorescent proteins for multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Salomonnson, Emma; Mihalko, Laura Anne; Verkhusha, Vladislav V.; Luker, Kathryn E.; Luker, Gary D.

    2012-09-01

    Multiphoton microscopy of cells and subcellular structures labeled with fluorescent proteins is the state-of-the-art technology for longitudinal imaging studies in tissues and living animals. Successful analysis of separate cell populations or signaling events by intravital microscopy requires optimal pairing of multiphoton excitation wavelengths with spectrally distinct fluorescent proteins. While prior studies have analyzed two photon absorption properties of isolated fluorescent proteins, there is limited information about two photon excitation and fluorescence emission profiles of fluorescent proteins expressed in living cells and intact tissues. Multiphoton microscopy was used to analyze fluorescence outputs of multiple blue, green, and red fluorescent proteins in cultured cells and orthotopic tumor xenografts of human breast cancer cells. It is shown that commonly used orange and red fluorescent proteins are excited efficiently by 750 to 760 nm laser light in living cells, enabling dual color imaging studies with blue or cyan proteins without changing excitation wavelength. It is also shown that small incremental changes in excitation wavelength significantly affect emission intensities from fluorescent proteins, which can be used to optimize multi-color imaging using a single laser wavelength. These data will direct optimal selection of fluorescent proteins for multispectral two photon microscopy.

  17. Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

    PubMed

    Farnum, C E; Turgai, J; Wilsman, N J

    1990-09-01

    The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

  18. Comparative Assessment of Liver Tumor Motion Using Cine–Magnetic Resonance Imaging Versus 4-Dimensional Computed Tomography

    SciTech Connect

    Fernandes, Annemarie T.; Apisarnthanarax, Smith; Yin, Lingshu; Zou, Wei; Rosen, Mark; Plastaras, John P.; Ben-Josef, Edgar; Metz, James M.; Teo, Boon-Keng

    2015-04-01

    Purpose: To compare the extent of tumor motion between 4-dimensional CT (4DCT) and cine-MRI in patients with hepatic tumors treated with radiation therapy. Methods and Materials: Patients with liver tumors who underwent 4DCT and 2-dimensional biplanar cine-MRI scans during simulation were retrospectively reviewed to determine the extent of target motion in the superior–inferior, anterior–posterior, and lateral directions. Cine-MRI was performed over 5 minutes. Tumor motion from MRI was determined by tracking the centroid of the gross tumor volume using deformable image registration. Motion estimates from 4DCT were performed by evaluation of the fiducial, residual contrast (or liver contour) positions in each CT phase. Results: Sixteen patients with hepatocellular carcinoma (n=11), cholangiocarcinoma (n=3), and liver metastasis (n=2) were reviewed. Cine-MRI motion was larger than 4DCT for the superior–inferior direction in 50% of patients by a median of 3.0 mm (range, 1.5-7 mm), the anterior–posterior direction in 44% of patients by a median of 2.5 mm (range, 1-5.5 mm), and laterally in 63% of patients by a median of 1.1 mm (range, 0.2-4.5 mm). Conclusions: Cine-MRI frequently detects larger differences in hepatic intrafraction tumor motion when compared with 4DCT most notably in the superior–inferior direction, and may be useful when assessing the need for or treating without respiratory management, particularly in patients with unreliable 4DCT imaging. Margins wider than the internal target volume as defined by 4DCT were required to encompass nearly all the motion detected by cine-MRI for some of the patients in this study.

  19. Determination of Internal Target Volume for Radiation Treatment Planning of Esophageal Cancer by Using 4-Dimensional Computed Tomography (4DCT)

    SciTech Connect

    Chen, Xiaojian; Lu, Haijun; Tai, An; Johnstone, Candice; Gore, Elizabeth; Li, X. Allen

    2014-09-01

    Purpose: To determine an efficient strategy for the generation of the internal target volume (ITV) for radiation treatment planning for esophageal cancer using 4-dimensional computed tomography (4DCT). Methods and Materials: 4DCT sets acquired for 20 patients with esophageal carcinoma were analyzed. Each of the 4DCT sets was binned into 10 respiratory phases. For each patient, the gross tumor volume (GTV) was delineated on the 4DCT set at each phase. Various strategies to derive ITV were explored, including the volume from the maximum intensity projection (MIP; ITV{sub M}IP), unions of the GTVs from selected multiple phases ITV2 (0% and 50% phases), ITV3 (ITV2 plus 80%), and ITV4 (ITV3 plus 60%), as well as the volumes expanded from ITV2 and ITV3 with a uniform margin. These ITVs were compared to ITV10 (the union of the GTVs for all 10 phases) and the differences were measured with the overlap ratio (OR) and relative volume ratio (RVR) relative to ITV10 (ITVx/ITV10). Results: For all patients studied, the average GTV from a single phase was 84.9% of ITV10. The average ORs were 91.2%, 91.3%, 94.5%, and 96.4% for ITV{sub M}IP, ITV2, ITV3, and ITV4, respectively. Low ORs were associated with irregular breathing patterns. ITV3s plus 1 mm uniform margins (ITV3+1) led to an average OR of 98.1% and an average RVR of 106.4%. Conclusions: The ITV generated directly from MIP underestimates the range of the respiration motion for esophageal cancer. The ITV generated from 3 phases (ITV3) may be used for regular breathers, whereas the ITV generated from 4 phases (ITV4) or ITV3 plus a 1-mm uniform margin may be applied for irregular breathers.

  20. On Darboux's approach to R-separability of variables. Classification of conformally flat 4-dimensional binary metrics

    NASA Astrophysics Data System (ADS)

    Szereszewski, A.; Sym, A.

    2015-09-01

    are binary metrics. In this paper we solve the following problem: to classify all conformally flat (of arbitrary signature) 4-dimensional binary metrics. Among them there are 1) those that are separable in the sense of SRE metrics Kalnins-Miller (1978 Trans. Am. Math. Soc. 244 241-61 1982 J. Phys. A: Math. Gen. 15 2699-709 1984 Adv. Math. 51 91-106 1983 SIAM J. Math. Anal. 14 126-37) and 2) new examples of non-Stäckel R-separability in 4 dimensions.

  1. Axial Plane Optical Microscopy

    PubMed Central

    Li, Tongcang; Ota, Sadao; Kim, Jeongmin; Wong, Zi Jing; Wang, Yuan; Yin, Xiaobo; Zhang, Xiang

    2014-01-01

    We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues. PMID:25434770

  2. Light sheet microscopy.

    PubMed

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-01-01

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

  3. Label-Free Determination of Hemodynamic Parameters in the Microcirculaton with Third Harmonic Generation Microscopy

    PubMed Central

    Dietzel, Steffen; Pircher, Joachim; Nekolla, A. Katharina; Gull, Mazhar; Brändli, André W.; Pohl, Ulrich; Rehberg, Markus

    2014-01-01

    Determination of blood flow velocity and related hemodynamic parameters is an important aspect of physiological studies which in many settings requires fluorescent labeling. Here we show that Third Harmonic Generation (THG) microscopy is a suitable tool for label-free intravital investigations of the microcirculation in widely-used physiological model systems. THG microscopy is a non-fluorescent multi-photon scanning technique combining the advantages of label-free imaging with restriction of signal generation to a focal spot. Blood flow was visualized and its velocity was measured in adult mouse cremaster muscle vessels, non-invasively in mouse ear vessels and in Xenopus tadpoles. In arterioles, THG line scanning allowed determination of the flow pulse velocity curve and hence the heart rate. By relocating the scan line we obtained velocity profiles through vessel diameters, allowing shear rate calculations. The cell free layer containing the glycocalyx was also visualized. Comparison of the current microscopic resolution with theoretical, diffraction limited resolution let us conclude that an about sixty-fold THG signal intensity increase may be possible with future improved optics, optimized for 1200–1300 nm excitation. THG microscopy is compatible with simultaneous two-photon excited fluorescence detection. It thus also provides the opportunity to determine important hemodynamic parameters in parallel to common fluorescent observations without additional label. PMID:24933027

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

    PubMed Central

    Bertlich, Mattis; Weiss, Bernhard; Dietzel, Steffen; Canis, Martin

    2015-01-01

    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. PMID:25883941

  5. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

    PubMed Central

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe

    2016-01-01

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  6. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies.

    PubMed

    Grandjean, Capucine L; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A; Bousso, Philippe

    2016-10-04

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

  7. Presence of soot in the respiratory tract and esophagus as an element of consultative process addressing intravital staying in fire atmosphere.

    PubMed

    Raczkowska, Zuzanna; Borowska-Solonynko, Aleksandra; Samojłowicz, Dorota

    2012-01-01

    This paper was prepared in view of consultative difficulties which occur during autopsies of bodies found at the seat of fire. The most difficult problem in such cases is establishing--in absence of typical signs of intravitality--whether the deceased was alive during the fire and inhaling the fire atmosphere; especially when the concentration of carboxyhemoglobin (COHb) in blood is higher than 10% and soot is found in the respiratory tract. The authors analyzed 241 reports of autopsies which had been performed at Institute of Forensic Medicine, Warsaw Medical University, between 2006 and 2011. The following data were analyzed: age, gender, the place where the body was found, blood concentration of COHb and alcohol and the presence of soot in the upper and lower respiratory tract, as well as in the esophagus. It was noted that if the concentration of COHb was higher, soot was more frequently present in the respiratory tract and esophagus. At the same time, the presence, as well as absence of soot was noted regardless of COHb concentration in blood, including 0% concentration. In cases of performing autopsies on bodies found at the seat of fire, examining the upper and down respiratory tract seems to be irrelevant in terms of its consultative usefulness; however, the presence of soot in the esophagus concomitant with low COHb concentration in blood is important in this context.

  8. Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin.

    PubMed

    Masedunskas, Andrius; Chen, Yun; Stussman, Rebecca; Weigert, Roberto; Mather, Ian H

    2017-04-01

    The lipid droplet (LD) fraction of milk has attracted special attention because it supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism. Because many aspects of this key process remain uncharacterized, we developed a facile method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. Using these techniques, we describe the first kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0-2 μm/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in apical nucleation centers, either close to or in association with the apical surface. Droplets even continued to expand as they were emerging from the cell. Contrary to expectations, LDs attached to the apical plasma membrane but still associated with the cytoplasm were released after oxytocin-mediated contraction of the myoepithelium. Thus milk LD secretion is an intermittently regulated process. This novel procedure will have broad application for investigating trafficking events within the mammary epithelium in real time.

  9. Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue

    PubMed Central

    Erami, Zahra; Herrmann, David; Warren, Sean C.; Nobis, Max; McGhee, Ewan J.; Lucas, Morghan C.; Leung, Wilfred; Reischmann, Nadine; Mrowinska, Agata; Schwarz, Juliane P.; Kadir, Shereen; Conway, James R.W.; Vennin, Claire; Karim, Saadia A.; Campbell, Andrew D.; Gallego-Ortega, David; Magenau, Astrid; Murphy, Kendelle J.; Ridgway, Rachel A.; Law, Andrew M.; Walters, Stacey N.; Grey, Shane T.; Croucher, David R.; Zhang, Lei; Herzog, Herbert; Hardeman, Edna C.; Gunning, Peter W.; Ormandy, Christopher J.; Evans, T.R. Jeffry; Strathdee, Douglas; Sansom, Owen J.; Morton, Jennifer P.; Anderson, Kurt I.; Timpson, Paul

    2015-01-01

    Summary E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments. PMID:26725115

  10. Complementarity of variable-magnification and spectral-separation fluorescence imaging systems for noninvasive detection of metastasis and intravital detection of single cancer cells in mouse models.

    PubMed

    Zhang, Yong; Hiroshima, Yukihiko; Ma, Huaiyu; Zhang, Nan; Zhao, Ming; Hoffman, Robert M

    2015-02-01

    Imaging of tumor growth, progression and metastasis with fluorescent proteins in mouse models is a powerful technology. A limit to fluorescent-protein imaging has been for non-invasive deep-seated tumors, such as those in the lung. In the present study, the Maestro spectral-separation fluorescence imaging system and the OV100 variable-magnification imaging system were compared for noninvasive detection of metastasis in fluorescent protein-expressing orthotopic lung, liver, pancreas, and colon cancer in nude mouse tumor models, as well as for intravital single-cell imaging. Sensitivity, multispectral capability, contrast, and single cell resolution were investigated. The Maestro system outperformed the OV100 for noninvasive imaging of primary and metastatic tumors. The Maestro system detected brain tumor metastasis five days earlier than did the OV100. The Maestro had greater depth of detection compared with the OV100. By separating skin and food autofluorescence, the Maestro provided high-contrast images. The Maestro system was able to produce composite images with more unmixed components and detected more different color signals simultaneously than did the OV100. However, the OV100 system had higher resolution and was able to detect single cells in vivo unlike the Maestro. The present study demonstrates that the two instruments are complementary for imaging of all stages of cancer in mice, including single-cell trafficking and the superiority of in vivo fluorescent-protein imaging over luciferase imaging.

  11. Knocking at the brain’s door: intravital two-photon imaging of autoreactive T cell interactions with CNS structures

    PubMed Central

    Flügel, Alexander

    2010-01-01

    Since the first applications of two-photon microscopy in immunology 10 years ago, the number of studies using this advanced technology has increased dramatically. The two-photon microscope allows long-term visualization of cell motility in the living tissue with minimal phototoxicity. Using this technique, we examined brain autoantigen-specific T cell behavior in experimental autoimmune encephalitomyelitis, the animal model of human multiple sclerosis. Even before disease symptoms appear, the autoreactive T cells arrive at their target organ. There they crawl along the intraluminal surface of central nervous system (CNS) blood vessels before they extravasate. In the perivascular environment, the T cells meet phagocytes that present autoantigens. This contact activates the T cells to penetrate deep into the CNS parenchyma, where the infiltrated T cells again can find antigen, be further activated, and produce cytokines, resulting in massive immune cell recruitment and clinical disease. PMID:20623286

  12. Lasers for nonlinear microscopy.

    PubMed

    Wise, Frank

    2013-03-01

    Various versions of nonlinear microscopy are revolutionizing the life sciences, almost all of which are made possible because of the development of ultrafast lasers. In this article, the main properties and technical features of short-pulse lasers used in nonlinear microscopy are summarized. Recent research results on fiber lasers that will impact future instruments are also discussed.

  13. Pulmonary Ventilation Imaging Based on 4-Dimensional Computed Tomography: Comparison With Pulmonary Function Tests and SPECT Ventilation Images

    SciTech Connect

    Yamamoto, Tokihiro; Kabus, Sven; Lorenz, Cristian; Mittra, Erik; Hong, Julian C.; Chung, Melody; Eclov, Neville; To, Jacqueline; Diehn, Maximilian; Loo, Billy W.; Keall, Paul J.

    2014-10-01

    Purpose: 4-dimensional computed tomography (4D-CT)-based pulmonary ventilation imaging is an emerging functional imaging modality. The purpose of this study was to investigate the physiological significance of 4D-CT ventilation imaging by comparison with pulmonary function test (PFT) measurements and single-photon emission CT (SPECT) ventilation images, which are the clinical references for global and regional lung function, respectively. Methods and Materials: In an institutional review board–approved prospective clinical trial, 4D-CT imaging and PFT and/or SPECT ventilation imaging were performed in thoracic cancer patients. Regional ventilation (V{sub 4DCT}) was calculated by deformable image registration of 4D-CT images and quantitative analysis for regional volume change. V{sub 4DCT} defect parameters were compared with the PFT measurements (forced expiratory volume in 1 second (FEV{sub 1}; % predicted) and FEV{sub 1}/forced vital capacity (FVC; %). V{sub 4DCT} was also compared with SPECT ventilation (V{sub SPECT}) to (1) test whether V{sub 4DCT} in V{sub SPECT} defect regions is significantly lower than in nondefect regions by using the 2-tailed t test; (2) to quantify the spatial overlap between V{sub 4DCT} and V{sub SPECT} defect regions with Dice similarity coefficient (DSC); and (3) to test ventral-to-dorsal gradients by using the 2-tailed t test. Results: Of 21 patients enrolled in the study, 18 patients for whom 4D-CT and either PFT or SPECT were acquired were included in the analysis. V{sub 4DCT} defect parameters were found to have significant, moderate correlations with PFT measurements. For example, V{sub 4DCT}{sup HU} defect volume increased significantly with decreasing FEV{sub 1}/FVC (R=−0.65, P<.01). V{sub 4DCT} in V{sub SPECT} defect regions was significantly lower than in nondefect regions (mean V{sub 4DCT}{sup HU} 0.049 vs 0.076, P<.01). The average DSCs for the spatial overlap with SPECT ventilation defect regions were only moderate (V

  14. Investigation of the Dynamical Structure and Diffusion in a System of Hamiltonian Type: 4-Dimensional Symplectic Map

    NASA Astrophysics Data System (ADS)

    Todorovic, N.

    2009-09-01

    The Nekhoroshev theorem (Nekhoroshev 1977) is one of the most important theorems in modern Hamiltonian dynamics. This theorem applies to quasi integrable Hamiltonian systems of type H(I,\\varphi)=h(I)+\\varepsilon f (I, \\varphi), where h(I) is the integrable approximation, f(I, \\varphi) the perturbing function, \\varepsilon is a small perturbing parameter, Iin R^n are the actions and \\varphi in T^n the angles of the system. With some additional geometrical and analytical properties, the theorem provides the stability of actions in exponentially long times. In recent years it has been shown that with some modifications the Nekhoroshev theorem can be applied to the problems in Solar system dynamics (Morbidelli and Guzzo 1997, Guzzo et al 2002, Efthymiopoulos and Sándor 2105, Pavlović and Guzzo 2008). In this work, we are interested to observe numerically a Nekhoroshev like behavior on a model given with a 4-dimensional symplectic map. The model is not in the quasi-integrable form, i.e. independently from the perturbation it contains some additional hyperbolic structures (they appear in the model as primary resonances). Since the hyperbolic structures exist even for zero perturbation, the system will belong to the class of the so called a priori unstable systems. The main numerical tool used here was the Fast Lyapunov Indicator- FLI, introduced in (Froschlé et al. 1997, 2000). As an indicator of chaotic motion, FLI gives very precise and fast information about the chaoticity of an orbit. Also, among regular orbits, FLI is able to differentiate resonant from nonresonant motions. This property of FLI allows us to visualize the studied system and to obtain the Arnold web of the model (Froschlé et al. 2000). In such a way it was possible to observe the transition from a stable Nekhoroshev like structure (regular orbits dominate) to a globally chaotic system where resonances overlap, also known as Chirikov regime. Numerically, this transition happens when between 50

  15. Clinical specular microscopy

    SciTech Connect

    Hirst, L.W.; Laing, R.A.

    1987-01-01

    This book provides the general ophthalmologist with a guide to the clinical applications of specular microscopy. Important material is included on laser injury, cataract surgery, corneal transplants, glaucoma, uveitis, and trauma.

  16. Photothermal imaging scanning microscopy

    DOEpatents

    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.

  17. Intravital multiphoton tomography as a novel tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Niemeyer, Verena; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.

    2010-02-01

    Atopic Dermatitis (AD) is an inflammatory disease of human skin. Its pathogenesis is still unknown; however, dysfunctions of the epidermal barrier and the immune response are regarded as key factors for the development of AD. In our study we applied intravital multiphoton tomography (5D-IVT), equipped with a spectral-FLIM module for in-vivo and ex-vivo analysis of human skin affected with AD. In addition to the morphologic skin analysis, FLIM technology gain access to the metabolic status of the epidermal cells referring to the NADH specific fluorescence lifetime. We evaluated a characteristic 5D-IVT skin pattern of AD in comparison to histological sections and detected a correlation with the disease activity measured by SCORAD. FLIM analysis revealed a shift of the mean fluorescence lifetime (taum) of NADH, indicating an altered metabolic activity. Within an ex-vivo approach we have investigated cryo-sections of human skin with or without barrier defects. Spectral-FLIM allows the detection of autofluorescent signals that reflect the pathophysiological conditions of the defect skin barrier. In our study the taum value was shown to be different between healthy and affected skin. Application of the 5D-IVT allows non-invasive in-vivo imaging of human skin with a penetration depth of 150 μm. We could show that affected skin could be distinguished from healthy skin by morphological criteria, by FLIM and by spectral-FLIM. Further studies will evaluate the application of the 5D-IVT technology as a diagnostic tool and to monitor the therapeutic efficacy.

  18. Intravital multiphoton tomography as an appropriate tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis

    NASA Astrophysics Data System (ADS)

    Huck, Volker; Gorzelanny, Christian; Thomas, Kai; Mess, Christian; Dimitrova, Valentina; Schwarz, Martin; Riemann, Iris; Niemeyer, Verena; Luger, Thomas A.; König, Karsten; Schneider, Stefan W.

    2011-03-01

    Increasing incidence of inflammatory skin diseases such as Atopic Dermatitis (AD) has been noted in the past years. According to recent estimations around 15% of newborn subjects are affected with a disease severity that requires medical treatment. Although its pathogenesis is multifactorial, recent reports indicate that an impaired physical skin barrier predispose for the development of AD. The major part of this barrier is formed by the stratum corneum (SC) wherein corneocytes are embedded in a complex matrix of proteins and lipids. Its components were synthesized in the stratum granulosum (SG) and secreted via lamellar bodies at the SC/SG interface. Within a clinical in vivo study we focused on the skin metabolism at the SC/SG interface in AD affected patients in comparison to healthy subjects. Measurement of fluorescence life-time of NADH provides access to the metabolic state of skin. Due to the application of a 5D intravital tomographic skin analysis we facilitate the non-invasive investigation of human epidermis in the longitudinal course of AD therapy. We could ascertain by blinded analysis of 40 skin areas of 20 patients in a three month follow-up that the metabolic status at the SC/SG interface was altered in AD compromised skin even in non-lesional, apparent healthy skin regions. This illustrates an impaired skin barrier formation even at non-affected skin of AD subjects appearing promotive for the development of acute skin inflammation. Therefore, our findings allow a deeper understanding of the individual disease development and the improved management of the therapeutic intervention in clinical application.

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  20. Imaging immune response of skin mast cells in vivo with two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Li, Chunqiang; Pastila, Riikka K.; Lin, Charles P.

    2012-02-01

    Intravital multiphoton microscopy has provided insightful information of the dynamic process of immune cells in vivo. However, the use of exogenous labeling agents limits its applications. There is no method to perform functional imaging of mast cells, a population of innate tissue-resident immune cells. Mast cells are widely recognized as the effector cells in allergy. Recently their roles as immunoregulatory cells in certain innate and adaptive immune responses are being actively investigated. Here we report in vivo mouse skin mast cells imaging with two-photon microscopy using endogenous tryptophan as the fluorophore. We studied the following processes. 1) Mast cells degranulation, the first step in the mast cell activation process in which the granules are released into peripheral tissue to trigger downstream reactions. 2) Mast cell reconstitution, a procedure commonly used to study mast cells functioning by comparing the data from wild type mice, mast cell-deficient mice, and mast-cell deficient mice reconstituted with bone marrow-derived mast cells (BMMCs). Imaging the BMMCs engraftment in tissue reveals the mast cells development and the efficiency of BMMCs reconstitution. We observed the reconstitution process for 6 weeks in the ear skin of mast cell-deficient Kit wsh/ w-sh mice by two-photon imaging. Our finding is the first instance of imaging mast cells in vivo with endogenous contrast.

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

    PubMed Central

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

    2007-01-01

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

  2. Nonlinear vibrational microscopy

    DOEpatents

    Holtom, Gary R.; Xie, Xiaoliang Sunney; Zumbusch, Andreas

    2000-01-01

    The present invention is a method and apparatus for microscopic vibrational imaging using coherent Anti-Stokes Raman Scattering or Sum Frequency Generation. Microscopic imaging with a vibrational spectroscopic contrast is achieved by generating signals in a nonlinear optical process and spatially resolved detection of the signals. The spatial resolution is attained by minimizing the spot size of the optical interrogation beams on the sample. Minimizing the spot size relies upon a. directing at least two substantially co-axial laser beams (interrogation beams) through a microscope objective providing a focal spot on the sample; b. collecting a signal beam together with a residual beam from the at least two co-axial laser beams after passing through the sample; c. removing the residual beam; and d. detecting the signal beam thereby creating said pixel. The method has significantly higher spatial resolution then IR microscopy and higher sensitivity than spontaneous Raman microscopy with much lower average excitation powers. CARS and SFG microscopy does not rely on the presence of fluorophores, but retains the resolution and three-dimensional sectioning capability of confocal and two-photon fluorescence microscopy. Complementary to these techniques, CARS and SFG microscopy provides a contrast mechanism based on vibrational spectroscopy. This vibrational contrast mechanism, combined with an unprecedented high sensitivity at a tolerable laser power level, provides a new approach for microscopic investigations of chemical and biological samples.

  3. Optical imaging. Expansion microscopy.

    PubMed

    Chen, Fei; Tillberg, Paul W; Boyden, Edward S

    2015-01-30

    In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~10(7) cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

  4. Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging

    PubMed Central

    Li, Hui; Cui, Quan; Zhang, Zhihong; Luo, Qingming

    2015-01-01

    Background The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector (AOD). However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring. Methods We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities. Results Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging. Conclusions This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events. PMID:25694951

  5. Light microscopy digital imaging.

    PubMed

    Joubert, James; Sharma, Deepak

    2011-10-01

    This unit presents an overview of digital imaging hardware used in light microscopy. CMOS, CCD, and EMCCDs are the primary sensors used. The strengths and weaknesses of each define the primary applications for these sensors. Sensor architecture and formats are also reviewed. Color camera design strategies and sensor window cleaning are also described in the unit.

  6. Video Telescope Operating Microscopy.

    PubMed

    Divers, Stephen J

    2015-09-01

    Exotic pet veterinarians frequently have to operate on small animals, and magnification is commonly used. Existing endoscopy equipment can be used with a mechanical arm and telescope to enable video telescope operating microscopy. The additional equipment items and their specifics are described, and several case examples are provided.

  7. Scanning ultrafast electron microscopy.

    PubMed

    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.

  8. Photoacoustic computed microscopy

    PubMed Central

    Yao, Lei; Xi, Lei; Jiang, Huabei

    2014-01-01

    Photoacoustic microscopy (PAM) is emerging as a powerful technique for imaging microvasculature at depths beyond the ~1 mm depth limit associated with confocal microscopy, two-photon microscopy and optical coherence tomography. PAM, however, is currently qualitative in nature and cannot quantitatively measure important functional parameters including oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), oxygen saturation (sO2), blood flow (BF) and rate of oxygen metabolism (MRO2). Here we describe a new photoacoustic microscopic method, termed photoacoustic computed microscopy (PACM) that combines current PAM technique with a model-based inverse reconstruction algorithm. We evaluate the PACM approach using tissue-mimicking phantoms and demonstrate its in vivo imaging ability of quantifying HbO2, HbR, sO2, cerebral BF and cerebral MRO2 at the small vessel level in a rodent model. This new technique provides a unique tool for neuroscience research and for visualizing microvasculature dynamics involved in tumor angiogenesis and in inflammatory joint diseases. PMID:24828539

  9. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

    NASA Astrophysics Data System (ADS)

    Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

    2015-11-01

    Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic.

  10. Line-scanning confocal microscopy for high-resolution imaging of upconverting rare-earth-based contrast agents

    PubMed Central

    Higgins, Laura M.; Zevon, Margot; Ganapathy, Vidya; Sheng, Yang; Tan, Mei Chee; Riman, Richard E.; Roth, Charles M.; Moghe, Prabhas V.; Pierce, Mark C.

    2015-01-01

    Abstract. Rare-earth (RE) doped nanocomposites emit visible luminescence when illuminated with continuous wave near-infrared light, making them appealing candidates for use as contrast agents in biomedical imaging. However, the emission lifetime of these materials is much longer than the pixel dwell times used in scanning intravital microscopy. To overcome this limitation, we have developed a line-scanning confocal microscope for high-resolution, optically sectioned imaging of samples labeled with RE-based nanomaterials. Instrument performance is quantified using calibrated test objects. NaYF4:Er,Yb nanocomposites are imaged in vitro, and in ex vivo tissue specimens, with direct comparison to point-scanning confocal microscopy. We demonstrate that the extended pixel dwell time of line-scanning confocal microscopy enables subcellular-level imaging of these nanomaterials while maintaining optical sectioning. The line-scanning approach thus enables microscopic imaging of this emerging class of contrast agents for preclinical studies, with the potential to be adapted for real-time in vivo imaging in the clinic. PMID:26603495

  11. Polarized Light Microscopy

    NASA Technical Reports Server (NTRS)

    Frandsen, Athela F.

    2016-01-01

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

  12. Laser-induced (endo)vascular photothermal effects studied by combined brightfield and fluorescence microscopy in hamster dorsal skin fold venules

    NASA Astrophysics Data System (ADS)

    Bezemer, R.; Heger, M.; van den Wijngaard, J. P. H.; Mordon, S. R.; van Gemert, M. J. C.; Beek, J. F.

    2007-07-01

    The putative features of the (endo)vascular photothermal response, characterized by laser-induced thermal denaturation of blood and vessel wall constituents, have been elucidated individually, but not simultaneously in dynamic, isolated in vivo systems. A hamster dorsal skin fold model in combination with brightfield/fluorescence intravital microscopy was used to examine the effect of laser pulse duration and blood flow velocity on the size of the thermal coagulum, its attachment behavior, and laser-mediated vasomotion. The size of the coagulum and the extent of vasoconstriction and latent vasodilation were proportional to the laser pulse duration, but pulse duration had no effect on coagulum attachment/dislodgement. Blood flow velocity exhibited no significant effect on the studied parameters. The (endo)vascular photothermal response is governed predominantly by laser energy deposition and to a marginal extent by blood flow velocity.

  13. Probing the immune and healing response of murine intestinal mucosa by time-lapse 2-photon microscopy of laser-induced lesions with real-time dosimetry

    PubMed Central

    Orzekowsky-Schroeder, Regina; Klinger, Antje; Freidank, Sebastian; Linz, Norbert; Eckert, Sebastian; Hüttmann, Gereon; Gebert, Andreas; Vogel, Alfred

    2014-01-01

    Gut mucosa is an important interface between body and environment. Immune response and healing processes of murine small intestinal mucosa were investigated by intravital time-lapse two-photon excited autofluorescence microscopy of the response to localized laser-induced damage. Epithelial lesions were created by 355-nm, 500-ps pulses from a microchip laser that produced minute cavitation bubbles. Size and dynamics of these bubbles were monitored using a novel interferometric backscattering technique with 80 nm resolution. Small bubbles (< 2.5 µm maximum radius) merely resulted in autofluorescence loss of the target cell. Larger bubbles (7-25 µm) affected several cells and provoked immigration of immune cells (polymorphonuclear leucocytes). Damaged cells were expelled into the lumen, and the epithelium healed within 2 hours by stretching and migration of adjacent epithelial cells. PMID:25360369

  14. Quantitative deconvolution microscopy.

    PubMed

    Goodwin, Paul C

    2014-01-01

    The light microscope is an essential tool for the study of cells, organelles, biomolecules, and subcellular dynamics. A paradox exists in microscopy whereby the higher the needed lateral resolution, the more the image is degraded by out-of-focus information. This creates a significant need to generate axial contrast whenever high lateral resolution is required. One strategy for generating contrast is to measure or model the optical properties of the microscope and to use that model to algorithmically reverse some of the consequences of high-resolution imaging. Deconvolution microscopy implements model-based methods to enable the full diffraction-limited resolution of the microscope to be exploited even in complex and living specimens.

  15. Ion photon emission microscopy

    NASA Astrophysics Data System (ADS)

    Rossi, P.; Doyle, B. L.; Banks, J. C.; Battistella, A.; Gennaro, G.; McDaniel, F. D.; Mellon, M.; Vittone, E.; Vizkelethy, G.; Wing, N. D.

    2003-09-01

    A new ion-induced emission microscopy has been invented and demonstrated, which is called ion photon emission microscopy (IPEM). It employs a low current, broad ion beam impinging on a sample, previously coated or simply covered with a few microns of a fast, highly efficient phosphor layer. The light produced at the single ion impact point is collected with an optical microscope and projected at high magnification onto a single photon position sensitive detector (PSD). This allows maps of the ion strike effects to be produced, effectively removing the need for a microbeam. Irradiation in air and even the use of alpha particle sources with no accelerator are possible. Potential applications include ion beam induced charge collection studies of semiconducting and insulating materials, single event upset studies on microchips and even biological cells in radiobiological effectiveness experiments. We describe the IPEM setup, including a 60× OM-40 microscope with a 1.5 mm hole for the beam transmission and a Quantar PSD with 60 μm pixel. Bicron plastic scintillator blades of 10 μm were chosen as a phosphor for their nanosecond time resolution, homogeneity, utility and commercial availability. The results given in this paper are for a prototype IPEM system. They indicate a resolution of ˜12 μm, the presence of a spatial halo and a He-ion efficiency of ˜20%. This marks the first time that nuclear microscopy has been performed with a radioactive source.

  16. Multimodal multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Légaré, François; Pfeffer, Christian P.; Ganikhanov, Feruz

    2009-02-01

    Multiphoton microscopy is a powerful technique for high spatial resolution thick tissue imaging. In its simple version, it uses a high repetition rate femtosecond oscillator laser source focussed and scanned across biological sample that contains fluorophores. However, not every biological structure is inherently fluorescent or can be stained without causing biochemical changes. To circumvent these limitations, other non-invasive nonlinear optical imaging approaches are currently being developed and investigated with regard to different applications. These techniques are: (1) second harmonic generation (SHG), (2) third harmonic generation (THG), and (3) coherent anti-Stokes Raman scattering (CARS) microscopy. The main advantage of the above mentioned techniques is that they derive their imaging contrast from optical nonlinearities that do not involve fluorescence process. As a particular application example we investigated collagen arrays. We show that combining SHG-THG-CARS onto a single imaging platform provides complementary information about the sub-micron architecture of the tissue. SHG microscopy reveals the fibrillar architecture of collagen arrays and confirm a rather high degree of heterogeneity of χ(2) within the focal volume, THG highlights the boundaries between the collagen sheets, and CH2 spectroscopic contrast with CARS.

  17. Electrochemical force microscopy

    DOEpatents

    Kalinin, Sergei V.; Jesse, Stephen; Collins, Liam F.; Rodriguez, Brian J.

    2017-01-10

    A system and method for electrochemical force microscopy are provided. The system and method are based on a multidimensional detection scheme that is sensitive to forces experienced by a biased electrode in a solution. The multidimensional approach allows separation of fast processes, such as double layer charging, and charge relaxation, and slow processes, such as diffusion and faradaic reactions, as well as capturing the bias dependence of the response. The time-resolved and bias measurements can also allow probing both linear (small bias range) and non-linear (large bias range) electrochemical regimes and potentially the de-convolution of charge dynamics and diffusion processes from steric effects and electrochemical reactivity.

  18. Fourier plane imaging microscopy

    SciTech Connect

    Dominguez, Daniel Peralta, Luis Grave de; Alharbi, Nouf; Alhusain, Mdhaoui; Bernussi, Ayrton A.

    2014-09-14

    We show how the image of an unresolved photonic crystal can be reconstructed using a single Fourier plane (FP) image obtained with a second camera that was added to a traditional compound microscope. We discuss how Fourier plane imaging microscopy is an application of a remarkable property of the obtained FP images: they contain more information about the photonic crystals than the images recorded by the camera commonly placed at the real plane of the microscope. We argue that the experimental results support the hypothesis that surface waves, contributing to enhanced resolution abilities, were optically excited in the studied photonic crystals.

  19. Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

    NASA Astrophysics Data System (ADS)

    Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

    2012-03-01

    Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

  20. Multi-pass microscopy

    NASA Astrophysics Data System (ADS)

    Juffmann, Thomas; Klopfer, Brannon B.; Frankort, Timmo L. I.; Haslinger, Philipp; Kasevich, Mark A.

    2016-09-01

    Microscopy of biological specimens often requires low light levels to avoid damage. This yields images impaired by shot noise. An improved measurement accuracy at the Heisenberg limit can be achieved exploiting quantum correlations. If sample damage is the limiting resource, an equivalent limit can be reached by passing photons through a specimen multiple times sequentially. Here we use self-imaging cavities and employ a temporal post-selection scheme to present full-field multi-pass polarization and transmission micrographs with variance reductions of 4.4+/-0.8 dB (11.6+/-0.8 dB in a lossless setup) and 4.8+/-0.8 dB, respectively, compared with the single-pass shot-noise limit. If the accuracy is limited by the number of detected probe particles, our measurements show a variance reduction of 25.9+/-0.9 dB. The contrast enhancement capabilities in imaging and in diffraction studies are demonstrated with nanostructured samples and with embryonic kidney 293T cells. This approach to Heisenberg-limited microscopy does not rely on quantum state engineering.

  1. Multi-pass microscopy

    PubMed Central

    Juffmann, Thomas; Klopfer, Brannon B.; Frankort, Timmo L.I.; Haslinger, Philipp; Kasevich, Mark A.

    2016-01-01

    Microscopy of biological specimens often requires low light levels to avoid damage. This yields images impaired by shot noise. An improved measurement accuracy at the Heisenberg limit can be achieved exploiting quantum correlations. If sample damage is the limiting resource, an equivalent limit can be reached by passing photons through a specimen multiple times sequentially. Here we use self-imaging cavities and employ a temporal post-selection scheme to present full-field multi-pass polarization and transmission micrographs with variance reductions of 4.4±0.8 dB (11.6±0.8 dB in a lossless setup) and 4.8±0.8 dB, respectively, compared with the single-pass shot-noise limit. If the accuracy is limited by the number of detected probe particles, our measurements show a variance reduction of 25.9±0.9 dB. The contrast enhancement capabilities in imaging and in diffraction studies are demonstrated with nanostructured samples and with embryonic kidney 293T cells. This approach to Heisenberg-limited microscopy does not rely on quantum state engineering. PMID:27670525

  2. Magnetic force microscopy

    PubMed Central

    Passeri, Daniele; Dong, Chunhua; Reggente, Melania; Angeloni, Livia; Barteri, Mario; Scaramuzzo, Francesca A; De Angelis, Francesca; Marinelli, Fiorenzo; Antonelli, Flavia; Rinaldi, Federica; Marianecci, Carlotta; Carafa, Maria; Sorbo, Angela; Sordi, Daniela; Arends, Isabel WCE; Rossi, Marco

    2014-01-01

    Magnetic force microscopy (MFM) is an atomic force microscopy (AFM) based technique in which an AFM tip with a magnetic coating is used to probe local magnetic fields with the typical AFM spatial resolution, thus allowing one to acquire images reflecting the local magnetic properties of the samples at the nanoscale. Being a well established tool for the characterization of magnetic recording media, superconductors and magnetic nanomaterials, MFM is finding constantly increasing application in the study of magnetic properties of materials and systems of biological and biomedical interest. After reviewing these latter applications, three case studies are presented in which MFM is used to characterize: (i) magnetoferritin synthesized using apoferritin as molecular reactor; (ii) magnetic nanoparticles loaded niosomes to be used as nanocarriers for drug delivery; (iii) leukemic cells labeled using folic acid-coated core-shell superparamagnetic nanoparticles in order to exploit the presence of folate receptors on the cell membrane surface. In these examples, MFM data are quantitatively analyzed evidencing the limits of the simple analytical models currently used. Provided that suitable models are used to simulate the MFM response, MFM can be used to evaluate the magnetic momentum of the core of magnetoferritin, the iron entrapment efficiency in single vesicles, or the uptake of magnetic nanoparticles into cells. PMID:25050758

  3. Silver stain for electron microscopy

    NASA Technical Reports Server (NTRS)

    Corbett, R. L.

    1972-01-01

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

  4. Image Force Microscopy

    NASA Astrophysics Data System (ADS)

    Rajapaksa, Indrajith

    In this thesis we describe an enhancement to the Atomic force microscope (AFM) to simultaneously gather topographic features and spectroscopic information .Compared to the current state of the art of near-field excitation and far-field detection AFM imaging techniques our system uses a radical new approach near-field excitation and near-field detection. By placing the detector in the near-field we achieve high signal to noise and single molecular resolution. The origin of our near-field detector signal is the image force gradient due to the interaction of the stimulated molecular dipole with its image on the metal probe. We designed and built an optical and electronic system to capture this signal and simultaneously image nano-scale surface topography and optical image force gradient. By varying the wavelength of the excitation beam we measure the induced optical image force gradient spectra of molecules on surface. These spectra show good agreement with the absorption spectra of the bulk molecules measured by conventional absorption spectroscopy. We show that image force gradient is directly proportional to the optical absorption dipole strength. Using Finite Element 3D electromagnetic simulations and using Lorentz model for the excited molecular dipole we showed that the image force gradient has a decay length of 1nm, making the theoretical resolution of this microscopy technique approximately 1 nm. This rapid decay was measured experimentally .This resolution was seen by the high contrasting spectroscopic images of molecules on the surface. In follow on experiments this technique was extended to provide surface Raman spectroscopy and microscopy at molecular resolution. We create an image force gradient interaction through optical parametric down conversion between stimulated Raman excited molecules on a surface and a cantilevered nanometer scale probe brought very close to it. Spectroscopy and microscopy on clusters of molecules have been performed. Single

  5. Applications of subsurface microscopy.

    PubMed

    Tetard, Laurene; Passian, Ali; Farahi, Rubye H; Voy, Brynn H; Thundat, Thomas

    2012-01-01

    Exploring the interior of a cell is of tremendous importance in order to assess the effects of nanomaterials on biological systems. Outside of a controlled laboratory environment, nanomaterials will most likely not be conveniently labeled or tagged so that their translocation within a biological system cannot be easily identified and quantified. Ideally, the characterization of nanomaterials within a cell requires a nondestructive, label-free, and subsurface approach. Subsurface nanoscale imaging represents a real challenge for instrumentation. Indeed the tools available for high resolution characterization, including optical, electron or scanning probe microscopies, mainly provide topography images or require taggants that fluoresce. Although the intercellular environment holds a great deal of information, subsurface visualization remains a poorly explored area. Recently, it was discovered that by mechanically perturbing a sample, it was possible to observe its response in time with nanoscale resolution by probing the surface with a micro-resonator such as a microcantilever probe. Microcantilevers are used as the force-sensing probes in atomic force microscopy (AFM), where the nanometer-scale probe tip on the microcantilever interacts with the sample in a highly controlled manner to produce high-resolution raster-scanned information of the sample surface. Taking advantage of the existing capabilities of AFM, we present a novel technique, mode synthesizing atomic force microscopy (MSAFM), which has the ability to probe subsurface structures such as non-labeled nanoparticles embedded in a cell. In MSAFM mechanical actuators (PZTs) excite the probe and the sample at different frequencies as depicted in the first figure of this chapter. The nonlinear nature of the tip-sample interaction, at the point of contact of the probe and the surface of the sample, in the contact mode AFM configuration permits the mixing of the elastic waves. The new dynamic system comprises new

  6. Hyperspectral light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Jahr, Wiebke; Schmid, Benjamin; Schmied, Christopher; Fahrbach, Florian O.; Huisken, Jan

    2015-09-01

    To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.

  7. Sensitivity of photoacoustic microscopy

    PubMed Central

    Yao, Junjie; Wang, Lihong V.

    2014-01-01

    Building on its high spatial resolution, deep penetration depth and excellent image contrast, 3D photoacoustic microscopy (PAM) has grown tremendously since its first publication in 2005. Integrating optical excitation and acoustic detection, PAM has broken through both the optical diffusion and optical diffraction limits. PAM has 100% relative sensitivity to optical absorption (i.e., a given percentage change in the optical absorption coefficient yields the same percentage change in the photoacoustic amplitude), and its ultimate detection sensitivity is limited only by thermal noise. Focusing on the engineering aspects of PAM, this Review discusses the detection sensitivity of PAM, compares the detection efficiency of different PAM designs, and summarizes the imaging performance of various endogenous and exogenous contrast agents. It then describes representative PAM applications with high detection sensitivity, and outlines paths to further improvement. PMID:25302158

  8. Snapshot Hyperspectral Volumetric Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

    2016-04-01

    The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens.

  9. Snapshot Hyperspectral Volumetric Microscopy

    PubMed Central

    Wu, Jiamin; Xiong, Bo; Lin, Xing; He, Jijun; Suo, Jinli; Dai, Qionghai

    2016-01-01

    The comprehensive analysis of biological specimens brings about the demand for capturing the spatial, temporal and spectral dimensions of visual information together. However, such high-dimensional video acquisition faces major challenges in developing large data throughput and effective multiplexing techniques. Here, we report the snapshot hyperspectral volumetric microscopy that computationally reconstructs hyperspectral profiles for high-resolution volumes of ~1000 μm × 1000 μm × 500 μm at video rate by a novel four-dimensional (4D) deconvolution algorithm. We validated the proposed approach with both numerical simulations for quantitative evaluation and various real experimental results on the prototype system. Different applications such as biological component analysis in bright field and spectral unmixing of multiple fluorescence are demonstrated. The experiments on moving fluorescent beads and GFP labelled drosophila larvae indicate the great potential of our method for observing multiple fluorescent markers in dynamic specimens. PMID:27103155

  10. In vivo microdissection and live embryo imaging by two-photon microscopy to study Drosophila melanogaster early development

    NASA Astrophysics Data System (ADS)

    Supatto, Willy; Brouzes, Eric; Farge, Emmanuel; Beaurepaire, Emmanuel

    2004-09-01

    Animal embryo development exhibits a complex choreography of cell movements highly regulated both in time and space. This sequence of morphogenetic movements is initiated at gastrulation and is tightly controlled by a cascade of developmental gene expression. We have recently reported that developmental gene expression can in turn be mechanically regulated by morphogenetic movements during Drosophila melanogaster early development. In order to study this phenomenon of mechanically induced gene expression, it is necessary to develop new techniques of in vivo investigation. We show that the combination of femtosecond pulse intratissue surgery and two-photon-excitation fluorescence (2PEF) microscopy is a powerful tool for (i) disrupting natural morphogenetic movements and (ii) imaging native and disrupted morphogenetic movements during Drosophila development. (i) First, non-linear-absorption-mediated photo-disruption makes it possible to perform controlled intra-vital micro-dissections resulting in the modulation of morphogenetic movements and subsequent mechano-sensitive gene expression. (ii) Second, in vivo 2PEF microscopy of transgenic GFP systems appears to be an excellent technique for long-term in vivo imaging of the complex morphogenetic movements involved in normal or perturbed Drosophila gastrulation. Together, these two techniques provide a powerful novel approach to study embryo development.

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

    SciTech Connect

    Ramanathan, Nathan Muruganathan; Darling, Seth B.

    2015-01-01

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

  12. Extraterrestrial optical microscopy.

    PubMed

    Soffen, G A

    1969-07-01

    An examination of the literature concerned with the use of microscopy for planetary investigation reveals a serious deficiency of current efforts. Many scientists have recommended the use of a microscope for planetary investigation [Biology and the Exploration of Mars, C. S. Pittendrigh, W. Vishniac, and J. P. T. Pearman, Eds. (National Academy of Science-National Research Council, Washington, D. C., 1966), (a) D. Mazia, p. 31; (b) J. Lederberg, p. 137; (c) S. Fox, pp. 219, 226; (d) D. Glaser, p. 326; (e) D. Glaser, J. McCarthy, and M. Minsky, pp. 333, 341; (f) D. G. Rea, pp. 347-426; (g) P. G. Conger, pp. 409-414; (h) M. H. Fernandez, pp. 414-425; (i) D. Schwartz, pp.425-426 . H. P. Klein, Some Biological Problems in the Search for Extraterrestrial Life (American Astronautical Society, Washington, D. C., 1968).] but few are involved in developing the experiment. Since this is a particularly timely period for the preparation of planetary lander experiments, the reasons for this lack of effort would appear to be limited resources or an unclear course of action, rather than lack of interest. Microscopy used for planetary investigation is chiefly the interest of the biologist and the mineralogist. In both cases the desire to use magnifying optics in order to observe objects of submillimeter size is based upon the rich body of knowledge we have acquired from observing the terrestrial microcosm. In addition to purely imaging, certain special optical techniques, e.g., polarimetry, colorimetry, phase contrast, etc., can be used to enhance the interpretation of microscopic imaging data. This interaction of the optical with the chemical or structural aspects of nature can be used to great advantage in the exploration of extraterrestrial biology and mineralogy.

  13. Ultrasonic Force Microscopies

    NASA Astrophysics Data System (ADS)

    Kolosov, Oleg; Briggs, Andrew

    Ultrasonic Force Microscopy, or UFM, allows combination of two apparently mutually exclusive requirements for the nanomechanical probe—high stiffness for the efficient indentation and high mechanical compliance that brings force sensitivity. Somewhat inventively, UFM allows to combine these two virtues in the same cantilever by using indention of the sample at high frequency, when cantilever is very rigid, but detecting the result of this indention at much lower frequency. That is made possible due to the extreme nonlinearity of the nanoscale tip-surface junction force-distance dependence, that acts as "mechanical diode" detecting ultrasound in AFM. After introducing UFM principles, we discuss features of experimental UFM implementation, and the theory of contrast in this mode, progressing to quantitative measurements of contact stiffness. A variety of UFM applications ranging from semiconductor quantum nanostructures, graphene, very large scale integrated circuits, and reinforced ceramics to polymer composites and biological materials is presented via comprehensive imaging gallery accompanied by the guidance for the optimal UFM measurements of these materials. We also address effects of adhesion and topography on the elasticity imaging and the approaches for reducing artifacts connected with these effects. This is complemented by another extremely useful feature of UFM—ultrasound induced superlubricity that allows damage free imaging of materials ranging from stiff solid state devices and graphene to biological materials. Finally, we proceed to the exploration of time-resolved nanoscale phenomena using nonlinear mixing of multiple vibration frequencies in ultrasonic AFM—Heterodyne Force Microscopy, or HFM, that also include mixing of ultrasonic vibration with other periodic physical excitations, eg. electrical, photothermal, etc. Significant section of the chapter analyzes the ability of UFM and HFM to detect subsurface mechanical inhomogeneities, as well as

  14. Virtual microscopy in pathology education.

    PubMed

    Dee, Fred R

    2009-08-01

    Technology for acquisition of virtual slides was developed in 1985; however, it was not until the late 1990s that desktop computers had enough processing speed to commercialize virtual microscopy and apply the technology to education. By 2000, the progressive decrease in use of traditional microscopy in medical student education had set the stage for the entry of virtual microscopy into medical schools. Since that time, it has been successfully implemented into many pathology courses in the United States and around the world, with surveys indicating that about 50% of pathology courses already have or expect to implement virtual microscopy. Over the last decade, in addition to an increasing ability to emulate traditional microscopy, virtual microscopy has allowed educators to take advantage of the accessibility, efficiency, and pedagogic versatility of the computer and the Internet. The cost of virtual microscopy in education is now quite reasonable after taking into account replacement cost for microscopes, maintenance of glass slides, and the fact that 1-dimensional microscope space can be converted to multiuse computer laboratories or research. Although the current technology for implementation of virtual microscopy in histopathology education is very good, it could be further improved upon by better low-power screen resolution and depth of field. Nevertheless, virtual microscopy is beginning to play an increasing role in continuing education, house staff education, and evaluation of competency in histopathology. As Z-axis viewing (focusing) becomes more efficient, virtual microscopy will also become integrated into education in cytology, hematology, microbiology, and urinalysis.

  15. Intrinsic Friction Microscopy

    NASA Astrophysics Data System (ADS)

    Knorr, Daniel; Overney, Rene

    2008-03-01

    A novel scanning probe methodology based on lateral force microscopy is presented wherein kinetic friction measurements, obtained as a function of velocity for various temperatures, are used to deduce apparent Arrhenius-type activation energies for surface and subsurface molecular mobilities. Depending on the coupling strength (cooperativity) between molecular mobilities involved the dissipation energy can carry a significant entropic energy contribution, accounting for the majority of the apparent Arrhenius activation energy. The intrinsic friction methodology also provides a means of directly separating enthalpic energy contributions from entropic ones by employing absolute rate theory. As such, the degree of cooperativity in the system is readily apparent. This methodology is illustrated with nanoscale tribological experiments on two systems, (1) monodisperse, atactic polystyrene and (2) self assembling molecular glassy chromophores. In polystyrene, dissipation was found to be a discrete function of loading, where the γ-relaxation (phenyl group rotation) was recovered for ultra low loads and the β-relaxation (local backbone translation) for higher loads in the same temperature range, indicating sensitivity to surface and subsurface mobilities. For self assembling glassy chromophores, the degree of intermolecular cooperativity was deduced using the methodology, resulting in an increased understanding of the interactions between self assembling molecules.

  16. Ultrafast scanning probe microscopy

    DOEpatents

    Weiss, Shimon; Chemla, Daniel S.; Ogletree, D. Frank; Botkin, David

    1995-01-01

    An ultrafast scanning probe microscopy method for achieving subpicosecond-temporal resolution and submicron-spatial resolution of an observation sample. In one embodiment of the present claimed invention, a single short optical pulse is generated and is split into first and second pulses. One of the pulses is delayed using variable time delay means. The first pulse is then directed at an observation sample located proximate to the probe of a scanning probe microscope. The scanning probe microscope produces probe-sample signals indicative of the response of the probe to characteristics of the sample. The second pulse is used to modulate the probe of the scanning probe microscope. The time delay between the first and second pulses is then varied. The probe-sample response signal is recorded at each of the various time delays created between the first and second pulses. The probe-sample response signal is then plotted as a function of time delay to produce a cross-correlation of the probe sample response. In so doing, the present invention provides simultaneous subpicosecond-temporal resolution and submicron-spatial resolution of the sample.

  17. Ultrafast scanning tunneling microscopy

    SciTech Connect

    Botkin, D.A. |

    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. NMR imaging microscopy

    SciTech Connect

    Not Available

    1986-10-01

    In the past several years, proton nuclear magnetic resonance (NMR) imaging has become an established technique in diagnostic medicine and biomedical research. Although much of the work in this field has been directed toward development of whole-body imagers, James Aguayo, Stephen Blackband, and Joseph Schoeninger of the Johns Hopkins University School of Medicine working with Markus Hintermann and Mark Mattingly of Bruker Medical Instruments, recently developed a small-bore NMR microscope with sufficient resolution to image a single African clawed toad cell (Nature 1986, 322, 190-91). This improved resolution should lead to increased use of NMR imaging for chemical, as well as biological or physiological, applications. The future of NMR microscopy, like that of many other newly emerging techniques, is ripe with possibilities. Because of its high cost, however, it is likely to remain primarily a research tool for some time. ''It's like having a camera,'' says Smith. ''You've got a way to look at things at very fine levels, and people are going to find lots of uses for it. But it is a very expensive technique - it costs $100,000 to add imaging capability once you have a high-resolution NMR, which itself is at least a $300,000 instrument. If it can answer even a few questions that can't be answered any other way, though, it may be well worth the cost.''

  19. Mueller polarimetric microscopy

    NASA Astrophysics Data System (ADS)

    Laude-Boulesteix, Blandine; De Martino, Antonello; Le Naour, Gilles; Genestie, Catherine; Schwartz, Laurent; Garcia-Caurel, Enric; Drevillon, Bernard

    2004-07-01

    We present a multispectral polarimetric imaging system well suited for complete Mueller matrix microscopy. The source is a spectrally filtered halogen light bulb, and the image is formed on a fast CCD camera The light polarization is modulated before the sample and analyzed after the sample by using nematic liquid crystal modulators.. The whole Mueller matrix image of the sample is typically measured over 5 seconds for a good signal-to-noise ratio. The instrument design, together with an original and easy-to-operate calibration procedure provides a high polarimetric accuracy over wide ranges of wavelengths and magnifications. Mueller polarimetry provides separate images of scalar and vector retardation and dichroism of the sample, together with its depolarizing power, while all these effects do contribute simultaneously to the contrasts observed in standard polarized microsopy. Polarimetric images of several samples, namely an unstained rabbit cornea, a picrosirius red stained hepatic biopsy, and a rat artery specifically stained for collagen III are shown and discussed

  20. Microscopy of semiconducting materials

    NASA Astrophysics Data System (ADS)

    Pennycook, S. J.

    1991-04-01

    The purpose of the trip was to present an invited talk at the 7th Oxford Conference on Microscopy of Semiconducting Materials entitled, High-Resolution Z-Contrast Imaging of Heterostructures and Superlattices, (Oxford, United Kingdom) and to visit VG Microscopes, East Grinstead, for discussions on the progress of the Oak Ridge National Laboratory (ORNL) 300-kV high-resolution scanning transmission electron microscope (STEM), which is currently on order. The traveler also visited three other institutions with 100-kV STEMs that either have or intend to purchase the necessary modifications to provide Z-contrast capability similar to that of the existing ORNL machine. Specifically, Max-Planck Institut fuer Metallforschung (Stuttgart, Germany); Cambridge University, Department of Materials Science and Metallurgy (Cambridge, United Kingdom); and Cavendish Laboratory, Cambridge University (Cambridge, United Kingdom) were visited. In addition, discussions were held with C. Humphreys on the possibility of obtaining joint funding for collaborative research involving electron beam writing and Z-contrast imaging in the Cambridge and Oak Ridge STEMs, respectively.

  1. Ultrafast scanning probe microscopy

    DOEpatents

    Weiss, S.; Chemla, D.S.; Ogletree, D.F.; Botkin, D.

    1995-05-16

    An ultrafast scanning probe microscopy method is described for achieving subpicosecond-temporal resolution and submicron-spatial resolution of an observation sample. In one embodiment of the present claimed invention, a single short optical pulse is generated and is split into first and second pulses. One of the pulses is delayed using variable time delay means. The first pulse is then directed at an observation sample located proximate to the probe of a scanning probe microscope. The scanning probe microscope produces probe-sample signals indicative of the response of the probe to characteristics of the sample. The second pulse is used to modulate the probe of the scanning probe microscope. The time delay between the first and second pulses is then varied. The probe-sample response signal is recorded at each of the various time delays created between the first and second pulses. The probe-sample response signal is then plotted as a function of time delay to produce a cross-correlation of the probe sample response. In so doing, the present invention provides simultaneous subpicosecond-temporal resolution and submicron-spatial resolution of the sample. 6 Figs.

  2. Sample preparation for STED microscopy.

    PubMed

    Wurm, Christian A; Neumann, Daniel; Schmidt, Roman; Egner, Alexander; Jakobs, Stefan

    2010-01-01

    Since the discovery of the diffraction barrier in the late nineteenth century, it has been commonly accepted that with far-field optical microscopy it is not possible to resolve structural details considerably finer than half the wavelength of light. The emergence of STED microscopy showed that, at least for fluorescence imaging, these limits can be overcome. Since STED microscopy is a far-field technique, in principle, the same sample preparation as for conventional confocal microscopy may be utilized. The increased resolution, however, requires additional precautions to ensure the structural preservation of the specimen. We present robust protocols to generate test samples for STED microscopy. These protocols for bead samples and immunolabeled mammalian cells may be used as starting points to adapt existing labeling strategies for the requirements of sub-diffraction resolution microscopy.

  3. Verification of Planning Target Volume Settings in Volumetric Modulated Arc Therapy for Stereotactic Body Radiation Therapy by Using In-Treatment 4-Dimensional Cone Beam Computed Tomography

    SciTech Connect

    Takahashi, Wataru; Yamashita, Hideomi; Kida, Satoshi; Masutani, Yoshitaka; Sakumi, Akira; Ohtomo, Kuni; Nakagawa, Keiichi; Haga, Akihiro

    2013-07-01

    Purpose: To evaluate setup error and tumor motion during beam delivery by using 4-dimensional cone beam computed tomography (4D CBCT) and to assess the adequacy of the planning target volume (PTV) margin for lung cancer patients undergoing volumetric modulated arc therapy for stereotactic body radiation therapy (VMAT-SBRT). Methods and Materials: Fifteen lung cancer patients treated by single-arc VMAT-SBRT were selected in this analysis. All patients were treated with an abdominal compressor. The gross tumor volumes were contoured on maximum inspiration and maximum expiration CT datasets from 4D CT respiratory sorting and merged into internal target volumes (ITVs). The PTV margin was isotropically taken as 5 mm. Registration was automatically performed using “pre-3D” CBCT. Treatment was performed with a D95 prescription of 50 Gy delivered in 4 fractions. The 4D tumor locations during beam delivery were determined using in-treatment 4D CBCT images acquired in each fraction. Then, the discrepancy between the actual tumor location and the ITV was evaluated in the lateral, vertical, and longitudinal directions. Results: Overall, 55 4D CBCT sets during VMAT-SBRT were successfully obtained. The amplitude of tumor motion was less than 10 mm in all directions. The average displacements between ITV and actual tumor location during treatment were 0.41 ± 0.93 mm, 0.15 ± 0.58 mm, and 0.60 ± 0.99 mm for the craniocaudal, left-right, and anteroposterior directions, respectively. The discrepancy in each phase did not exceed 5 mm in any direction. Conclusions: With in-treatment 4D CBCT, we confirmed the required PTV margins when the registration for moving target was performed using pre-3D CBCT. In-treatment 4D CBCT is a direct method for quantitatively assessing the intrafractional location of a moving target.

  4. The Regional Ocean Modeling System (ROMS) 4-dimensional variational data assimilation systems . Part II - Performance and application to the California Current System

    NASA Astrophysics Data System (ADS)

    Moore, Andrew M.; Arango, Hernan G.; Broquet, Gregoire; Edwards, Chris; Veneziani, Milena; Powell, Brian; Foley, Dave; Doyle, James D.; Costa, Dan; Robinson, Patrick

    2011-10-01

    The Regional Ocean Modeling System (ROMS) 4-dimensional variational (4D-Var) data assimilation systems have been systematically applied to the mesoscale circulation environment of the California Current to demonstrate the performance and practical utility of the various components of ROMS 4D-Var. In particular, we present a comparison of three approaches to 4D-Var, namely: the primal formulation of the incremental strong constraint approach; the dual formulation “physical-space statistical analysis system”; and the dual formulation indirect representer approach. In agreement with theoretical considerations all three approaches converge to the same ocean circulation estimate when using the same observations and prior information. However, the rate of convergence of the dual formulation was found to be inferior to that of the primal formulation. Other aspects of the 4D-Var performance that relate to the use of multiple outer-loops, preconditioning, and the weak constraint are also explored. A systematic evaluation of the impact of the various components of the 4D-Var control vector (i.e. the initial conditions, surface forcing and open boundary conditions) is also presented. It is shown that correcting for uncertainties in the model initial conditions exerts the largest influence on the ability of the model to fit the available observations. Various important diagnostics of 4D-Var are also examined, including estimates of the posterior error, the information content of the observation array, and innovation-based consistency checks on the prior error assumptions. Using these diagnostic tools, we find that more than 90% of the observations assimilated into the model provide redundant information. This is a symptom of the large percentage of satellite data that are used and to some extent the nature of the data processing employed. This is the second in a series of three papers describing the ROMS 4D-Var systems.

  5. A Pilot Evaluation of a 4-Dimensional Cone-Beam Computed Tomographic Scheme Based on Simultaneous Motion Estimation and Image Reconstruction

    SciTech Connect

    Dang, Jun; Gu, Xuejun; Pan, Tinsu; Wang, Jing

    2015-02-01

    Purpose: To evaluate the performance of a 4-dimensional (4-D) cone-beam computed tomographic (CBCT) reconstruction scheme based on simultaneous motion estimation and image reconstruction (SMEIR) through patient studies. Methods and Materials: The SMEIR algorithm contains 2 alternating steps: (1) motion-compensated CBCT reconstruction using projections from all phases to reconstruct a reference phase 4D-CBCT by explicitly considering the motion models between each different phase and (2) estimation of motion models directly from projections by matching the measured projections to the forward projection of the deformed reference phase 4D-CBCT. Four lung cancer patients were scanned for 4 to 6 minutes to obtain approximately 2000 projections for each patient. To evaluate the performance of the SMEIR algorithm on a conventional 1-minute CBCT scan, the number of projections at each phase was reduced by a factor of 5, 8, or 10 for each patient. Then, 4D-CBCTs were reconstructed from the down-sampled projections using Feldkamp-Davis-Kress, total variation (TV) minimization, prior image constrained compressive sensing (PICCS), and SMEIR. Using the 4D-CBCT reconstructed from the fully sampled projections as a reference, the relative error (RE) of reconstructed images, root mean square error (RMSE), and maximum error (MaxE) of estimated tumor positions were analyzed to quantify the performance of the SMEIR algorithm. Results: The SMEIR algorithm can achieve results consistent with the reference 4D-CBCT reconstructed with many more projections per phase. With an average of 30 to 40 projections per phase, the MaxE in tumor position detection is less than 1 mm in SMEIR for all 4 patients. Conclusion: The results from a limited number of patients show that SMEIR is a promising tool for high-quality 4D-CBCT reconstruction and tumor motion modeling.

  6. Electronic Blending in Virtual Microscopy

    ERIC Educational Resources Information Center

    Maybury, Terrence S.; Farah, Camile S.

    2010-01-01

    Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

  7. Multi-contrast Photoacoustic Microscopy

    NASA Astrophysics Data System (ADS)

    Yao, Junjie

    Photoacoustic microscopy is a hybrid imaging modality with high spatial resolution, moderate imaging depth, excellent imaging contrast and functional imaging capability. Taking full advantage of this powerful weapon, we have investigated different anatomical, functional, flow dynamic and metabolic parameter measurements using photoacoustic microscopy. Specifically, Evans-blue dye was used to enhance photoacoustic microscopy of capillaries; label-free transverse and axial blood flow was measured based on bandwidth broadening and time shift of the photoacoustic signals; metabolic rate of oxygen was quantified in vivo from all the five parameters measured by photoacoustic microcopy; whole cross-sectional imaging of small intestine was achieved on a double-illumination photoacoustic microscopy with extended depth of focus and imaging depth; hemodynamic imaging was performed on a MEMS-mirror enhanced photoacoustic microscopy with a cross-sectional imaging rate of 400 Hz. As a maturing imaging technique, PAM is expected to find new applications in both fundamental life science and clinical practice.

  8. Studies in scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Sarid, Dror

    1995-06-01

    The following is a final report on our work in the field of Scanning Probe Microscopy (SPM), which has been funded by the AFOSR under Contract #F49620-92-J-0164. The AFOSR funding was instrumental in the establishment of a multi-lab facility at the Optical Sciences Center, which performs research in SPM using two ultrahigh vacuum (UHV) STM facilities, and several Atomic Force Microscopy (AFM) facilities. The fabrication and characterization work performed in the SPM Laboratory is supplemented by infrared (IR) spectroscopy, high resolution transmission electron microscopy (HRTEM), and scanning electron microscopy (SEM), available in other departments on campus. The report covers the following areas: (1) GaAs and CdSe Structures, (2) Optical Interactions on a nm and nsec Scales, (3) Fullerenes on Gold, (4) Fullerenes on MoS2, (5) Fullerenes on Si, (6) SiC, (7) Nanotubes, (8) Scanning Force Microscopy, and (9) Biology.

  9. Chip-scale microscopy imaging.

    PubMed

    Zheng, Guoan

    2012-08-01

    Chip-scale microscopy imaging platforms are pivotal for improving the efficiency of modern biomedical and bioscience experiments. Their integration with other lab-on-a-chip techniques would allow rapid, reliable and high-throughput sample analysis for applications in diverse disciplines. In typical chip-scale microscopy imaging platforms, the light path can be generalized to the following steps: photons leave the light source, interact with the sample and finally are detected by the sensor. Based on the light path of these platforms, the current review aims to provide some insights on design strategies for chip-scale microscopy. Specifically, we analyze current chip-scale microscopy approaches from three aspects: illumination design, sample manipulation and substrate/imager modification. We also discuss some opportunities for future developments of chip-scale microscopy, such as time multiplexed structured illumination and hydrodynamic focusing for high throughput sample manipulation.

  10. Atomic force microscopy combined with optical microscopy for cells investigation.

    PubMed

    Cascione, Mariafrancesca; de Matteis, Valeria; Rinaldi, Rosaria; Leporatti, Stefano

    2017-01-01

    This review reports on the combined use of the atomic force microscopy (AFM) and several type of optical/fluorescence/laser scanning microscopy for investigating cells. It is shown that the hybrid systems of AFM with optical-derived microscopies enable to study in detail cell surface properties (such as topography), their mechanical properties (e.g., Young's modulus) mechanotransduction phenomena and allow to gain insight into biological-related pathways and mechanisms in the complex nanoworld of cells. Microsc. Res. Tech. 80:109-123, 2017. © 2016 Wiley Periodicals, Inc.

  11. Microscopy techniques in flavivirus research.

    PubMed

    Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

    2014-04-01

    The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.

  12. Horizontal microscopy in square capillaries

    NASA Astrophysics Data System (ADS)

    Moroz, Pavel E.

    1992-07-01

    Intracellular protoplasmic movements may, due to gravity, have a vertical component greater or different from the horizontal one. This makes horizontal microscopy indispensable in the search for the cellular sensor of gravity. The possibility of the latter being a cell organelle assigns special significance to high-resolution microscopy. A horizontal suction device for picking up a cell and its high-resolution horizontal microscopy in a rectangular capillary may be helpful for detection of gravity-related shifts of cellular organelles in vivo.

  13. Research With Scanning Tip Microscopy

    DTIC Science & Technology

    1991-12-31

    08ro P noiwe bae?041Le Research With Scanning Tip Microscopy AFOSR-89-0498 V AUTHOS)i Professor Dror Sarid 7. PFOUImNG 00ANIZATION NAMEIS) AND...forces and (b) surfaces. UNCLASS UNCLASS UNCLASS UL FINAL REPORT TO THE AFOSR ൱-, to J4ti. r Aat io Research in Scanning Tip Microscopy Dror Sarid Dtst...microscopy have been used to investigate (a) forces and (b) surfaces. a. Forces 1. Dror Sarid , Douglas lams, Volker Weissenberger, and L. Stephen Bell

  14. Soil microstructure and electron microscopy

    NASA Technical Reports Server (NTRS)

    Smart, P.; Fryer, J. R.

    1988-01-01

    As part of the process of comparing Martian soils with terrestial soils, high resolution electron microscopy and associated techniques should be used to examine the finer soil particles, and various techniques of electron and optical microscopy should be used to examine the undisturbed structure of Martian soils. To examine the structure of fine grained portions of the soil, transmission electron microscopy may be required. A striking feature of many Martian soils is their red color. Although the present-day Martian climate appears to be cold, this color is reminiscent of terrestial tropical red clays. Their chemical contents are broadly similar.

  15. Audio-Visual Biofeedback Does Not Improve the Reliability of Target Delineation Using Maximum Intensity Projection in 4-Dimensional Computed Tomography Radiation Therapy Planning

    SciTech Connect

    Lu, Wei; Neuner, Geoffrey A.; George, Rohini; Wang, Zhendong; Sasor, Sarah; Huang, Xuan; Regine, William F.; Feigenberg, Steven J.; D'Souza, Warren D.

    2014-01-01

    Purpose: To investigate whether coaching patients' breathing would improve the match between ITV{sub MIP} (internal target volume generated by contouring in the maximum intensity projection scan) and ITV{sub 10} (generated by combining the gross tumor volumes contoured in 10 phases of a 4-dimensional CT [4DCT] scan). Methods and Materials: Eight patients with a thoracic tumor and 5 patients with an abdominal tumor were included in an institutional review board-approved prospective study. Patients underwent 3 4DCT scans with: (1) free breathing (FB); (2) coaching using audio-visual (AV) biofeedback via the Real-Time Position Management system; and (3) coaching via a spirometer system (Active Breathing Coordinator or ABC). One physician contoured all scans to generate the ITV{sub 10} and ITV{sub MIP}. The match between ITV{sub MIP} and ITV{sub 10} was quantitatively assessed with volume ratio, centroid distance, root mean squared distance, and overlap/Dice coefficient. We investigated whether coaching (AV or ABC) or uniform expansions (1, 2, 3, or 5 mm) of ITV{sub MIP} improved the match. Results: Although both AV and ABC coaching techniques improved frequency reproducibility and ABC improved displacement regularity, neither improved the match between ITV{sub MIP} and ITV{sub 10} over FB. On average, ITV{sub MIP} underestimated ITV{sub 10} by 19%, 19%, and 21%, with centroid distance of 1.9, 2.3, and 1.7 mm and Dice coefficient of 0.87, 0.86, and 0.88 for FB, AV, and ABC, respectively. Separate analyses indicated a better match for lung cancers or tumors not adjacent to high-intensity tissues. Uniform expansions of ITV{sub MIP} did not correct for the mismatch between ITV{sub MIP} and ITV{sub 10}. Conclusions: In this pilot study, audio-visual biofeedback did not improve the match between ITV{sub MIP} and ITV{sub 10}. In general, ITV{sub MIP} should be limited to lung cancers, and modification of ITV{sub MIP} in each phase of the 4DCT data set is recommended.

  16. TH-E-17A-06: Anatomical-Adaptive Compressed Sensing (AACS) Reconstruction for Thoracic 4-Dimensional Cone-Beam CT

    SciTech Connect

    Shieh, C; Kipritidis, J; OBrien, R; Cooper, B; Kuncic, Z; Keall, P

    2014-06-15

    Purpose: The Feldkamp-Davis-Kress (FDK) algorithm currently used for clinical thoracic 4-dimensional (4D) cone-beam CT (CBCT) reconstruction suffers from noise and streaking artifacts due to projection under-sampling. Compressed sensing theory enables reconstruction of under-sampled datasets via total-variation (TV) minimization, but TV-minimization algorithms such as adaptive-steepest-descent-projection-onto-convex-sets (ASD-POCS) often converge slowly and are prone to over-smoothing anatomical details. These disadvantages can be overcome by incorporating general anatomical knowledge via anatomy segmentation. Based on this concept, we have developed an anatomical-adaptive compressed sensing (AACS) algorithm for thoracic 4D-CBCT reconstruction. Methods: AACS is based on the ASD-POCS framework, where each iteration consists of a TV-minimization step and a data fidelity constraint step. Prior to every AACS iteration, four major thoracic anatomical structures - soft tissue, lungs, bony anatomy, and pulmonary details - were segmented from the updated solution image. Based on the segmentation, an anatomical-adaptive weighting was applied to the TV-minimization step, so that TV-minimization was enhanced at noisy/streaky regions and suppressed at anatomical structures of interest. The image quality and convergence speed of AACS was compared to conventional ASD-POCS using an XCAT digital phantom and a patient scan. Results: For the XCAT phantom, the AACS image represented the ground truth better than the ASD-POCS image, giving a higher structural similarity index (0.93 vs. 0.84) and lower absolute difference (1.1*10{sup 4} vs. 1.4*10{sup 4}). For the patient case, while both algorithms resulted in much less noise and streaking than FDK, the AACS image showed considerably better contrast and sharpness of the vessels, tumor, and fiducial marker than the ASD-POCS image. In addition, AACS converged over 50% faster than ASD-POCS in both cases. Conclusions: The proposed AACS

  17. Studies in Scanning Probe Microscopy.

    DTIC Science & Technology

    2007-11-02

    refereed journals, as well as two books titled Scanning Force Microscopy, With Applications to Electric, Magnetic, and Atomic Forces published by Oxford University Press in 1991 and a revised edition in 1994.

  18. Fluorescence Microscopy of Single Molecules

    ERIC Educational Resources Information Center

    Zimmermann, Jan; van Dorp, Arthur; Renn, Alois

    2004-01-01

    The investigation of photochemistry and photophysics of individual quantum systems is described with the help of a wide-field fluorescence microscopy approach. The fluorescence single molecules are observed in real time.

  19. En face coherence microscopy [Invited

    PubMed Central

    Thouvenin, Olivier; Grieve, Kate; Xiao, Peng; Apelian, Clement; Boccara, A. Claude

    2017-01-01

    En face coherence microscopy or flying spot or full field optical coherence tomography or microscopy (FF-OCT/FF-OCM) belongs to the OCT family because the sectioning ability is mostly linked to the source coherence length. In this article we will focus our attention on the advantages and the drawbacks of the following approaches: en face versus B scan tomography in terms of resolution, coherent versus incoherent illumination and influence of aberrations, and scanning versus full field imaging. We then show some examples to illustrate the diverse applications of en face coherent microscopy and show that endogenous or exogenous contrasts can add valuable information to the standard morphological image. To conclude we discuss a few domains that appear promising for future development of en face coherence microscopy. PMID:28270972

  20. Computer microscopy in lymphoma diagnostics

    NASA Astrophysics Data System (ADS)

    Mozhenkova, A. V.; Tupitsin, N. N.; Frenkel, M. A.; Falaleeva, N. A.; Nikitaev, V. G.; Polyakov, E. V.

    2017-01-01

    The article describes the application of computer microscopy with multi-spectral camera for the comparative characteristics of normal lymphocytes and lymphoid cells in follicular lymphoma. Wavelet functions are used to quantify parameters of the cells nuclei images.

  1. Vertically scanned laser sheet microscopy.

    PubMed

    Dong, Di; Arranz, Alicia; Zhu, Shouping; Yang, Yujie; Shi, Liangliang; Wang, Jun; Shen, Chen; Tian, Jie; Ripoll, Jorge

    2014-01-01

    Laser sheet microscopy is a widely used imaging technique for imaging the three-dimensional distribution of a fluorescence signal in fixed tissue or small organisms. In laser sheet microscopy, the stripe artifacts caused by high absorption or high scattering structures are very common, greatly affecting image quality. To solve this problem, we report here a two-step procedure which consists of continuously acquiring laser sheet images while vertically displacing the sample, and then using the variational stationary noise remover (VSNR) method to further reduce the remaining stripes. Images from a cleared murine colon acquired with a vertical scan are compared with common stitching procedures demonstrating that vertically scanned light sheet microscopy greatly improves the performance of current light sheet microscopy approaches without the need for complex changes to the imaging setup and allows imaging of elongated samples, extending the field of view in the vertical direction.

  2. Magnetic Force Microscopy in Liquids.

    PubMed

    Ares, Pablo; Jaafar, Miriam; Gil, Adriana; Gómez-Herrero, Julio; Asenjo, Agustina

    2015-09-01

    In this work, the use of magnetic force microscopy (MFM) to acquire images of magnetic nanostructures in liquid environments is presented. Optimization of the MFM signal acquisition in liquid media is performed and it is applied to characterize the magnetic signal of magnetite nanoparticles. The ability for detecting magnetic nanostructures along with the well-known capabilities of atomic force microscopy in liquids suggests potential applications in fields such as nanomedicine, nanobiotechnology, or nanocatalysis.

  3. Confocal microscopy in transmitted light

    NASA Astrophysics Data System (ADS)

    Dodt, Hans-Ulrich; Becker, Klaus

    2003-10-01

    We developed a confocal microscope for transmitted light to visualize fine details in phase objects like unstained biological specimens. The main difficulty of confocal microscopy in transmission is the alignment of illumination and detector pinholes. This alignment was achieved by using "electronic pinholes" on the detector side. As a first step, we were able to image cells in onion skin at greater depths and with higher resolution than by using conventional microscopy.

  4. Low Voltage Scanning Electron Microscopy

    DTIC Science & Technology

    1988-10-01

    Microscopy List of Keywords ,Scanning electron microscopy SEM X -ray .Micoranalysis EDX/EDS -%Low voltage , High resolution -Ceramic surfaces Supported...energy component normal to the surface). (a) Applications to x -ray microanalysis The essential problem leading to the specification of a LVSEM is...illustrated (Fig.l), for a conventional microprobe operated with 20nA probe current, by the contrast of the alumunium (K) x -ray signal as the probe is scanned

  5. Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

    PubMed

    Haynes-Gimore, Nikesha; Banach, Maureen; Brown, Edward; Dawes, Ryan; Edholm, Eva-Stina; Kim, Minsoo; Robert, Jacques

    2015-12-15

    Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

  6. Systemic and Microvascular Effects of Resuscitation with Blood Products After Severe Hemorrhage in Rats

    DTIC Science & Technology

    2014-01-01

    thickness, blood flow, and microvascular permeability were studied using intravital microscopy. Hemodynamics and coagulation tests (rotational...Microcirculation; packed red blood cells; intravital microscopy; ROTEM; plasma proteins; rats. Current US military guidance for the resuscitation of warfighters...was used for coagulation assays (rotational thromboelast- ometry [ROTEM], TEM Innovations GmbH, Germany). Intravital Microscopy and Animal Preparation

  7. Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos

    NASA Astrophysics Data System (ADS)

    Chetty, S. Shashank; Praneetha, S.; Basu, Sandeep; Sachidanandan, Chetana; Murugan, A. Vadivel

    2016-05-01

    Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 μg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 μg/mL. Hence, we propose that the rapid, low-cost, large-scale “sustainable” MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI).

  8. Accuracy of Routine Treatment Planning 4-Dimensional and Deep-Inspiration Breath-Hold Computed Tomography Delineation of the Left Anterior Descending Artery in Radiation Therapy

    SciTech Connect

    White, Benjamin M.; Vennarini, Sabina; Lin, Lilie; Freedman, Gary; Santhanam, Anand; Low, Daniel A.; Both, Stefan

    2015-03-15

    Purpose: To assess the feasibility of radiation therapy treatment planning 4-dimensional computed tomography (4DCT) and deep-inspiration breath-hold (DIBH) CT to accurately contour the left anterior descending artery (LAD), a primary indicator of radiation-induced cardiac toxicity for patients undergoing radiation therapy. Methods and Materials: Ten subjects were prospectively imaged with a cardiac-gated MRI protocol to determine cardiac motion effects, including the displacement of a region of interest comprising the LAD. A series of planar views were obtained and resampled to create a 3-dimensional (3D) volume. A 3D optical flow deformable image registration algorithm determined tissue displacement during the cardiac cycle. The measured motion was then used as a spatial boundary to characterize motion blurring of the radiologist-delineated LAD structure for a cohort of 10 consecutive patients enrolled prospectively on a breast study including 4DCT and DIBH scans. Coronary motion–induced blurring artifacts were quantified by applying an unsharp filter to accentuate the LAD structure despite the presence of motion blurring. The 4DCT maximum inhalation and exhalation respiratory phases were coregistered to determine the LAD displacement during tidal respiration, as visualized in 4DCT. Results: The average 90th percentile heart motion for the region of interest was 0.7 ± 0.1 mm (left–right [LR]), 1.3 ± 0.6 mm (superior–inferior [SI]), and 0.6 ± 0.2 mm (anterior–posterior [AP]) in the cardiac-gated MRI cohort. The average relative increase in the number of voxels comprising the LAD contour was 69.4% ± 4.5% for the DIBH. The LAD volume overestimation had the dosimetric impact of decreasing the reported mean LAD dose by 23% ± 9% on average in the DIBH. During tidal respiration the average relative LAD contour increase was 69.3% ± 5.9% and 67.9% ± 4.6% for inhalation and exhalation respiratory phases, respectively. The average 90th

  9. Structured line illumination Raman microscopy

    PubMed Central

    Watanabe, Kozue; Palonpon, Almar F.; Smith, Nicholas I.; Chiu, Liang-da; Kasai, Atsushi; Hashimoto, Hitoshi; Kawata, Satoshi; Fujita, Katsumasa

    2015-01-01

    In the last couple of decades, the spatial resolution in optical microscopy has increased to unprecedented levels by exploiting the fluorescence properties of the probe. At about the same time, Raman imaging techniques have emerged as a way to image inherent chemical information in a sample without using fluorescent probes. However, in many applications, the achievable resolution is limited to about half the wavelength of excitation light. Here we report the use of structured illumination to increase the spatial resolution of label-free spontaneous Raman microscopy, generating highly detailed spatial contrast from the ensemble of molecular information in the sample. Using structured line illumination in slit-scanning Raman microscopy, we demonstrate a marked improvement in spatial resolution and show the applicability to a range of samples, including both biological and inorganic chemical component mapping. This technique is expected to contribute towards greater understanding of chemical component distributions in organic and inorganic materials. PMID:26626144

  10. Computational 'microscopy' of cellular membranes.

    PubMed

    Ingólfsson, Helgi I; Arnarez, Clément; Periole, Xavier; Marrink, Siewert J

    2016-01-15

    Computational 'microscopy' refers to the use of computational resources to simulate the dynamics of a molecular system. Tuned to cell membranes, this computational 'microscopy' technique is able to capture the interplay between lipids and proteins at a spatio-temporal resolution that is unmatched by other methods. Recent advances allow us to zoom out from individual atoms and molecules to supramolecular complexes and subcellular compartments that contain tens of millions of particles, and to capture the complexity of the crowded environment of real cell membranes. This Commentary gives an overview of the main concepts of computational 'microscopy' and describes the state-of-the-art methods used to model cell membrane processes. We illustrate the power of computational modelling approaches by providing a few in-depth examples of large-scale simulations that move up from molecular descriptions into the subcellular arena. We end with an outlook towards modelling a complete cell in silico.

  11. Correlative microscopy of detergent granules.

    PubMed

    van Dalen, G; Nootenboom, P; Heussen, P C M

    2011-03-01

    The microstructure of detergent products for textile cleaning determines to a large extent the physical properties of these products. Correlative microscopy was used to reveal the microstructure by reconciling images obtained by scanning electron microscopy with energy dispersive X-ray analysis, X-ray microtomography and Fourier transform infrared microscopy. These techniques were applied on the same location of a subsample of a spray-dried detergent base powder embedded in polyacrylate. In this way, the three-dimensional internal and external structure of detergent granules could be investigated from milli to nano scale with detailed spatial information about the components present. This will generate knowledge how to design optimal microstructures for laundry products to obtain product properties demanded by the market. This method is also very useful for other powder systems used in a large variety of industries (e.g. for pharmaceutical, food, ceramic and metal industries).

  12. Confocal microscopy and exfoliative cytology

    PubMed Central

    Reddy, Shyam Prasad; Ramani, Pratibha; Nainani, Purshotam

    2013-01-01

    Context: Early detection of potentially malignant lesions and invasive squamous-cell carcinoma in the oral cavity could be greatly improved through techniques that permit visualization of subtle cellular changes indicative of the neoplastic transformation process. One such technique is confocal microscopy. Combining rapidity with reliability, an innovative idea has been put forward using confocal microscope in exfoliative cytology. Aims: The main objective of this study was to assess confocal microscopy for cytological diagnosis and the results were compared with that of the standard PAP stain. Settings and Design: Confocal microscope, acridine orange (AO) stain, PAP (Papanicolaou) stain. The study was designed to assess confocal microscopy for cytological diagnosis. In the process, smears of patients with (clinically diagnosed and/or suspected) oral squamous cell carcinoma as well as those of controls (normal people) were stained with acridine orange and observed under confocal microscope. The results were compared with those of the standard PAP method. Materials and Methods: Samples of buccal mucosa smears from normal patients and squamous cell carcinoma patients were made, fixed in 100% alcohol, followed by AO staining. The corresponding set of smears was stained with PAP stain using rapid PAP stain kit. The results obtained were compared with those obtained with AO confocal microscopy. Results: The study had shown nuclear changes (malignant cells) in the smears of squamous cell carcinoma patients as increased intensity of fluorescence of the nucleus, when observed under confocal microscope. Acridine orange confocal microscopy showed good amount of sensitivity and specificity (93%) in identifying malignant cells in exfoliative cytological smears. Conclusion: Confocal microscopy was found to have good sensitivity in the identification of cancer (malignant) cells in exfoliative cytology, at par with the PAP method. The rapidity of processing and screening a

  13. DHMI: dynamic holographic microscopy interface

    NASA Astrophysics Data System (ADS)

    He, Xuefei; Zheng, Yujie; Lee, Woei Ming

    2016-12-01

    Digital holographic microscopy (DHM) is a powerful in-vitro biological imaging tool. In this paper, we report a fully automated off-axis digital holographic microscopy system completed with a graphical user interface in the Matlab environment. The interface primarily includes Fourier domain processing, phase reconstruction, aberration compensation and autofocusing. A variety of imaging operations such as region of interest selection, de-noising mode (filtering and averaging), low frame rate imaging for immediate reconstruction and high frame rate imaging routine ( 27 fps) are implemented to facilitate ease of use.

  14. The future of electron microscopy

    SciTech Connect

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifies to the importance of modern microscopy.

  15. The future of electron microscopy

    DOE PAGES

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifiesmore » to the importance of modern microscopy.« less

  16. Optical microscopy versus scanning electron microscopy in urolithiasis.

    PubMed

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

    2009-10-01

    Stone analysis is incompletely done in many clinical centers. Identification of the stone component is essential for deciding future prophylaxis. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) still remains a distant dream for routine hospital work. It is in this context that optical microscopy is suggested as an alternate procedure. The objective of this article was to assess the utility of an optical microscope which gives magnification of up to 40x and gives clear picture of the surface of the stones. In order to authenticate the morphological analysis of urinary stones, SEM and elemental distribution analysis were performed. A total of 250 urinary stones of different compositions were collected from stone clinic, photographed, observed under an optical microscope, and optical photographs were taken at different angles. Twenty-five representative samples among these were gold sputtered to make them conductive and were fed into the SEM machine. Photographs of the samples were taken at different angles at magnifications up to 4,000. Elemental distribution analysis (EDAX) was done to confirm the composition. The observations of the two studies were compared. The different appearances of the stones under optical illuminated microscopy were mostly standardized appearances, namely bosselations of pure whewellite, spiculations of weddellite, bright yellow colored appearance of uric acid, and dirty white amorphous appearance of phosphates. SEM and EDAX gave clearer pictures and gave added confirmation of the stone composition. From the references thus obtained, it was possible to confirm the composition by studying the optical microscopic pictures. Higher magnification capacity of the SEM and the EDAX patterns are useful to give reference support for performing optical microscopy work. After standardization, routine analysis can be performed with optical microscopy. The advantage of the optical microscope is that, it

  17. Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization.

    PubMed

    Xiang, Jiayun; Cai, Xiaolei; Lou, Xiaoding; Feng, Guangxue; Min, Xuehong; Luo, Wenwen; He, Bairong; Goh, Chi Ching; Ng, Lai Guan; Zhou, Jian; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong

    2015-07-15

    Fluorescent organic dots are emerging as promising bioimaging reagents because of their high brightness, good photostability, excellent biocompatibility, and facile surface functionalization. Organic dots with large two-photon absorption (TPA) cross sections are highly desired for two-photon fluorescence microscopy. In this work, we report two biocompatible and photostable organic dots fabricated by encapsulating tetraphenylethene derivatives within DSPE-PEG matrix. The two organic dots show absorption maxima at 425 and 483 nm and emit green and red fluorescence at 560 and 645 nm, with high fluorescence quantum yields of 64% and 22%, respectively. Both organic dots exhibit excellent TPA property in the range of 800-960 nm, affording upon excitation at 820 nm remarkably large TPA cross sections of 1.2×10(6) and 2.5×10(6) GM on the basis of dot concentration. The bare fluorophores and their organic dots are biocompatible and have been used to stain living cells for one- and two-photon fluorescence bioimagings. The cRGD-modified organic dots can selectively target integrin αvβ3 overexpressing breast cancer cells for targeted imaging. The organic dots are also applied for real-time two-photon fluorescence in vivo visualization of the blood vasculature of mouse ear, providing the spatiotemporal information about the whole blood vascular network. These results demonstrate that the present fluorescent organic dots are promising candidates for living cell and tissue imaging.

  18. Color-televised medical microscopy

    NASA Technical Reports Server (NTRS)

    Heath, M. A.; Peck, J. C.

    1968-01-01

    Color television microscopy used at laboratory range magnifications, reproduces a slide image with sufficient fidelity for medical laboratory and instructional use. The system is used for instant pathological reporting between operating room and remotely located pathologist viewing a biopsy through this medium.

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

    PubMed

    Davis, Brynmor J; Marks, Daniel L; Ralston, Tyler S; Carney, P Scott; Boppart, Stephen A

    2008-06-01

    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.

  20. Sustainable, Rapid Synthesis of Bright-Luminescent CuInS2-ZnS Alloyed Nanocrystals: Multistage Nano-xenotoxicity Assessment and Intravital Fluorescence Bioimaging in Zebrafish-Embryos

    PubMed Central

    Chetty, S. Shashank; Praneetha, S.; Basu, Sandeep; Sachidanandan, Chetana; Murugan, A. Vadivel

    2016-01-01

    Near-infrared (NIR) luminescent CuInS2-ZnS alloyed nanocrystals (CIZS-NCs) for highly fluorescence bioimaging have received considerable interest in recent years. Owing, they became a desirable alternative to heavy-metal based-NCs and organic dyes with unique optical properties and low-toxicity for bioimaging and optoelectronic applications. In the present study, bright and robust CIZS-NCs have been synthesized within 5 min, as-high-as 230 °C without requiring any inert-gas atmosphere via microwave-solvothermal (MW-ST) method. Subsequently, the in vitro and in vivo nano-xenotoxicity and cellular uptake of the MUA-functionalized CIZS-NCs were investigated in L929, Vero, MCF7 cell lines and zebrafish-embryos. We observed minimal toxicity and acute teratogenic consequences upto 62.5 μg/ml of the CIZS-NCs in zebrafish-embryos. We also observed spontaneous uptake of the MUA-functionalized CIZS-NCs by 3 dpf older zebrafish-embryos that are evident through bright red fluorescence-emission at a low concentration of 7.8 μg/mL. Hence, we propose that the rapid, low-cost, large-scale “sustainable” MW-ST synthesis of CIZS-NCs, is an ideal bio-nanoprobe with good temporal and spatial resolution for rapid labeling, long-term in vivo tracking and intravital-fluorescence-bioimaging (IVBI). PMID:27188464

  1. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2016-07-12

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

  2. Materials science through electron microscopy

    NASA Astrophysics Data System (ADS)

    Fujita, Hiroshi

    1992-03-01

    Electron microscopy has greatly contributed as a powerful tool in both the characterization and identification of materials in the atomic scale. In these contributions, the most important advantage is it's ability for dynamic study of phenomena, i.e., in situ experiments. This research has been carried out using high voltage electron microscopes, but some results have been obtained with high resolution electron microscopes under critical conditions. Electron microscopy has been improved further to become an indispensable ?Micro-Laboratory? in which formation of various advance materials can also be carried out precisely in the atomic scale. Electron beam science and engineering is a typical example in this research field, and detailed processes of crystalline-amorphous transition and electron irradiation induced foreign atom implantation have been clarified by this method. Recently, new applications to the research fields of non-linear material behavior, such as the behavior of atom clusters and the role of electric dipoles on diffusion, have been carried out.

  3. Electron microscopy of electromagnetic waveforms

    NASA Astrophysics Data System (ADS)

    Ryabov, A.; Baum, P.

    2016-07-01

    Rapidly changing electromagnetic fields are the basis of almost any photonic or electronic device operation. We report how electron microscopy can measure collective carrier motion and fields with subcycle and subwavelength resolution. A collimated beam of femtosecond electron pulses passes through a metamaterial resonator that is previously excited with a single-cycle electromagnetic pulse. If the probing electrons are shorter in duration than half a field cycle, then time-frozen Lorentz forces distort the images quasi-classically and with subcycle time resolution. A pump-probe sequence reveals in a movie the sample’s oscillating electromagnetic field vectors with time, phase, amplitude, and polarization information. This waveform electron microscopy can be used to visualize electrodynamic phenomena in devices as small and fast as available.

  4. Contact microscopy with synchrotron radiation

    SciTech Connect

    Panessa-Warren, B.J.

    1985-10-01

    Soft x-ray contact microscopy with synchrotron radiation offers the biologist and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM or SEM methods (i.e. hydrated samples, samples easily damaged by an electron beam, electron dense samples, thick specimens, unstained low contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash x-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of x-ray wavelengths or specific individual wavelengths which optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of x-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples. 24 refs., 10 figs.

  5. Dynamic imaging with electron microscopy

    SciTech Connect

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-02-20

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

  6. Rotational scanning atomic force microscopy.

    PubMed

    Ulčinas, A; Vaitekonis, Š

    2017-03-10

    A non-raster scanning technique for atomic force microscopy (AFM) imaging which combines rotational and translational motion is presented. The use of rotational motion for the fast scan axis allows us to significantly increase the scanning speed while imaging a large area (diameter > 30 μm). An image reconstruction algorithm and the factors influencing the resolution of the technique are discussed. The experimental results show the potential of the rotational scanning technique for high-throughput large area AFM investigation.

  7. Hyperspectral holographic Fourier-microscopy

    SciTech Connect

    Kalenkov, G S; Kalenkov, S G; Shtan'ko, A E

    2015-04-30

    A detailed theory of the method of holographic recording of hyperspectral wave fields is developed. New experimentally obtained hyperspectral holographic images of microscopic objects are presented. The possibilities of the method are demonstrated experimentally using the examples of urgent microscopy problems: speckle noise suppression, obtaining hyperspectral image of a microscopic object, as well as synthesis of a colour image and obtaining an optical profile of a phase object. (holography)

  8. Rotational scanning atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Ulčinas, A.; Vaitekonis, Š.

    2017-03-01

    A non-raster scanning technique for atomic force microscopy (AFM) imaging which combines rotational and translational motion is presented. The use of rotational motion for the fast scan axis allows us to significantly increase the scanning speed while imaging a large area (diameter > 30 μm). An image reconstruction algorithm and the factors influencing the resolution of the technique are discussed. The experimental results show the potential of the rotational scanning technique for high-throughput large area AFM investigation.

  9. A history of urine microscopy.

    PubMed

    Cameron, J Stewart

    2015-11-01

    The naked-eye appearance of the urine must have been studied by shamans and healers since the Stone Age, and an elaborate interpretation of so-called Uroscopy began around 600 AD as a form of divination. A 1000 years later, the first primitive monocular and compound microscopes appeared in the Netherlands, and along with many other objects and liquids, urine was studied from around 1680 onwards as the enlightenment evolved. However, the crude early instruments did not permit fine study because of chromatic and linear/spherical blurring. Only after complex multi-glass lenses which avoided these problems had been made and used in the 1820s in London by Lister, and in Paris by Chevalier and Amici, could urinary microscopy become a practical, clinically useful tool in the 1830s. Clinical urinary microscopy was pioneered by Rayer and his pupils in Paris (especially Vigla), in the late 1830s, and spread to UK and Germany in the 1840s, with detailed descriptions and interpretations of cells and formed elements of the urinary sediment by Nasse, Henle, Robinson and Golding Bird. Classes were held, most notably by Donné in Paris. After another 50 years, optical microscopy had reached its apogee, with magnifications of over 1000 times obtainable free of aberration, using immersion techniques. Atlases of the urinary sediment were published in all major European countries and in the US. Polarised light and phase contrast was used also after 1900 to study urine, and by the early 20th century, photomicroscopy (pioneered by Donné and Daguerre 50 years previously, but then ignored) became usual for teaching and recording. In the 1940s electron microscopy began, followed by detection of specific proteins and cells using immunofluorescent antibodies. All this had been using handheld methodology. Around 1980, machine-assisted observations began, and have dominated progress since.

  10. Mitochondrial Permeability Transition in Pathogenesis of Hemorrhagic Injury: Targeted Therapy with Minocycline

    DTIC Science & Technology

    2012-03-01

    minocy- cline treatment (Figures 1-4). Minocycline also improved mitochondrial function as assessed by intravital multiphoton imaging of the...will make direct measurements by intravital multiphoton microscopy to determine whether onset of the mitochondrial permeability transition and...oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization were assessed by intravital microscopy. After H/R with vehicle or

  11. Paleomagnetic Analysis Using SQUID Microscopy

    NASA Technical Reports Server (NTRS)

    Weiss, Benjamin P.; Lima, Eduardo A.; Fong, Luis E.; Baudenbacher, Franz J.

    2007-01-01

    Superconducting quantum interference device (SQUID) microscopes are a new generation of instruments that map magnetic fields with unprecedented spatial resolution and moment sensitivity. Unlike standard rock magnetometers, SQUID microscopes map magnetic fields rather than measuring magnetic moments such that the sample magnetization pattern must be retrieved from source model fits to the measured field data. In this paper, we presented the first direct comparison between paleomagnetic analyses on natural samples using joint measurements from SQUID microscopy and moment magnetometry. We demonstrated that in combination with apriori geologic and petrographic data, SQUID microscopy can accurately characterize the magnetization of lunar glass spherules and Hawaiian basalt. The bulk moment magnitude and direction of these samples inferred from inversions of SQUID microscopy data match direct measurements on the same samples using moment magnetometry. In addition, these inversions provide unique constraints on the magnetization distribution within the sample. These measurements are among the most sensitive and highest resolution quantitative paleomagnetic studies of natural remanent magnetization to date. We expect that this technique will be able to extend many other standard paleomagnetic techniques to previously inaccessible microscale samples.

  12. Multi-photon excitation microscopy

    PubMed Central

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-01-01

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments. PMID:16756664

  13. Multi-photon excitation microscopy.

    PubMed

    Diaspro, Alberto; Bianchini, Paolo; Vicidomini, Giuseppe; Faretta, Mario; Ramoino, Paola; Usai, Cesare

    2006-06-06

    Multi-photon excitation (MPE) microscopy plays a growing role among microscopical techniques utilized for studying biological matter. In conjunction with confocal microscopy it can be considered the imaging workhorse of life science laboratories. Its roots can be found in a fundamental work written by Maria Goeppert Mayer more than 70 years ago. Nowadays, 2PE and MPE microscopes are expected to increase their impact in areas such biotechnology, neurobiology, embryology, tissue engineering, materials science where imaging can be coupled to the possibility of using the microscopes in an active way, too. As well, 2PE implementations in noninvasive optical bioscopy or laser-based treatments point out to the relevance in clinical applications. Here we report about some basic aspects related to the phenomenon, implications in three-dimensional imaging microscopy, practical aspects related to design and realization of MPE microscopes, and we only give a list of potential applications and variations on the theme in order to offer a starting point for advancing new applications and developments.

  14. Holographic opto-fluidic microscopy.

    PubMed

    Bishara, Waheb; Zhu, Hongying; Ozcan, Aydogan

    2010-12-20

    Over the last decade microfluidics has created a versatile platform that has significantly advanced the ways in which micro-scale organisms and objects are controlled, processed and investigated, by improving the cost, compactness and throughput aspects of analysis. Microfluidics has also expanded into optics to create reconfigurable and flexible optical devices such as reconfigurable lenses, lasers, waveguides, switches, and on-chip microscopes. Here we present a new opto-fluidic microscopy modality, i.e., Holographic Opto-fluidic Microscopy (HOM), based on lensless holographic imaging. This imaging modality complements the miniaturization provided by microfluidics and would allow the integration of microscopy into existing on-chip microfluidic devices with various functionalities. Our imaging modality utilizes partially coherent in-line holography and pixel super-resolution to create high-resolution amplitude and phase images of the objects flowing within micro-fluidic channels, which we demonstrate by imaging C. elegans, Giardia lamblia, and Mulberry pollen. HOM does not involve complicated fabrication processes or precise alignment, nor does it require a highly uniform flow of objects within microfluidic channels.

  15. Multiphoton microscopy of atheroslcerotic plaques

    NASA Astrophysics Data System (ADS)

    Lilledahl, Magnus B.; de Lange Davies, Catharina; Haugen, Olav A.; Svaasand, Lars O.

    2007-02-01

    Multiphoton microscopy is a techniques that fascilitates three dimensional imaging of intact, unstained tissue. Especially connective tissue has a relatively strong nonlinear optical response and can easily be imaged. Atherosclerosis is a disease where lipids accumulate in the vessel wall and there is a thickening of the intima by growth of a cap of connective tissue. The mechanical strength of this fibrous cap is of clinically importance. If the cap ruptures a thrombosis forms which can block a coronary vessel and therby causing myocardial infarction. Multiphoton microscopy can be used to image the fibrous cap and thereby determine the thickness of the cap and the structure of the connective fibres. This could possibly be developed into a diagnostic and clincal tool to monitor the vulnerability of a plaque and also to better understand the development of a plaque and effects of treatment. We have collected multiphoton microscopy images from atherosclerotic plaque in human aorta, both two photon excited fluorescens and second harmonic generated signal. The feasability of using this technique to determine the state of the plaque is explored.

  16. 3D structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Dougherty, William M.; Goodwin, Paul C.

    2011-03-01

    Three-dimensional structured illumination microscopy achieves double the lateral and axial resolution of wide-field microscopy, using conventional fluorescent dyes, proteins and sample preparation techniques. A three-dimensional interference-fringe pattern excites the fluorescence, filling in the "missing cone" of the wide field optical transfer function, thereby enabling axial (z) discrimination. The pattern acts as a spatial carrier frequency that mixes with the higher spatial frequency components of the image, which usually succumb to the diffraction limit. The fluorescence image encodes the high frequency content as a down-mixed, moiré-like pattern. A series of images is required, wherein the 3D pattern is shifted and rotated, providing down-mixed data for a system of linear equations. Super-resolution is obtained by solving these equations. The speed with which the image series can be obtained can be a problem for the microscopy of living cells. Challenges include pattern-switching speeds, optical efficiency, wavefront quality and fringe contrast, fringe pitch optimization, and polarization issues. We will review some recent developments in 3D-SIM hardware with the goal of super-resolved z-stacks of motile cells.

  17. Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

    PubMed Central

    Liv, Nalan; van Oosten Slingeland, Daan S. B.; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W.; Hoogenboom, Jacob P.

    2016-01-01

    We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

  18. Fluorescence-integrated transmission electron microscopy images: integrating fluorescence microscopy with transmission electron microscopy.

    PubMed

    Sims, Paul A; Hardin, Jeff D

    2007-01-01

    This chapter describes high-pressure freezing (HPF) techniques for correlative light and electron microscopy on the same sample. Laser scanning confocal microscopy (LSCM) is exploited for its ability to collect fluorescent, as well as transmitted and back scattered light (BSL) images at the same time. Fluorescent information from a whole mount (preembedding) or from thin sections (post-embedding) can be displayed as a color overlay on transmission electron microscopy (TEM) images. Fluorescence-integrated TEM (F-TEM) images provide a fluorescent perspective to TEM images. The pre-embedding method uses a thin two-part agarose pad to immobilize live Caenorhabditis elegans embryos for LSCM, HPF, and TEM. Pre-embedding F-TEM images display fluorescent information collected from a whole mount of live embryos onto all thin sections collected from that sample. In contrast, the postembedding method uses HPF and freeze substitution with 1% paraformaldehyde in 95% ethanol followed by low-temperature embedding in methacrylate resin. This procedure preserves the structure and function of green fluorescent protein (GFP) as determined by immunogold labeling of GFP, when compared with GFP expression, both demonstrated in the same thin section.

  19. Combining fluorescence and bioluminescence microscopy.

    PubMed

    Goda, Kazuhito; Hatta-Ohashi, Yoko; Akiyoshi, Ryutaro; Sugiyama, Takashi; Sakai, Ikuko; Takahashi, Takeo; Suzuki, Hirobumi

    2015-08-01

    Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

  20. Twin-photon confocal microscopy.

    PubMed

    Simon, D S; Sergienko, A V

    2010-10-11

    A recently introduced two-channel confocal microscope with correlated detection promises up to 50% improvement in transverse spatial resolution [Simon, Sergienko, Optics Express 18, 9765 (2010)] via the use of photon correlations. Here we achieve similar results in a different manner, introducing a triple-confocal correlated microscope which exploits the correlations present in optical parametric amplifiers. It is based on tight focusing of pump radiation onto a thin sample positioned in front of a nonlinear crystal, followed by coincidence detection of signal and idler photons, each focused onto a pinhole. This approach offers further resolution enhancement in confocal microscopy.

  1. Superresolved spatially multiplexed interferometric microscopy.

    PubMed

    Picazo-Bueno, José Ángel; Zalevsky, Zeev; García, Javier; Micó, Vicente

    2017-03-01

    Superresolution capability by angular and time multiplexing is implemented onto a regular microscope. The technique, named superresolved spatially multiplexed interferometric microscopy (S2MIM), follows our previously reported SMIM technique [Opt. Express22, 14929 (2014)OPEXFF1094-408710.1364/OE.22.014929, J. Biomed. Opt.21, 106007 (2016)JBOPFO1083-366810.1117/1.JBO.21.10.106007] improved with superresolved imaging. All together, S2MIM updates a commercially available non-holographic microscope into a superresolved holographic one. Validation is presented for an Olympus BX-60 upright microscope with resolution test targets.

  2. Confocal microscopy in microgravity research.

    PubMed

    Goede, A P; Brakenhoff, G J; Woldringh, C L; Aalders, J W; Imhof, J P; van Kralingen, P; Mels, W A; Schreinemakers, P; Zegers, A

    1992-01-01

    We have studied the application and the feasibility of confocal scanning laser microscopy (CSLM) in microgravity research. Its superior spatial resolution and 3D imaging capabilities and its use of light as a probe, render this instrument ideally suited for the study of living biological material on a (sub-)cellular level. In this paper a number of pertinent biological microgravity experiments is listed, concentrating on the direct observation of developing cells and cellular structures under microgravity condition. A conceptual instrument design is also presented, aimed at sounding rocket application followed by Biorack/Biolab application at a later stage.

  3. Scanning probe microscopy in catalysis.

    PubMed

    Yeung, King Lun; Yao, Nan

    2004-09-01

    This review discusses the recent progress in the application of scanning probe microscopy (SPM) in catalysis. SPM proves to be an invaluable technique for imaging catalytic surfaces and interfaces. Most SPM research is related to the structural and morphological transformation associated with catalyst preparation and use. Real-time SPM observation of surface dynamics including adsorption, diffusion and reaction, provides invaluable insights to the mechanism of catalysis. SPM is also used to shape and manipulate surfaces and surface processes. Fabrication of nanostructured catalysts, direct manipulation of adsorbed atoms and molecules and tip-mediated reactions are some examples of new SPM approach in catalyst research.

  4. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E.; Parvin, Bahram

    2009-06-09

    The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time: quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

  5. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E; Parvin, Bahram

    2013-10-01

    The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

  6. Visual-servoing optical microscopy

    DOEpatents

    Callahan, Daniel E.; Parvin, Bahram

    2011-05-24

    The present invention provides methods and devices for the knowledge-based discovery and optimization of differences between cell types. In particular, the present invention provides visual servoing optical microscopy, as well as analysis methods. The present invention provides means for the close monitoring of hundreds of individual, living cells over time; quantification of dynamic physiological responses in multiple channels; real-time digital image segmentation and analysis; intelligent, repetitive computer-applied cell stress and cell stimulation; and the ability to return to the same field of cells for long-term studies and observation. The present invention further provides means to optimize culture conditions for specific subpopulations of cells.

  7. Aperture scanning Fourier ptychographic microscopy

    PubMed Central

    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

  8. Image scanning microscopy with radially polarized light

    NASA Astrophysics Data System (ADS)

    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.

  9. Virtual intraoperative surgical photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Changho; Lee, Donghyun; Zhou, Qifa; Kim, Jeehyun; Kim, Chulhong

    2015-07-01

    A virtual intraoperative surgical photoacoustic microscopy at 1064 nm wavelength (VISPAM) system was designed and fabricated by integrating a commercial type surgical microscope and laser scanning photoacoustic microscopy (PAM) with a 1064 nm pulsed laser. Based on simple augmented reality device, VISPAM could simultaneously provide 2D depth-resolved photoacoustic and magnified microscope images of surgery regions on the same vision of surgeon via an eyepiece of the microscope. The invisible 1064 nm laser removed the interruption of surgical sight due to visible laser scanning of previous report, and decreased the danger of tissue damage caused by over irradiated laser. In addition, to approach the real practical surgery application, a needle-type transducer was utilized without a water bath for PA signal coupling. In order to verify our system's performance, we conducted needle guiding as ex vivo phantom study and needle guiding and injection of carbon particles mixtures into a melanoma tumor region as in vivo study. We expect that VISPAM can be essential tool of brain and ophthalmic microsurgery.

  10. Developing Photo Activated Localization Microscopy

    NASA Astrophysics Data System (ADS)

    Hess, Harald

    2015-03-01

    Photo Activated Localization Microscopy, PALM, acquires super-resolution images by activating a subset of activatable fluorescent labels and estimating the center of the each molecular label to sub-diffractive accuracy. When this process is repeated thousands of times for different subsets of molecules, then an image can be rendered from all the center coordinates of the molecules. I will describe the circuitous story of its development that began with another super-resolution technique, NSOM, developed by my colleague Eric Betzig, who imaged single molecules at room temperature, and later we spectrally resolved individual luminescent centers of quantum wells. These two observations inspired a generalized path to localization microscopy, but that path was abandoned because no really useful fluorescent labels were available. After a decade of nonacademic industrial pursuits and the subsequent freedom of unemployment, we came across a class of genetically expressible fluorescent proteins that were switchable or convertible that enabled the concept to be implemented and be biologically promising. The past ten years have been very active with many groups exploring applications and enhancements of this concept. Demonstrating significant biological relevance will be the metric if its success.

  11. RGB digital lensless holographic microscopy

    NASA Astrophysics Data System (ADS)

    Garcia-Sucerquia, Jorge

    2013-11-01

    The recent introduction of color digital lensless holographic microscopy (CDLHM) has shown the possibility of imaging microscopic specimens at full color without the need of lenses. Owing to the simplicity, robustness, and compactness of the digital lensless holographic microscopes (DLHM), they have been presented as the ideal candidates to being developed into portable holographic microscopes. However, in the case of CDLHM the utilization of three independent lasers hinders the portability option for this microscope. In this contribution an alternative to reduce the complexity of CDLHM aimed to recover the portability of this microscopy technology is presented. A super-bright white-light light-emitting diode (LED) is spectrally and spatially filtered to produce the needed illumination by CDLHM to work. CDLHM with LED illumination is used to image at full color a section of the head of a drosophila melanogaster fly (fruit fly). The LED-CDLHM method shows the capability of imaging objects of 2μm size in comparison with the micrometer resolution reported for LASER-CDLHM.

  12. Light Sheet Fluorescence Microscopy (LSFM)

    PubMed Central

    Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

    2015-01-01

    The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

  13. Atomic force microscopy as nanorobot.

    PubMed

    Xi, Ning; Fung, Carmen Kar Man; Yang, Ruiguo; Lai, King Wai Chiu; Wang, Donna H; Seiffert-Sinha, Kristina; Sinha, Animesh A; Li, Guangyong; Liu, Lianqing

    2011-01-01

    Atomic force microscopy (AFM) is a powerful and widely used imaging technique that can visualize single molecules under physiological condition at the nanometer scale. In this chapter, an AFM-based nanorobot for biological studies is introduced. Using the AFM tip as an end effector, the AFM can be modified into a nanorobot that can manipulate biological objects at the single-molecule level. By functionalizing the AFM tip with specific antibodies, the nanorobot is able to identify specific types of receptors on the cell membrane. It is similar to the fluorescent optical microscopy but with higher resolution. By locally updating the AFM image based on interaction force information and objects' model during nanomanipulation, real-time visual feedback is obtained through the augmented reality interface. The development of the AFM-based nanorobotic system enables us to conduct in situ imaging, sensing, and manipulation simultaneously at the nanometer scale (e.g., protein and DNA levels). The AFM-based nanorobotic system offers several advantages and capabilities for studying structure-function relationships of biological specimens. As a result, many biomedical applications can be achieved by the AFM-based nanorobotic system.

  14. Cluster computing for digital microscopy.

    PubMed

    Carrington, Walter A; Lisin, Dimitri

    2004-06-01

    Microscopy is becoming increasingly digital and dependent on computation. Some of the computational tasks in microscopy are computationally intense, such as image restoration (deconvolution), some optical calculations, image segmentation, and image analysis. Several modern microscope technologies enable the acquisition of very large data sets. 3D imaging of live cells over time, multispectral imaging, very large tiled 3D images of thick samples, or images from high throughput biology all can produce extremely large images. These large data sets place a very large burden on laboratory computer resources. This combination of computationally intensive tasks and larger data sizes can easily exceed the capability of single personal computers. The large multiprocessor computers that are the traditional technology for larger tasks are too expensive for most laboratories. An alternative approach is to use a number of inexpensive personal computers as a cluster; that is, use multiple networked computers programmed to run the problem in parallel on all the computers in the cluster. By the use of relatively inexpensive over-the-counter hardware and open source software, this approach can be much more cost effective for many tasks. We discuss the different computer architectures available, and their advantages and disadvantages.

  15. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    Selective labelling, identification and spatial distribution of cell surface biomarkers can provide important clinical information, such as distinction between healthy and diseased cells, evolution of a disease and selection of the optimal patient-specific treatment. Immunofluorescence is the gold standard for efficient detection of biomarkers expressed by cells. However, antibodies (Abs) conjugated to fluorescent dyes remain limited by their photobleaching, high sensitivity to the environment, low light intensity, and wide absorption and emission spectra. Immunoplasmonics is a novel microscopy method based on the visualization of Abs-functionalized plasmonic nanoparticles (fNPs) targeting cell surface biomarkers. Tunable fNPs should provide higher multiplexing capacity than immunofluorescence since NPs are photostable over time, strongly scatter light at their plasmon peak wavelengths and can be easily functionalized. In this article, we experimentally demonstrate accurate multiplexed detection based on the immunoplasmonics approach. First, we achieve the selective labelling of three targeted cell surface biomarkers (cluster of differentiation 44 (CD44), epidermal growth factor receptor (EGFR) and voltage-gated K+ channel subunit KV1.1) on human cancer CD44+ EGFR+ KV1.1+ MDA-MB-231 cells and reference CD44- EGFR- KV1.1+ 661W cells. The labelling efficiency with three stable specific immunoplasmonics labels (functionalized silver nanospheres (CD44-AgNSs), gold (Au) NSs (EGFR-AuNSs) and Au nanorods (KV1.1-AuNRs)) detected by reflected light microscopy (RLM) is similar to the one with immunofluorescence. Second, we introduce an improved method for 3D localization and spectral identification of fNPs based on fast z-scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed

  16. Multiphoton fluorescence microscopy in biology

    NASA Astrophysics Data System (ADS)

    Heikal, Ahmed A.; Webb, Watt W.

    2002-11-01

    The inherent advantages of nonlinear excitation make multiphoton fluorescence microscopy (MPFM) awell-suited imaging technique for extracting valuable information from turbid and thick biological samples. These advantages include high three-dimensional spatial resolution, large penetration depth, minimum out-of-focus cellular photodamage, and high signal-to-noise contrast. We have investigated the nonlinear spectroscopy of biologically important molecules such as NADH, flavins, and intrinsically fluorescent proteins. Fundamental understanding of the molecular spectroscopy and dynamics of these biomolecules is essential for advancing their applications in biological research. MPFM has been utilized for monitoring a large spectrum of biological processes including metabolic activity and exocytosis. We will discuss two-photon (2P) redox fluorescence microscopy of NADH, which gives a quantitative measure of the respiratory chain activity, thus allowing functional imaging of energy metabolism in neurons and native brain tissue. Finally, a rational design strategy, based on donor-acceptor-donor configuration, will be elucidated for fluorescent probes with large 2P-excitation cross-section. These dyes are water-soluble, yet possess a high affinity to organic phases with site-specific labeling and Ca+2 sensitivity (Kd ~ 350 nM). A brief account on the biological application of nanocrystals and second harmonic imaging will be reviewed.

  17. Nanocharacterization: Atomic Scale Visualization with Microscopy

    NASA Astrophysics Data System (ADS)

    Broadbridge, Christine

    2007-10-01

    This workshop will include an overview presentation of nanotechnology and nanocharacterization tools (electron microscopy and atomic force microscopy) as well as examples of curricular components for middle and high school teachers. Tours/demonstrations of microscopy facilities in the IMS facility at UConn will be provided.

  18. Kelvin Probe Force Microscopy in liquid using Electrochemical Force Microscopy

    DOE PAGES

    Collins, Liam; Jesse, Stephen; Kilpatrick, J.; ...

    2015-01-01

    Conventional closed loop-Kelvin probe force microscopy (KPFM) has emerged as a powerful technique for probing electric and transport phenomena at the solid-gas interface. The extension of KPFM capabilities to probe electrostatic and electrochemical phenomena at the solid–liquid interface is of interest for a broad range of applications from energy storage to biological systems. However, the operation of KPFM implicitly relies on the presence of a linear lossless dielectric in the probe-sample gap, a condition which is violated for ionically-active liquids (e.g., when diffuse charge dynamics are present). Here, electrostatic and electrochemical measurements are demonstrated in ionically-active (polar isopropanol, milli-Q watermore » and aqueous NaCl) and ionically-inactive (non-polar decane) liquids by electrochemical force microscopy (EcFM), a multidimensional (i.e., bias- and time-resolved) spectroscopy method. In the absence of mobile charges (ambient and non-polar liquids), KPFM and EcFM are both feasible, yielding comparable contact potential difference (CPD) values. In ionically-active liquids, KPFM is not possible and EcFM can be used to measure the dynamic CPD and a rich spectrum of information pertaining to charge screening, ion diffusion, and electrochemical processes (e.g., Faradaic reactions). EcFM measurements conducted in isopropanol and milli-Q water over Au and highly ordered pyrolytic graphite electrodes demonstrate both sample- and solvent-dependent features. Finally, the feasibility of using EcFM as a local force-based mapping technique of material-dependent electrostatic and electrochemical response is investigated. The resultant high dimensional dataset is visualized using a purely statistical approach that does not require a priori physical models, allowing for qualitative mapping of electrostatic and electrochemical material properties at the solid–liquid interface.« less

  19. Kelvin Probe Force Microscopy in liquid using Electrochemical Force Microscopy

    SciTech Connect

    Collins, Liam; Jesse, Stephen; Kilpatrick, J.; Tselev, Alexander; Okatan, Mahmut Baris; Kalinin, Sergei V.; Rodriguez, Brian

    2015-01-01

    Conventional closed loop-Kelvin probe force microscopy (KPFM) has emerged as a powerful technique for probing electric and transport phenomena at the solid-gas interface. The extension of KPFM capabilities to probe electrostatic and electrochemical phenomena at the solid–liquid interface is of interest for a broad range of applications from energy storage to biological systems. However, the operation of KPFM implicitly relies on the presence of a linear lossless dielectric in the probe-sample gap, a condition which is violated for ionically-active liquids (e.g., when diffuse charge dynamics are present). Here, electrostatic and electrochemical measurements are demonstrated in ionically-active (polar isopropanol, milli-Q water and aqueous NaCl) and ionically-inactive (non-polar decane) liquids by electrochemical force microscopy (EcFM), a multidimensional (i.e., bias- and time-resolved) spectroscopy method. In the absence of mobile charges (ambient and non-polar liquids), KPFM and EcFM are both feasible, yielding comparable contact potential difference (CPD) values. In ionically-active liquids, KPFM is not possible and EcFM can be used to measure the dynamic CPD and a rich spectrum of information pertaining to charge screening, ion diffusion, and electrochemical processes (e.g., Faradaic reactions). EcFM measurements conducted in isopropanol and milli-Q water over Au and highly ordered pyrolytic graphite electrodes demonstrate both sample- and solvent-dependent features. Finally, the feasibility of using EcFM as a local force-based mapping technique of material-dependent electrostatic and electrochemical response is investigated. The resultant high dimensional dataset is visualized using a purely statistical approach that does not require a priori physical models, allowing for qualitative mapping of electrostatic and electrochemical material properties at the solid–liquid interface.

  20. Algorithmic methods in diffraction microscopy

    NASA Astrophysics Data System (ADS)

    Thibault, Pierre

    Recent diffraction imaging techniques use properties of coherent sources (most notably x-rays and electrons) to transfer a portion of the imaging task to computer algorithms. "Diffraction microscopy" is a method which consists in reconstructing the image of a specimen from its diffraction pattern. Because only the amplitude of a wavefield incident on a detector is measured, reconstruction of the image entails to recovering the lost phases. This extension of the 'phase problem" commonly met in crystallography is solved only if additional information is available. The main topic of this thesis is the development of algorithmic techniques in diffraction microscopy. In addition to introducing new methods, it is meant to be a review of the algorithmic aspects of the field of diffractive imaging. An overview of the scattering approximations used in the interpretation of diffraction datasets is first given, as well as a numerical propagation tool useful in conditions where known approximations fail. Concepts central to diffraction microscopy---such as oversampling---are then introduced and other similar imaging techniques described. A complete description of iterative reconstruction algorithms follows, with a special emphasis on the difference map, the algorithm used in this thesis. The formalism, based on constraint sets and projection onto these sets, is then defined and explained. Simple projections commonly used in diffraction imaging are then described. The various ways experimental realities can affect reconstruction methods will then be enumerated. Among the diverse sources of algorithmic difficulties, one finds that noise, missing data and partial coherence are typically the most important. Other related difficulties discussed are the detrimental effects of crystalline domains in a specimen, and the convergence problems occurring when the support of a complex-valued specimen is not well known. The last part of this thesis presents reconstruction results; an

  1. Multiphoton microscopy in life sciences.

    PubMed

    König, K

    2000-11-01

    Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm(-2) to GW cm(-2), which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via

  2. Differential Multiphoton Laser Scanning Microscopy

    SciTech Connect

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2012-01-01

    Multifocal multiphoton laser scanning microscopy (mfMPLSM) in the biological and medical sciences has the potential to become a ubiquitous tool for obtaining high-resolution images at video rates. While current implementations of mfMPLSM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for mfMPLSM in which whole-field detection with a single detector, rather than detection with a matrix of detectors, such as a charge-coupled device (CCD) camera, is implemented. This advance makes mfMPLSM fully compatible for use in imaging through scattering media. Further, we demonstrate that this method makes it possible to simultaneously obtain multiple images and view differences in excitation parameters in a single scan of the specimen.

  3. Superresolution microscopy with transient binding.

    PubMed

    Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

    2016-06-01

    For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution.

  4. Scanning Electrochemical Microscopy in Neuroscience

    NASA Astrophysics Data System (ADS)

    Schulte, Albert; Nebel, Michaela; Schuhmann, Wolfgang

    2010-07-01

    This article reviews recent work involving the application of scanning electrochemical microscopy (SECM) to the study of individual cultured living cells, with an emphasis on topographical and functional imaging of neuronal and secretory cells of the nervous and endocrine system. The basic principles of biological SECM and associated negative amperometric-feedback and generator/collector-mode SECM imaging are discussed, and successful use of the methodology for screening soft and fragile membranous objects is outlined. The drawbacks of the constant-height mode of probe movement and the benefits of the constant-distance mode of SECM operation are described. Finally, representative examples of constant-height and constant-distance mode SECM on a variety of live cells are highlighted to demonstrate the current status of single-cell SECM in general and of SECM in neuroscience in particular.

  5. Phase Aberrations in Diffraction Microscopy

    SciTech Connect

    Marchesini, S; Chapman, H N; Barty, A; Howells, M R; Spence, J H; Cui, C; Weierstall, U; Minor, A M

    2005-09-29

    In coherent X-ray diffraction microscopy the diffraction pattern generated by a sample illuminated with coherent x-rays is recorded, and a computer algorithm recovers the unmeasured phases to synthesize an image. By avoiding the use of a lens the resolution is limited, in principle, only by the largest scattering angles recorded. However, the imaging task is shifted from the experiment to the computer, and the algorithm's ability to recover meaningful images in the presence of noise and limited prior knowledge may produce aberrations in the reconstructed image. We analyze the low order aberrations produced by our phase retrieval algorithms. We present two methods to improve the accuracy and stability of reconstructions.

  6. Differential Multiphoton Laser Scanning Microscopy

    PubMed Central

    Field, Jeffrey J.; Sheetz, Kraig E.; Chandler, Eric V.; Hoover, Erich E.; Young, Michael D.; Ding, Shi-you; Sylvester, Anne W.; Kleinfeld, David; Squier, Jeff A.

    2016-01-01

    Multifocal multiphoton microscopy (MMM) in the biological and medical sciences has become an important tool for obtaining high resolution images at video rates. While current implementations of MMM achieve very high frame rates, they are limited in their applicability to essentially those biological samples that exhibit little or no scattering. In this paper, we report on a method for MMM in which imaging detection is not necessary (single element point detection is implemented), and is therefore fully compatible for use in imaging through scattering media. Further, we demonstrate that this method leads to a new type of MMM wherein it is possible to simultaneously obtain multiple images and view differences in excitation parameters in a single shot. PMID:27390511

  7. Multifocal interferometric synthetic aperture microscopy

    PubMed Central

    Xu, Yang; Chng, Xiong Kai Benjamin; Adie, Steven G.; Boppart, Stephen A.; Scott Carney, P.

    2014-01-01

    There is an inherent trade-off between transverse resolution and depth of field (DOF) in optical coherence tomography (OCT) which becomes a limiting factor for certain applications. Multifocal OCT and interferometric synthetic aperture microscopy (ISAM) each provide a distinct solution to the trade-off through modification to the experiment or via post-processing, respectively. In this paper, we have solved the inverse problem of multifocal OCT and present a general algorithm for combining multiple ISAM datasets. Multifocal ISAM (MISAM) uses a regularized combination of the resampled datasets to bring advantages of both multifocal OCT and ISAM to achieve optimal transverse resolution, extended effective DOF and improved signal-to-noise ratio. We present theory, simulation and experimental results. PMID:24977909

  8. Interference techniques in fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Dogan, Mehmet

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

  9. Resolution enhancement techniques in microscopy

    NASA Astrophysics Data System (ADS)

    Cremer, Christoph; Masters, Barry R.

    2013-05-01

    We survey the history of resolution enhancement techniques in microscopy and their impact on current research in biomedicine. Often these techniques are labeled superresolution, or enhanced resolution microscopy, or light-optical nanoscopy. First, we introduce the development of diffraction theory in its relation to enhanced resolution; then we explore the foundations of resolution as expounded by the astronomers and the physicists and describe the conditions for which they apply. Then we elucidate Ernst Abbe's theory of optical formation in the microscope, and its experimental verification and dissemination to the world wide microscope communities. Second, we describe and compare the early techniques that can enhance the resolution of the microscope. Third, we present the historical development of various techniques that substantially enhance the optical resolution of the light microscope. These enhanced resolution techniques in their modern form constitute an active area of research with seminal applications in biology and medicine. Our historical survey of the field of resolution enhancement uncovers many examples of reinvention, rediscovery, and independent invention and development of similar proposals, concepts, techniques, and instruments. Attribution of credit is therefore confounded by the fact that for understandable reasons authors stress the achievements from their own research groups and sometimes obfuscate their contributions and the prior art of others. In some cases, attribution of credit is also made more complex by the fact that long term developments are difficult to allocate to a specific individual because of the many mutual connections often existing between sometimes fiercely competing, sometimes strongly collaborating groups. Since applications in biology and medicine have been a major driving force in the development of resolution enhancing approaches, we focus on the contribution of enhanced resolution to these fields.

  10. Scanning Tunneling Optical Resonance Microscopy

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila; Wilt, Dave; Raffaelle, Ryne; Gennett, Tom; Tin, Padetha; Lau, Janice; Castro, Stephanie; Jenkins, Philip; Scheiman, Dave

    2003-01-01

    Scanning tunneling optical resonance microscopy (STORM) is a method, now undergoing development, for measuring optoelectronic properties of materials and devices on the nanoscale by means of a combination of (1) traditional scanning tunneling microscopy (STM) with (2) tunable laser spectroscopy. In STORM, an STM tip probing a semiconductor is illuminated with modulated light at a wavelength in the visible-to-near-infrared range and the resulting photoenhancement of the tunneling current is measured as a function of the illuminating wavelength. The photoenhancement of tunneling current occurs when the laser photon energy is sufficient to excite charge carriers into the conduction band of the semiconductor. Figure 1 schematically depicts a proposed STORM apparatus. The light for illuminating the semiconductor specimen at the STM would be generated by a ring laser that would be tunable across the wavelength range of interest. The laser beam would be chopped by an achromatic liquid-crystal modulator. A polarization-maintaining optical fiber would couple the light to the tip/sample junction of a commercial STM. An STM can be operated in one of two modes: constant height or constant current. A STORM apparatus would be operated in the constant-current mode, in which the height of the tip relative to the specimen would be varied in order to keep the tunneling current constant. In this mode, a feedback control circuit adjusts the voltage applied to a piezoelectric actuator in the STM that adjusts the height of the STM tip to keep the tunneling current constant. The exponential relationship between the tunneling current and tip-to-sample distance makes it relatively easy to implement this mode of operation. The choice of method by which the photoenhanced portion of the tunneling current would be measured depends on choice of the frequency at which the input illumination would be modulated (chopped). If the frequency of modulation were low enough (typically < 10 Hz) that the

  11. Developments in optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Rolland, J. P.; Meemon, P.; Thompson, K. P.; Murali, S.; Lee, K. S.

    2010-11-01

    Optical Coherence Microscopy (OCM) utilizes a high NA microscope objective in the sample arm to achieve an axially and laterally high resolution OCT image. An increase in NA, however, leads to a dramatically decreased depth of focus (DOF), and hence shortens the imaging depth range so that high lateral resolution is maintained only within a small depth region around the focal plane. One solution to increase the depth of imaging while keeping a high lateral resolution is dynamic-focusing. Utilizing the voltage controlled refocus capability of a liquid lens, we have recently presented a solution for invariant high resolution imaging using the liquid lens embedded within a fixed optics hand-held custom microscope designed specifically for optical imaging systems using a broadband light source centered at 800 nm with a 120 nm bandwidth. Subsequently, we have developed a Gabor-Domain Optical Coherence Microscopy (GD-OCM) that utilizes the high speed imaging of spectral domain OCT, the high lateral resolution of OCM, and the ability of real time refocusing of our custom design variable focus objective. Finally, key developments in Phase-Resolved Doppler OCT (PR-DOCT) are key enablers to combine high-resolution structural imaging with functional imaging. In this paper we review achievements in GD-OCM and detail how portions of in-focus cross-sectional images can be extracted and fused to form an invariant lateral resolution image with multiple cross-sectional images acquired corresponding to a discrete refocusing step along depth enabled by the varifocal device. We demonstrate sub-cellular resolution imaging of an African frog tadpole (Xenopus Laevis) taken from a 500 μm × 500 μm cross-section as well as cellular imaging in in vivo skin. Finally, A novel dual-detection full-range Fourier-domain optical coherence tomography system was developed that provides 7 μm axial resolution (in air) at about 90 kHz axial scan rate for mirror-image phase resolved Doppler imaging

  12. Spectroscopic imaging in electron microscopy

    SciTech Connect

    Pennycook, Stephen J; Colliex, C.

    2012-01-01

    In the scanning transmission electron microscope, multiple signals can be simultaneously collected, including the transmitted and scattered electron signals (bright field and annular dark field or Z-contrast images), along with spectroscopic signals such as inelastically scattered electrons and emitted photons. In the last few years, the successful development of aberration correctors for the electron microscope has transformed the field of electron microscopy, opening up new possibilities for correlating structure to functionality. Aberration correction not only allows for enhanced structural resolution with incident probes into the sub-angstrom range, but can also provide greater probe currents to facilitate mapping of intrinsically weak spectroscopic signals at the nanoscale or even the atomic level. In this issue of MRS Bulletin, we illustrate the power of the new generation of electron microscopes with a combination of imaging and spectroscopy. We show the mapping of elemental distributions at atomic resolution and also the mapping of electronic and optical properties at unprecedented spatial resolution, with applications ranging from graphene to plasmonic nanostructures, and oxide interfaces to biology.

  13. Nanorheology by atomic force microscopy

    SciTech Connect

    Li, Tai-De; Chiu, Hsiang-Chih; Ortiz-Young, Deborah; Riedo, Elisa

    2014-12-15

    We present an Atomic Force Microscopy (AFM) based method to investigate the rheological properties of liquids confined within a nanosize gap formed by an AFM tip apex and a solid substrate. In this method, a conventional AFM cantilever is sheared parallel to a substrate surface by means of a lock-in amplifier while it is approaching and retracting from the substrate in liquid. The normal solvation forces and lateral viscoelastic shear forces experienced by the AFM tip in liquid can be simultaneously measured as a function of the tip-substrate distance with sub-nanometer vertical resolution. A new calibration method is applied to compensate for the linear drift of the piezo transducer and substrate system, leading to a more precise determination of the tip-substrate distance. By monitoring the phase lag between the driving signal and the cantilever response in liquid, the frequency dependent viscoelastic properties of the confined liquid can also be derived. Finally, we discuss the results obtained with this technique from different liquid-solid interfaces. Namely, octamethylcyclotetrasiloxane and water on mica and highly oriented pyrolytic graphite.

  14. Electron microscopy and forensic practice

    NASA Astrophysics Data System (ADS)

    Kotrlý, Marek; Turková, Ivana

    2013-05-01

    Electron microanalysis in forensic practice ranks among basic applications used in investigation of traces (latents, stains, etc.) from crime scenes. Applying electron microscope allows for rapid screening and receiving initial information for a wide range of traces. SEM with EDS/WDS makes it possible to observe topography surface and morphology samples and examination of chemical components. Physical laboratory of the Institute of Criminalistics Prague use SEM especially for examination of inorganic samples, rarely for biology and other material. Recently, possibilities of electron microscopy have been extended considerably using dual systems with focused ion beam. These systems are applied mainly in study of inner micro and nanoparticles , thin layers (intersecting lines in graphical forensic examinations, analysis of layers of functional glass, etc.), study of alloys microdefects, creating 3D particles and aggregates models, etc. Automated mineralogical analyses are a great asset to analysis of mineral phases, particularly soils, similarly it holds for cathode luminescence, predominantly colour one and precise quantitative measurement of their spectral characteristics. Among latest innovations that are becoming to appear also at ordinary laboratories are TOF - SIMS systems and micro Raman spectroscopy with a resolution comparable to EDS/WDS analysis (capable of achieving similar level as through EDS/WDS analysis).

  15. Lensfree microscopy on a cellphone.

    PubMed

    Tseng, Derek; Mudanyali, Onur; Oztoprak, Cetin; Isikman, Serhan O; Sencan, Ikbal; Yaglidere, Oguzhan; Ozcan, Aydogan

    2010-07-21

    We demonstrate lensfree digital microscopy on a cellphone. This compact and light-weight holographic microscope installed on a cellphone does not utilize any lenses, lasers or other bulky optical components and it may offer a cost-effective tool for telemedicine applications to address various global health challenges. Weighing approximately 38 grams (<1.4 ounces), this lensfree imaging platform can be mechanically attached to the camera unit of a cellphone where the samples are loaded from the side, and are vertically illuminated by a simple light-emitting diode (LED). This incoherent LED light is then scattered from each micro-object to coherently interfere with the background light, creating the lensfree hologram of each object on the detector array of the cellphone. These holographic signatures captured by the cellphone permit reconstruction of microscopic images of the objects through rapid digital processing. We report the performance of this lensfree cellphone microscope by imaging various sized micro-particles, as well as red blood cells, white blood cells, platelets and a waterborne parasite (Giardia lamblia).

  16. Disposable optics for microscopy diagnostics.

    PubMed

    Vilmi, Pauliina; Varjo, Sami; Sliz, Rafal; Hannuksela, Jari; Fabritius, Tapio

    2015-11-20

    The point-of-care testing (POCT) is having increasing role on modern health care systems due to a possibility to perform tests for patients conveniently and immediately. POCT includes lot of disposable devices because of the environment they are often used. For a disposable system to be reasonably utilized, it needs to be high in quality but low in price. Optics based POCT systems are interesting approach to be developed, and here we describe a low-cost fabrication process for microlens arrays for microscopy. Lens arrays having average lens diameter of 222 μm with 300 μm lens pitch were fabricated. The lenses were characterized to have standard deviation of 0.06 μm in height and 4.61 μm in diameter. The resolution limit of 3.9μm is demonstrated with real images, and the images were compared with ones made with glass and polycarbonate lens arrays. The image quality is at the same level than with the glass lenses and the manufacturing costs are very low, thus making them suitable for POCT applications.

  17. Liquid Cell Transmission Electron Microscopy.

    PubMed

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-27

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  18. Liquid Cell Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  19. Disposable optics for microscopy diagnostics

    PubMed Central

    Vilmi, Pauliina; Varjo, Sami; Sliz, Rafal; Hannuksela, Jari; Fabritius, Tapio

    2015-01-01

    The point-of-care testing (POCT) is having increasing role on modern health care systems due to a possibility to perform tests for patients conveniently and immediately. POCT includes lot of disposable devices because of the environment they are often used. For a disposable system to be reasonably utilized, it needs to be high in quality but low in price. Optics based POCT systems are interesting approach to be developed, and here we describe a low-cost fabrication process for microlens arrays for microscopy. Lens arrays having average lens diameter of 222 μm with 300 μm lens pitch were fabricated. The lenses were characterized to have standard deviation of 0.06 μm in height and 4.61 μm in diameter. The resolution limit of 3.9μm is demonstrated with real images, and the images were compared with ones made with glass and polycarbonate lens arrays. The image quality is at the same level than with the glass lenses and the manufacturing costs are very low, thus making them suitable for POCT applications. PMID:26586153

  20. Holographic microscopy in low coherence

    NASA Astrophysics Data System (ADS)

    Chmelík, Radim; Petráček, Jiří; Slabá, Michala; Kollárová, Věra; Slabý, Tomáš; Čolláková, Jana; Komrska, Jiří; Dostál, Zbyněk.; Veselý, Pavel

    2016-03-01

    Low coherence of the illumination substantially improves the quality of holographic and quantitative phase imaging (QPI) by elimination of the coherence noise and various artefacts and by improving the lateral resolution compared to the coherent holographic microscopy. Attributes of coherence-controlled holographic microscope (CCHM) designed and built as an off-axis holographic system allowing QPI within the range from complete coherent to incoherent illumination confirmed these expected advantages. Low coherence illumination also furnishes the coherence gating which constraints imaging of some spatial frequencies of an object axially thus forming an optical section in the wide sense. In this way the depth discrimination capability of the microscope is introduced at the price of restricting the axial interval of possible numerical refocusing. We describe theoretically these effects for the whole range of illumination coherence. We also show that the axial refocusing constraints can be overcome using advanced mode of imaging based on mutual lateral shift of reference and object image fields in CCHM. Lowering the spatial coherence of illumination means increasing its numerical aperture. We study how this change of the illumination geometry influences 3D objects QPI and especially the interpretation of live cells QPI in terms of the dry mass density measurement. In this way a strong dependence of the imaging process on the light coherence is demonstrated. The theoretical calculations and numerical simulations are supported by experimental data including a chance of time-lapse watching of live cells even in optically turbid milieu.

  1. Advances in quantitative Kerr microscopy

    NASA Astrophysics Data System (ADS)

    Soldatov, I. V.; Schäfer, R.

    2017-01-01

    An advanced wide-field Kerr microscopy approach to the vector imaging of magnetic domains is demonstrated. Utilizing the light from eight monochrome light emitting diodes, guided to the microscope by glass fibers, and being properly switched in synchronization with the camera exposure, domain images with orthogonal in-plane sensitivity are obtained simultaneously at real time. After calibrating the Kerr contrast under the same orthogonal sensitivity conditions, the magnetization vector field of complete magnetization cycles along the hysteresis loop can be calculated and plotted as a coded color or vector image. In the pulsed mode also parasitic, magnetic field-dependent Faraday rotations in the microscope optics are eliminated, thus increasing the accuracy of the measured magnetization angles to better than 5∘. The method is applied to the investigation of the magnetization process in a patterned Permalloy film element. Furthermore it is shown that the effective magnetic anisotropy axes in a GaMnAs semiconducting film can be quantitatively measured by vectorial analysis of the domain structure.

  2. Nanorheology by atomic force microscopy.

    PubMed

    Li, Tai-De; Chiu, Hsiang-Chih; Ortiz-Young, Deborah; Riedo, Elisa

    2014-12-01

    We present an Atomic Force Microscopy (AFM) based method to investigate the rheological properties of liquids confined within a nanosize gap formed by an AFM tip apex and a solid substrate. In this method, a conventional AFM cantilever is sheared parallel to a substrate surface by means of a lock-in amplifier while it is approaching and retracting from the substrate in liquid. The normal solvation forces and lateral viscoelastic shear forces experienced by the AFM tip in liquid can be simultaneously measured as a function of the tip-substrate distance with sub-nanometer vertical resolution. A new calibration method is applied to compensate for the linear drift of the piezo transducer and substrate system, leading to a more precise determination of the tip-substrate distance. By monitoring the phase lag between the driving signal and the cantilever response in liquid, the frequency dependent viscoelastic properties of the confined liquid can also be derived. Finally, we discuss the results obtained with this technique from different liquid-solid interfaces. Namely, octamethylcyclotetrasiloxane and water on mica and highly oriented pyrolytic graphite.

  3. Photoacoustic microscopy of human teeth

    NASA Astrophysics Data System (ADS)

    Rao, Bin; Cai, Xin; Favazza, Christopher; Yao, Junjie; Li, Li; Duong, Steven; Liaw, Lih-Huei; Holtzman, Jennifer; Wilder-Smith, Petra; Wang, Lihong V.

    2011-03-01

    Photoacoustic microscopy (PAM) utilizes short laser pulses to deposit energy into light absorbers and sensitively detects the ultrasonic waves the absorbers generate in response. PAM directly renders a three-dimensional spatial distribution of sub-surface optical absorbers. Unlike other optical imaging technologies, PAM features label-free optical absorption contrast and excellent imaging depths. Standard dental imaging instruments are limited to X-ray and CCD cameras. Subsurface optical dental imaging is difficult due to the highly-scattering enamel and dentin tissue. Thus, very few imaging methods can detect dental decay or diagnose dental pulp, which is the innermost part of the tooth, containing the nerves, blood vessels, and other cells. Here, we conducted a feasibility study on imaging dental decay and dental pulp with PAM. Our results showed that PAM is sensitive to the color change associated with dental decay. Although the relative PA signal distribution may be affected by surface contours and subsurface reflections from deeper dental tissue, monitoring changes in the PA signals (at the same site) over time is necessary to identify the progress of dental decay. Our results also showed that deep-imaging, near-infrared (NIR) PAM can sensitively image blood in the dental pulp of an in vitro tooth. In conclusion, PAM is a promising tool for imaging both dental decay and dental pulp.

  4. Enhanced effects with scanning force microscopy

    NASA Astrophysics Data System (ADS)

    Howells, S.; Chen, T.; Gallagher, M.; Yi, L.; Sarid, D.

    1991-05-01

    A general theory that describes the operation of scanning force microscopy in the contact force regime is presented. It is shown that force derivatives along the surface of a sample produce images that can be dramatically enhanced relative to those of surface topography. For scanning tunneling microscopy atomic force microscopy configurations, the spring constant of the cantilever and the force derivatives perpendicular to the surface of the sample determine the enhancement, respectively.

  5. Scanning Tip Microscopy With Applications To Biology

    NASA Astrophysics Data System (ADS)

    Sarid, Dror; Thall, Edmond H.; Iams, Douglas A.; Ingle, Jeffery T.; Henson, Tammy D.; Lee, Y. C.; Bell, L. Stephen

    1989-06-01

    Scanning tunneling microscopy and atomic force microscopy, denoted here scanning tip microscopy, are two powerful novel techniques for imaging surfaces with atomic resolution. We describe the underlying principles of these two techniques with special emphasis on an instrument developed in our laboratory that uses a laser diode to detect minute deflections of a tip as it raster scans the surface of a sample. Applications of these techniques to research in biology are assessed and their relative merits discussed.

  6. Wavelength Independent Optical Microscopy and Lithography

    DTIC Science & Technology

    1987-10-31

    Leviatan , Y., J. Appl. Phys. 60, 1577 (1986). 7. Harootunian, A., Near-Field Scanning Optical Microscopy and Raman Microscopy, Cornell University Ph.D...although the approach used may not be valid in the Another potential problem concerns the effect of the near field. More recently, Leviatan 21...Massey, "Microscopy and Pattern Generation With Scanned Evanescent Waves," AppL. Opt. 23, 658 (1984). The authors wish to thank Yehuda Leviatan for 21

  7. Scanning Tunneling Microscopy Studies of Quasicrystals

    NASA Astrophysics Data System (ADS)

    Becker, Russell S.; Kortan, A. Refik

    The following sections are included: * INTRODUCTION * EXPERIMENTAL * X-RAY DIFFRACTION * SCANNING TUNNELING MICROSCOPY * STRUCTURE MODELLING BASED ON STM * COMPARISON WITH MODELS BASED ON BULK STUDIES * CONCLUSION * REFERENCES

  8. Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

    DTIC Science & Technology

    2015-02-01

    Optical imaging , metabolism, tumor microenvironment, NADH, FAD, intravital imaging , collagen, metastasis 3.Overall Project Summary Our preliminary...Keely, KW Eliceiri. Novel Intravital Imaging Approaches to Characterize Collagen Alignment in Defined Mammary Tumor Models. Microscopy and...fixturing for intravital FLIM imaging through a rodent mammary imaging window. Stage is raised to accommodate tall 20xW objective. 14     Figure

  9. Computing myocardial motion in 4-dimensional echocardiography.

    PubMed

    Mukherjee, Ryan; Sprouse, Chad; Pinheiro, Aurélio; Abraham, Theodore; Burlina, Philippe

    2012-07-01

    We describe a novel method for computing dense 3D myocardial motion with high accuracy in four-dimensional (4D) echocardiography (3 dimensions spatial plus time). The method is based on a classic variational optical flow technique but exploits modern developments in optical flow research to utilize the full capabilities of 4D echocardiography. Using a variety of metrics, we present an in-depth performance evaluation of the method on synthetic, phantom, and intraoperative 4D transesophageal echocardiographic data. When compared with state-of-the-art optical flow and speckle tracking techniques currently found in 4D echocardiography, the method we present shows notable improvements in error rates. We believe the performance improvements shown can have a positive impact when the method is used as input for various applications, such as strain computation, biomechanical modeling, and automated diagnostics.

  10. The 2015 super-resolution microscopy roadmap

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  11. Digital microscopy. Bringing new technology into focus.

    PubMed

    2010-06-01

    Digital microscopy enables the scanning of microscope slides so that they can be viewed, analyzed, and archived on a computer. While the technology is not yet widely accepted by pathologists, a switch to digital microscopy systems seems to be inevitable in the near future.

  12. Microscopy & microanalysis 2016 in Columbus, Ohio

    DOE PAGES

    Michael, Joseph R.

    2016-01-08

    The article provides information about an upcoming conference from the program chair. The Microscopy Society of America (MSA), the Microanalysis Society (MAS), and the International Metallographic Society (IMS) invite participation in Microscopy & Microanalysis 2016 in Columbus, Ohio, July 24 through July 28, 2016.

  13. Super-resolution microscopy of mitochondria.

    PubMed

    Jakobs, Stefan; Wurm, Christian A

    2014-06-01

    Mitochondria, the powerhouses of the cell, are essential organelles in eukaryotic cells. With their complex inner architecture featuring a smooth outer and a highly convoluted inner membrane, they are challenging objects for microscopy. The diameter of mitochondria is generally close to the resolution limit of conventional light microscopy, rendering diffraction-unlimited super-resolution light microscopy (nanoscopy) for imaging submitochondrial protein distributions often mandatory. In this review, we discuss what can be expected when imaging mitochondria with conventional diffraction-limited and diffraction-unlimited microscopy. We provide an overview on recent studies using super-resolution microscopy to investigate mitochondria and discuss further developments and challenges in mitochondrial biology that might by addressed with these technologies in the future.

  14. Optical super-resolution microscopy in neurobiology.

    PubMed

    Sigrist, Stephan J; Sabatini, Bernardo L

    2012-02-01

    Understanding the highly plastic nature of neurons requires the dynamic visualization of their molecular and cellular organization in a native context. However, due to the limited resolution of standard light microscopy, many of the structural specializations of neurons cannot be resolved. A recent revolution in light microscopy has given rise to several super-resolution light microscopy methods yielding 2-10-fold higher resolution than conventional microscopy. We here describe the principles behind these techniques as well as their application to the analysis of the molecular architecture of the synapse. Furthermore, we discuss the potential for continued development of super-resolution microscopy as necessary for live imaging of neuronal structure and function in the brain.

  15. What a couple of dimensions can do for you: Comparative developmental studies using 4D microscopy--examples from tardigrade development.

    PubMed

    Hejnol, Andreas; Schnabel, Ralf

    2006-04-01

    The development of an organism consists of processes occurring in space and time. To analyze this 4-dimensional development in embryogenesis, an appropriate method should be chosen. We present here a sophisticated method, 4D microscopy (3D time-lapse microscopy), initially developed to analyze the cell lineage of wild-type and mutant embryos of the nematode Caenorhabditis elegans. Our method records the entire development of an embryo and allows detailed analyses of events such as cleavage, cell migration, cell death (apoptosis), and cell differentiation during development. The 4D microscopy system has 3 main parts: a motorized microscope, trigger software, and a database that facilitates the analysis of recordings. Adopting the 4D microscopy technique for uses beyond the analysis of C. elegans makes it possible to discern the cell lineage of other small embryos. Our method fills a gap in the study of the development of diverse organisms that are impossible to observe with fluorescent labeling techniques using single blastomeres. The use of this technique to investigate the development of organisms such as tardigrades, acoelomorphs, rotifers, and gastrotrichs provides fresh insight into the evolution of developmental processes and the phylogenetic relationships between such taxa. Using tardigrade development as an example, we demonstrate that the use of 4D microscopy can reveal new characters and corroborate or disapprove old characters. We discuss the results in the light of recent phylogenetic hypotheses regarding the Arthropoda and their probable sister group, the Cycloneuralia, which together form the Ecdysozoa.

  16. Gabor domain optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Murali, Supraja

    Time domain Optical Coherence Tomography (TD-OCT), first reported in 1991, makes use of the low temporal coherence properties of a NIR broadband laser to create depth sectioning of up to 2mm under the surface using optical interferometry and point to point scanning. Prior and ongoing work in OCT in the research community has concentrated on improving axial resolution through the development of broadband sources and speed of image acquisition through new techniques such as Spectral domain OCT (SD-OCT). In SD-OCT, an entire depth scan is acquired at once with a low numerical aperture (NA) objective lens focused at a fixed point within the sample. In this imaging geometry, a longer depth of focus is achieved at the expense of lateral resolution, which is typically limited to 10 to 20 mum. Optical Coherence Microscopy (OCM), introduced in 1994, combined the advantages of high axial resolution obtained in OCT with high lateral resolution obtained by increasing the NA of the microscope placed in the sample arm. However, OCM presented trade-offs caused by the inverse quadratic relationship between the NA and the DOF of the optics used. For applications requiring high lateral resolution, such as cancer diagnostics, several solutions have been proposed including the periodic manual re-focusing of the objective lens in the time domain as well as the spectral domain C-mode configuration in order to overcome the loss in lateral resolution outside the DOF. In this research, we report for the first time, high speed, sub-cellular imaging (lateral resolution of 2 mum) in OCM using a Gabor domain image processing algorithm with a custom designed and fabricated dynamic focus microscope interfaced to a Ti:Sa femtosecond laser centered at 800 nm within an SD-OCM configuration. It is envisioned that this technology will provide a non-invasive replacement for the current practice of multiple biopsies for skin cancer diagnosis. The research reported here presents three important advances

  17. Data assimilation in a coupled physical-biogeochemical model of the California current system using an incremental lognormal 4-dimensional variational approach: Part 3-Assimilation in a realistic context using satellite and in situ observations

    NASA Astrophysics Data System (ADS)

    Song, Hajoon; Edwards, Christopher A.; Moore, Andrew M.; Fiechter, Jerome

    2016-10-01

    A fully coupled physical and biogeochemical ocean data assimilation system is tested in a realistic configuration of the California Current System using the Regional Ocean Modeling System. In situ measurements for sea surface temperature and salinity as well as satellite observations for temperature, sea level and chlorophyll are used for the year 2000. Initial conditions of the combined physical and biogeochemical state are adjusted at the start of each 3-day assimilation cycle. Data assimilation results in substantial reduction of root-mean-square error (RMSE) over unconstrained model output. RMSE for physical variables is slightly lower when assimilating only physical variables than when assimilating both physical variables and surface chlorophyll. Surface chlorophyll RMSE is lowest when assimilating both physical variables and surface chlorophyll. Estimates of subsurface, nitrate and chlorophyll show modest improvements over the unconstrained model run relative to independent, unassimilated in situ data. Assimilation adjustments to the biogeochemical initial conditions are investigated within different regions of the California Current System. The incremental, lognormal 4-dimensional data assimilation method tested here represents a viable approach to coupled physical biogeochemical state estimation at practical computational cost.

  18. Data assimilation in a coupled physical-biogeochemical model of the California Current System using an incremental lognormal 4-dimensional variational approach: Part 1-Model formulation and biological data assimilation twin experiments

    NASA Astrophysics Data System (ADS)

    Song, Hajoon; Edwards, Christopher A.; Moore, Andrew M.; Fiechter, Jerome

    2016-10-01

    A quadratic formulation for an incremental lognormal 4-dimensional variational assimilation method (incremental L4DVar) is introduced for assimilation of biogeochemical observations into a 3-dimensional ocean circulation model. L4DVar assumes that errors in the model state are lognormally rather than Gaussian distributed, and implicitly ensures that state estimates are positive definite, making this approach attractive for biogeochemical variables. The method is made practical for a realistic implementation having a large state vector through linear assumptions that render the cost function quadratic and allow application of existing minimization techniques. A simple nutrient-phytoplankton-zooplankton-detritus (NPZD) model is coupled to the Regional Ocean Modeling System (ROMS) and configured for the California Current System. Quadratic incremental L4DVar is evaluated in a twin model framework in which biological fields only are in error and compared to G4DVar which assumes Gaussian distributed errors. Five-day assimilation cycles are used and statistics from four years of model integration analyzed. The quadratic incremental L4DVar results in smaller root-mean-squared errors and better statistical agreement with reference states than G4DVar while maintaining a positive state vector. The additional computational cost and implementation effort are trivial compared to the G4DVar system, making quadratic incremental L4DVar a practical and beneficial option for realistic biogeochemical state estimation in the ocean.

  19. Visualizing quantitative microscopy data: History and challenges.

    PubMed

    Sailem, Heba Z; Cooper, Sam; Bakal, Chris

    2016-01-01

    Data visualization is a fundamental aspect of science. In the context of microscopy-based studies, visualization typically involves presentation of the images themselves. However, data visualization is challenging when microscopy experiments entail imaging of millions of cells, and complex cellular phenotypes are quantified in a high-content manner. Most well-established visualization tools are inappropriate for displaying high-content data, which has driven the development of new visualization methodology. In this review, we discuss how data has been visualized in both classical and high-content microscopy studies; as well as the advantages, and disadvantages, of different visualization methods.

  20. Soft x-ray holographic microscopy

    SciTech Connect

    Stickler, Daniel; Froemter, Robert; Stillrich, Holger; Menk, Christian; Oepen, Hans Peter; Tieg, Carsten; Streit-Nierobisch, Simone; Sprung, Michael; Gutt, Christian; Stadler, Lorenz-M.; Leupold, Olaf; Gruebel, Gerhard

    2010-01-25

    We present a new x-ray microscopy technique based on Fourier transform holography (FTH), where the sample is separate from the optics part of the setup. The sample can be shifted with respect to the holography optics, thus large-scale or randomly distributed objects become accessible. As this extends FTH into a true microscopy technique, we call it x-ray holographic microscopy (XHM). FTH allows nanoscale imaging without the need for nanometer-size beams. Simple Fourier transform yields an unambiguous image reconstruction. We demonstrate XHM by studying the magnetic domain evolution of a Co/Pt multilayer film as function of locally varied iron overlayer thickness.

  1. Latest advances in commercially available STED microscopy

    NASA Astrophysics Data System (ADS)

    Fouquet, Wernher; Giske, Arnold

    2012-02-01

    STimulated Emission Depletion (STED) microscopy enables imaging of biological samples combining significantly improved optical resolution with all benefits of confocal microscopy. Especially, by combining multi-channel image acquisition with high spatial resolution opens up a new understanding of co-localization experiments on nanoscales. Such a microscope provides new insights in various fields of biology, such as cell and membrane biology, neurobiology and physiology. We present new developments and a variety of biological examples for STED microscopy, showing structural details on scales well below 70nm and give an overview of possible field of applications, mainly focused on live cell imaging.

  2. Using hydrogels in microscopy: A tutorial.

    PubMed

    Flood, Peter; Page, Henry; Reynaud, Emmanuel G

    2016-05-01

    Sample preparation for microscopy is a crucial step to ensure the best experimental outcome. It often requires the use of specific mounting media that have to be tailored to not just the sample but the chosen microscopy technique. The media must not damage the sample or impair the optical path, and may also have to support the correct physiological function/development of the sample. For decades, researchers have used embedding media such as hydrogels to maintain samples in place. Their ease of use and transparency has promoted them as mainstream mounting media. However, they are not as straightforward to implement as assumed. They can contain contaminants, generate forces on the sample, have complex diffusion and structural properties that are influenced by multiple factors and are generally not designed for microscopy in mind. This short review will discuss the advantages and disadvantages of using hydrogels for microscopy sample preparation and highlight some of the less obvious problems associated with the area.

  3. Rotary-scanning optical resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    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.

  4. Saturated pattern-illuminated Fourier ptychography microscopy

    NASA Astrophysics Data System (ADS)

    Fang, Yue; Chen, Youhua; Kuang, Cuifang; Xiu, Peng; Liu, Qiulan; Ge, Baoliang; Liu, Xu

    2017-01-01

    We report a series of simulation studies which extends pattern-illuminated Fourier ptychography microscopy by integrating with the nonlinearity arising from saturation of the fluorophore excited state for super-resolution fluorescence imaging. This extended technique, termed Saturated pattern-illuminated Fourier ptychography (SpiFP) microscopy, could achieve a resolution four times that of wide field when the illuminating light intensity approaches the saturation threshold in simulations. Increasing light intensity leads to further resolution enhancement. In order to demonstrate the performance of SpiFP, we make a comparison between SpiFP and saturated structure illumination microscopy in simulations, and prove that the SpiFP exhibits superior robustness to noise, aberration correcting ability, and pattern’s flexibility. Introducing the saturation of the fluorescent emission brings in notable improvements in imaging performance, implying its potential in nanoscale-sized biological observations by wide-field microscopy.

  5. Lensless Digital Holographic Microscopy for Life Detection

    NASA Astrophysics Data System (ADS)

    Serabyn, E.; Liewer, K.; Wallace, J. K.; Rider, S.; Lindensmith, C.; Nadeau, J.

    2016-10-01

    Microscopy capable of volume imaging can be used to search for microbial life on ocean worlds. Here we discuss our recent digital holographic microscope (DHM) systems, which provide micron-scale resolution in a very compact package.

  6. Atomic resolution 3D electron diffraction microscopy

    SciTech Connect

    Miao, Jianwei; Ohsuna, Tetsu; Terasaki, Osamu; O'Keefe, Michael A.

    2002-03-01

    Electron lens aberration is the major barrier limiting the resolution of electron microscopy. Here we describe a novel form of electron microscopy to overcome electron lens aberration. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a 2 x 2 x 2 unit cell nano-crystal (framework of LTA [Al12Si12O48]8) can be ab initio determined at the resolution of 1 Angstrom from a series of simulated noisy diffraction pattern projections with rotation angles ranging from -70 degrees to +70 degrees in 5 degrees increments along a single rotation axis. This form of microscopy (which we call 3D electron diffraction microscopy) does not require any reference waves, and can image the 3D structure of nanocrystals, as well as non-crystalline biological and materials science samples, with the resolution limited only by the quality of sample diffraction.

  7. Multiphoton microscopy: an introduction to gastroenterologists.

    PubMed

    Cho, Hye Jin; Chun, Hoon Jai; Kim, Eun Sun; Cho, Bong Rae

    2011-10-28

    Multiphoton microscopy, relying on the simultaneous absorption of two or more photons by a fluorophore, has come to occupy a prominent place in modern biomedical research with its ability to allow real-time observation of a single cell and molecules in intact tissues. Multiphoton microscopy exhibits nonlinear optical contrast properties, which can make it possible to provide an exceptionally large depth penetration with less phototoxicity. This system becomes more and more an inspiring tool for a non-invasive imaging system to realize "optical biopsy" and to examine the functions of living cells. In this review, we briefly present the physical principles and properties of multiphoton microscopy as well as the current applications in biological fields. In addition, we address what we see as the future potential of multiphoton microscopy for gastroenterologic research.

  8. Theoretical simulation of scanning probe microscopy.

    PubMed

    Tsukada, Masaru

    2011-01-01

    Methods of theoretical simulation of scanning probe microscopy, including scanning tunneling microscopy (STM), atomic force microscopy(AFM) and Kelvin prove force microscopy (KPFM) have been reviewed with recent topics as case studies. For the case of the STM simulation, the importance of the tip electronic states is emphasized and some advanced formalism is presented based on the non-equilibrium Green's function theory beyond Bardeen's perturbation theory. For the simulation of AFM, we show examples of 3D-force map for AFM in water, and theoretical analyses for a nano-mechanical experiment on a protein molecule. An attempt to simulate KPFM images based on the electrostatic multi-pole interaction between a tip and a sample is also introduced.

  9. Multiphoton microscopy in defining liver function

    NASA Astrophysics Data System (ADS)

    Thorling, Camilla A.; Crawford, Darrell; Burczynski, Frank J.; Liu, Xin; Liau, Ian; Roberts, Michael S.

    2014-09-01

    Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

  10. Polarization-sensitive interferometric synthetic aperture microscopy

    PubMed Central

    South, Fredrick A.; Liu, Yuan-Zhi; Xu, Yang; Shemonski, Nathan D.; Carney, P. Scott; Boppart, Stephen A.

    2015-01-01

    Three-dimensional optical microscopy suffers from the well-known compromise between transverse resolution and depth-of-field. This is true for both structural imaging methods and their functional extensions. Interferometric synthetic aperture microscopy (ISAM) is a solution to the 3D coherent microscopy inverse problem that provides depth-independent transverse resolution. We demonstrate the extension of ISAM to polarization sensitive imaging, termed polarization-sensitive interferometric synthetic aperture microscopy (PS-ISAM). This technique is the first functionalization of the ISAM method and provides improved depth-of-field for polarization-sensitive imaging. The basic assumptions of polarization-sensitive imaging are explored, and refocusing of birefringent structures is experimentally demonstrated. PS-ISAM enables high-resolution volumetric imaging of birefringent materials and tissue. PMID:26648593

  11. Magnetic resonance microscopy in biomedical research.

    PubMed

    Serša, I

    2012-01-01

    Magnetic resonance (MR) microscopy is a special modality of MRI with an emphasis on high spatial resolution. While its main principle is identical to conventional clinical MRI, there are several differences between the two that are mainly associated with a use of stronger magnets and gradients. MR microscopy has numerous interesting applications in material and bio sciences in which high spatial resolution is demanded and long experiment times are allowed.

  12. Coatings and alternatives for SEM microscopy

    SciTech Connect

    Lee, R.H.

    1995-03-01

    Several methods of preparing samples of low electrical conductivity for conventional scanning electron microscopy are reviewed. Two new methods are chromium sputter-coating and low-voltage electron microscopy with a field emission gun. Photomicrographs of different coatings at high magnification show the structure of each coating. Advantages and disadvantages of each material are presented. Results with sputtered coatings are compared to an evaporated carbon coating.

  13. Advanced electron microscopy for advanced materials.

    PubMed

    Van Tendeloo, Gustaaf; Bals, Sara; Van Aert, Sandra; Verbeeck, Jo; Van Dyck, Dirk

    2012-11-08

    The idea of this Review is to introduce newly developed possibilities of advanced electron microscopy to the materials science community. Over the last decade, electron microscopy has evolved into a full analytical tool, able to provide atomic scale information on the position, nature, and even the valency atoms. This information is classically obtained in two dimensions (2D), but can now also be obtained in 3D. We show examples of applications in the field of nanoparticles and interfaces.

  14. Imaging DNA Structure by Atomic Force Microscopy.

    PubMed

    Pyne, Alice L B; Hoogenboom, Bart W

    2016-01-01

    Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine superstructure and secondary structure of surface-bound DNA. The method is also readily applicable to probe DNA-DNA interactions and DNA-protein complexes.

  15. Investigation of wear phenomena by microscopy

    NASA Technical Reports Server (NTRS)

    Buckley, D. H.

    1982-01-01

    The various wear mechanisms involved in the loss of material from metallic and nonmetallic surfaces are discussed. The results presented indicate how various microscopy techniques used in conjunction with other analytical tools can assist in the elucidation of a wear mechanism. Without question, microscopy is the single most important tool for the study of the wear of surfaces, to assess and address inherent mechanisms of the material removal process.

  16. Subwavelength optical microscopy in the far field

    SciTech Connect

    Sun Qingqing; Zubairy, M. Suhail; Al-Amri, M.; Scully, Marlan O.

    2011-06-15

    We present a procedure for subwavelength optical microscopy. The identical atoms are distributed on a plane and shined with a standing wave. We rotate the plane to different angles and record the resonant fluorescence spectra in the far field, from which we can obtain their distance and location information. This procedure also works for atomic separation above one wavelength and therefore provides a seamless microscopy.

  17. Bessel light sheet structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Noshirvani Allahabadi, Golchehr

    Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in

  18. Ovarian Tumor-Stroma Interactions in an In Vivo Orthotopic Model

    DTIC Science & Technology

    2011-08-01

    cancer cells to the novel environment. We have devised an Intravital Video Microscopy approach to this problem in which MOVCAR cells labeled with green...Ovarian cancer, gene expression, metastasis, intravital video microscopy 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER...placed in a dorsal skin-fold chamber for Intravital Video Microscopy (IVM). The minced pseudo-organ tissue revascularizes and recapitulates some of the

  19. Cryo-scanning electron microscopy and light microscopy for the study of fungi interactions.

    PubMed

    Sempere, F; Santamarina, M P

    2011-03-01

    The application of the cryo-scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations.

  20. Exploring Scanning Probe Microscopy with Mathematica

    NASA Astrophysics Data System (ADS)

    Sarid, Dror

    1997-10-01

    This book/software edition provides a complete set of computational models that describe the physical phenomena associated with scanning tunneling microscopy, atomic force microscopy, and related technologies. Its self-contained presentation spares researchers the valuable time spent hunting through the technical literature in search of prior theoretical results required to understand the models presented. Mathematica code for all examples is included both in the book and at the accompanying ftp site, affording the freedom to change, at will, the values and parameters of specific problems or even modify the programs themselves to suit various modeling needs. Exploring Scanning Probe Microscopy with Mathematica is both a solid professional reference and an advanced-level text, beginning with scanning probe microscopy basics and moving on to cutting-edge techniques, experiments, and theory. In the section devoted to atomic force microscopy, Dr. Sarid describes the mechanical properties of cantilevers, atomic force microscope tip-sample interactions, and cantilever vibration characteristics. This is followed by an in-depth treatment of theoretical and practical aspects of tunneling phenomena, including metal-insulator-metal tunneling and Fowler-Nordheim field emission. The final section features chapters covering density of states in arbitrary dimensions, quantum wells and dots, and electrostatics.

  1. X-ray microscopy of human malaria

    SciTech Connect

    Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W.

    1997-04-01

    Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.

  2. Photoacoustic microscopy: superdepth, superresolution, and superb contrast.

    PubMed

    Yao, Junjie; Song, Liang; Wang, Lihing V

    2015-01-01

    Since its invention in the 17th century, optical microscopy has revolutionized biomedical studies by scrutinizing the biological realm on cellular levels, taking advantage of its excellent light-focusing capability. However, most biological tissues scatter light highly. As light travels in tissue, cumulative scattering events cause the photons to lose their original propagation direction and, thus, their ability to be focused, which has largely limited the penetration depth of optical microscopy. Conventional planar optical microscopy can provide penetration of only ~100 ?m before photons begin to be scattered. The penetration of modern optical microcopy, such as confocal microscopy and multiphoton microscopy, is still limited to approximately the optical diffusion limit (~1 mm in the skin as approximated by one optical transport mean free path), where scattered photons retain a strong memory of the original propagation direction. So far, it still remains a challenge for pure optical methods to achieve high-resolution in vivo imaging beyond the diffusion limit (i.e., superdepth imaging).

  3. Functional photoacoustic microscopy of pH

    NASA Astrophysics Data System (ADS)

    Chatni, M. Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P.; Maslov, Konstantin I.; Wang, Lihong V.

    2012-02-01

    pH is a tightly regulated indicator of metabolic activity. In mammalian systems, imbalance of pH regulation may result from or result in serious illness. Even though the regulation system of pH is very robust, tissue pH can be altered in many diseases such as cancer, osteoporosis and diabetes mellitus. Traditional high-resolution optical imaging techniques, such as confocal microscopy, routinely image pH in cells and tissues using pH sensitive fluorescent dyes, which change their fluorescence properties with the surrounding pH. Since strong optical scattering in biological tissue blurs images at greater depths, high-resolution pH imaging is limited to penetration depths of 1mm. Here, we report photoacoustic microscopy (PAM) of commercially available pH-sensitive fluorescent dye in tissue phantoms. Using both opticalresolution photoacoustic microscopy (OR-PAM), and acoustic resolution photoacoustic microscopy (AR-PAM), we explored the possibility of recovering the pH values in tissue phantoms. In this paper, we demonstrate that PAM was capable of recovering pH values up to a depth of 2 mm, greater than possible with other forms of optical microscopy.

  4. CARS microscopy of Alzheimer's diseased brain tissue

    NASA Astrophysics Data System (ADS)

    Enejder, Annika; Kiskis, Juris; Fink, Helen; Nyberg, Lena; Thyr, Jakob; Li, Jia-Yi

    2014-02-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

  5. Ultrafast Science Opportunities with Electron Microscopy

    SciTech Connect

    Durr, Hermann

    2016-04-28

    X-rays and electrons are two of the most fundamental probes of matter. When the Linac Coherent Light Source (LCLS), the world’s first x-ray free electron laser, began operation in 2009, it transformed ultrafast science with the ability to generate laser-like x-ray pulses from the manipulation of relativistic electron beams. This document describes a similar future transformation. In Transmission Electron Microscopy, ultrafast relativistic (MeV energy) electron pulses can achieve unsurpassed spatial and temporal resolution. Ultrafast temporal resolution will be the next frontier in electron microscopy and can ideally complement ultrafast x-ray science done with free electron lasers. This document describes the Grand Challenge science opportunities in chemistry, material science, physics and biology that arise from an MeV ultrafast electron diffraction & microscopy facility, especially when coupled with linac-based intense THz and X-ray pump capabilities.

  6. Fluorescence microscopy: A tool to study autophagy

    NASA Astrophysics Data System (ADS)

    Rai, Shashank; Manjithaya, Ravi

    2015-08-01

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

  7. Spectroscopy and atomic force microscopy of biomass.

    PubMed

    Tetard, L; Passian, A; Farahi, R H; Kalluri, U C; Davison, B H; Thundat, T

    2010-05-01

    Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.

  8. Near-infrared hyperspectral reflective confocal microscopy

    NASA Astrophysics Data System (ADS)

    Huang, Wei; Zhang, Yunhai; Miao, Xin; Xue, Xiaojun; Xiao, Yun

    2016-10-01

    A Near-Infrared HyperSpectral Reflective Confocal Microscopy (NIHS-RCM) is proposed in order to get high resolution images of deep biological tissues such as skin. The microscopy system uses a super-continuum laser for illumination, an acousto-optic tunable filter (AOTF) for rapid selection of near-infrared spectrum, a resonant galvanometer scanner for high speed imaging (15f/s) and near-infrared avalanche diode as detector. Porcine skin and other experiments show that the microscopy system could get deep tissue images (180 μm), and show the different ingredients of tissue with different wavelength of illumination. The system has the ability of selectively imaging of multiple ingredients at deep tissue which can be used in skin diseases diagnosis and other fields.

  9. Cryogenics with cement microscopy redefines cement behavior

    SciTech Connect

    Mehta, S.; Jones, R. ); Caveny, B. )

    1994-10-03

    Cement microscopy (CM), cryogenics, environmental scanning microscopy (ESM), scanning electron microscopy (SEM), and other technologies are leading investigators to change their views on cement gelation, hydration, and retardation. Cement samples frozen in a nitrogen slush and viewed with an SEM present a more accurate picture of the setting process. Observations made through this technique have revolutionized ARCO Exploration and Production Technology's and Halliburton Energy Services' oil field cement procurement and slurry design. Findings from this joint study are expected to lead to: optimized waiting on cement (WOC) times; reduced planning and design time; optimized slurry retarder additions; optimized gel times to fit given situations; especially applicable to squeeze operations; improved cement selection (from vendors) for peak performance; and improved cement manufacture. The paper discusses the measuring methods and the findings on the following: cement voids, cement gelation, and retardation mechanisms. It also briefly discusses the impact these discoveries have on operations.

  10. Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

    PubMed Central

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; Xie, X. Sunney

    2012-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  11. Coherent nonlinear optical imaging: beyond fluorescence microscopy.

    PubMed

    Min, Wei; Freudiger, Christian W; Lu, Sijia; Xie, X Sunney

    2011-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.

  12. Ultrahigh resolution photoacoustic microscopy via transient absorption

    PubMed Central

    Shelton, Ryan L.; Applegate, Brian E.

    2010-01-01

    We have developed a novel, hybrid imaging modality, Transient Absorption Ultrasonic Microscopy (TAUM), which takes advantage of the optical nonlinearities afforded by transient absorption to achieve ultrahigh-resolution photoacoustic microscopy. The theoretical point spread function for TAUM is functionally equivalent to confocal and two-photon fluorescence microscopy, potentially enabling cellular/subcellular photoacoustic imaging. A prototype TAUM system was designed, built, and used to image a cross-section through several capillaries in the excised cheek pouch of a Syrian Hamster. The well-resolved capillaries in the TAUM image provided experimental evidence of the spatial resolution. These results suggest that TAUM has excellent potential for producing volumetric images with cellular/subcellular resolution in three dimensions deep inside living tissue. PMID:21258499

  13. Space station microscopy: Beyond the box

    NASA Astrophysics Data System (ADS)

    Hunter, N. R.; Pierson, Duane L.; Mishra, S. K.

    Microscopy aboard Space Station Freedom poses many unique challenges for in-flight investigations. Disciplines such as material processing, plant and animal research, human reseach, enviromental monitoring, health care, and biological processing have diverse microscope requirements. The typical microscope not only does not meet the comprehensive needs of these varied users, but also tends to require excessive crew time. To assess user requirements, a comprehensive survey was conducted among investigators with experiments requiring microscopy. The survey examined requirements such as light sources, objectives, stages, focusing systems, eye pieces, video accessories, etc. The results of this survey and the application of an Intelligent Microscope Imaging System (IMIS) may address these demands for efficient microscopy service in space. The proposed IMIS can accommodate multiple users with varied requirements, operate in several modes, reduce crew time needed for experiments, and take maximum advantage of the restrictive data/ instruction transmission environment on Freedom.

  14. Light microscopy: an ongoing contemporary revolution

    NASA Astrophysics Data System (ADS)

    Weisenburger, Siegfried; Sandoghdar, Vahid

    2015-04-01

    The optical microscope is one of the oldest scientific instruments that is still used in forefront research. Ernst Abbe's nineteenth century formulation of the resolution limit in microscopy let generations of scientists believe that optical studies of individual molecules and resolving subwavelength structures were not feasible. The Nobel Prize in 2014 for super-resolution fluorescence microscopy marks a clear recognition that the old beliefs have to be revisited. In this article, we present a critical overview of various recent developments in optical microscopy. In addition to the popular super-resolution fluorescence methods, we discuss the prospects of various other techniques and imaging contrasts and consider some of the fundamental and practical challenges that lie ahead.

  15. A near-field optical microscopy nanoarray

    SciTech Connect

    Semin, D.J.; Ambrose, W.P.; Goodwin, P.M.; Kwller, A.; Wendt, J.R.

    1996-12-31

    Multiplexing near-field scanning optical microscopy (NSOM) by the use of a nanoarray with parallel imaging is studied. The fabrication, characterization, and utilization of nanoarrays with {approximately} 100 nm diameter apertures spaced 500 nm center-to- center is presented. Extremely uniform nanoarrays with {approximately} 10{sup 8} apertures were fabricated by electron beam lithography and reactive ion etching. The nanoarrays were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). In this paper we utilize these nanoarrays in a laser-illuminated microscope with parallel detection on a charge- coupled device (CCD). Detection of B-phycoerythrin (B-PE) molecules using near-field illumination is presented. In principle, our system can be used to obtain high lateral resolution NSOM images over a wide-field of view (e.g. 50-100 {mu}m) within seconds.

  16. Environmental scanning electron microscopy in cell biology.

    PubMed

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

    2013-01-01

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

  17. Space station microscopy: Beyond the box

    NASA Technical Reports Server (NTRS)

    Hunter, N. R.; Pierson, Duane L.; Mishra, S. K.

    1993-01-01

    Microscopy aboard Space Station Freedom poses many unique challenges for in-flight investigations. Disciplines such as material processing, plant and animal research, human reseach, enviromental monitoring, health care, and biological processing have diverse microscope requirements. The typical microscope not only does not meet the comprehensive needs of these varied users, but also tends to require excessive crew time. To assess user requirements, a comprehensive survey was conducted among investigators with experiments requiring microscopy. The survey examined requirements such as light sources, objectives, stages, focusing systems, eye pieces, video accessories, etc. The results of this survey and the application of an Intelligent Microscope Imaging System (IMIS) may address these demands for efficient microscopy service in space. The proposed IMIS can accommodate multiple users with varied requirements, operate in several modes, reduce crew time needed for experiments, and take maximum advantage of the restrictive data/ instruction transmission environment on Freedom.

  18. Applications of microscopy in Salmonella research.

    PubMed

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

    2015-01-01

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

  19. Magnetic exchange force microscopy with atomic resolution.

    PubMed

    Kaiser, Uwe; Schwarz, Alexander; Wiesendanger, Roland

    2007-03-29

    The ordering of neighbouring atomic magnetic moments (spins) leads to important collective phenomena such as ferromagnetism and antiferromagnetism. A full understanding of magnetism on the nanometre scale therefore calls for information on the arrangement of spins in real space and with atomic resolution. Spin-polarized scanning tunnelling microscopy accomplishes this but can probe only conducting materials. Force microscopy can be used on any sample independent of its conductivity. In particular, magnetic force microscopy is well suited to exploring ferromagnetic domain structures. However, atomic resolution cannot be achieved because data acquisition involves the sensing of long-range magnetostatic forces between tip and sample. Magnetic exchange force microscopy has been proposed for overcoming this limitation: by using an atomic force microscope with a magnetic tip, it should be possible to detect the short-range magnetic exchange force between tip and sample spins. Here we show for a prototypical antiferromagnetic insulator, the (001) surface of nickel oxide, that magnetic exchange force microscopy can indeed reveal the arrangement of both surface atoms and their spins simultaneously. In contrast with previous attempts to implement this method, we use an external magnetic field to align the magnetic polarization at the tip apex so as to optimize the interaction between tip and sample spins. This allows us to observe the direct magnetic exchange coupling between the spins of the tip atom and sample atom that are closest to each other, and thereby demonstrate the potential of magnetic exchange force microscopy for investigations of inter-spin interactions at the atomic level.

  20. Advanced Electron Microscopy in Materials Physics

    SciTech Connect

    Zhu, Y.; Jarausch, K.

    2009-06-01

    Aberration correction has opened a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes and extending information limits. The imaging and analytical performance of these corrector-equipped microscopes affords an unprecedented opportunity to study structure-property relationships of matter at the atomic scale. This new generation of microscopes is able to retrieve high-quality structural information comparable to neutron and synchrotron x-ray experiments, but with local atomic resolution. These advances in instrumentation are accelerating the research and development of various functional materials ranging from those for energy generation, conversion, transportation and storage to those for catalysis and nano-device applications. The dramatic improvements in electron-beam illumination and detection also present a host of new challenges for the interpretation and optimization of experiments. During 7-9 November 2007, a workshop, entitled 'Aberration Corrected Electron Microscopy in Material Physics', was convened at the Center for Functional Nanomaterials, Brookhaven National Laboratories (BNL) to address these opportunities and challenges. The workshop was co-sponsored by Hitachi High Technologies, a leader in electron microscopy instrumentation, and BNL's Institute of Advanced Electron Microscopy, a leader in materials physics research using electron microscopy. The workshop featured presentations by internationally prominent scientists working at the frontiers of electron microscopy, both on developing instrumentation and applying it in materials physics. The meeting, structured to stimulate scientific exchanges and explore new capabilities, brought together {approx}100 people from over 10 countries. This special issue complies many of the advances in instrument performance and materials physics reported by the invited speakers and attendees at the workshop.

  1. Improved Interference configuration for structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Houkai; Wei, Shibiao; Wu, Xiaojing; Yang, Yong; Zhang, Yuquan; Du, Luping; Liu, Jun; Zhu, Siwei; Yuan, Xiaocong

    2017-02-01

    We present an improved structured illumination configuration for structured illumination microscopy (SIM) based on spatial light modulator. Precise phase shifts and rotation of illumination fringes can be dynamically controlled using a spatial light modulator. The method is different from the conventional illumination configuration that are based on interference of ±1 diffractive order light. The experimental setup requires less optical elements making it compact, reliable, and suitable for integration. The method has been applied in the standing-wave total internal reflection fluorescent microscopy. High lateral resolution of sub-100 nm was achieved in single directional resolution enhancement experiments.

  2. Photobleaching imprinting microscopy: seeing clearer and deeper.

    PubMed

    Gao, Liang; Garcia-Uribe, Alejandro; Liu, Yan; Li, Chiye; Wang, Lihong V

    2014-01-15

    We present a generic sub-diffraction-limited imaging method - photobleaching imprinting microscopy (PIM) - for biological fluorescence imaging. A lateral resolution of 110 nm was measured, more than a twofold improvement over the optical diffraction limit. Unlike other super-resolution imaging techniques, PIM does not require complicated illumination modules or specific fluorescent dyes. PIM is expected to facilitate the conversion of super-resolution imaging into a routine lab tool, making it accessible to a much broader biological research community. Moreover, we show that PIM can increase the image contrast of biological tissue, effectively extending the fundamental depth limit of multi-photon fluorescence microscopy.

  3. Aberration corrected Lorentz scanning transmission electron microscopy.

    PubMed

    McVitie, S; McGrouther, D; McFadzean, S; MacLaren, D A; O'Shea, K J; Benitez, M J

    2015-05-01

    We present results from an aberration corrected scanning transmission electron microscope which has been customised for high resolution quantitative Lorentz microscopy with the sample located in a magnetic field free or low field environment. We discuss the innovations in microscope instrumentation and additional hardware that underpin the imaging improvements in resolution and detection with a focus on developments in differential phase contrast microscopy. Examples from materials possessing nanometre scale variations in magnetisation illustrate the potential for aberration corrected Lorentz imaging as a tool to further our understanding of magnetism on this lengthscale.

  4. Electron Microscopy of Natural and Epitaxial Diamond

    NASA Technical Reports Server (NTRS)

    Posthill, J. B.; George, T.; Malta, D. P.; Humphreys, T. P.; Rudder, R. A.; Hudson, G. C.; Thomas, R. E.; Markunas, R. J.

    1993-01-01

    Semiconducting diamond films have the potential for use as a material in which to build active electronic devices capable of operating at high temperatures or in high radiation environments. Ultimately, it is preferable to use low-defect-density single crystal diamond for device fabrication. We have previously investigated polycrystalline diamond films with transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and homoepitaxial films with SEM-based techniques. This contribution describes some of our most recent observations of the microstructure of natural diamond single crystals and homoepitaxial diamond thin films using TEM.

  5. Phase imaging with intermodulation atomic force microscopy.

    PubMed

    Platz, Daniel; Tholén, Erik A; Hutter, Carsten; von Bieren, Arndt C; Haviland, David B

    2010-05-01

    Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.

  6. Super-resolution optical microscopy: multiple choices.

    PubMed

    Huang, Bo

    2010-02-01

    The recent invention of super-resolution optical microscopy enables the visualization of fine features in biological samples with unprecedented clarity. It creates numerous opportunities in biology because vast amount of previously obscured subcellular processes now can be directly observed. Rapid development in this field in the past two years offers many imaging modalities that address different needs but they also complicates the choice of the 'perfect' method for answering a specific question. Here I will briefly describe the principles of super-resolution optical microscopy techniques and then focus on comparing their characteristics in various aspects of practical applications.

  7. Fidelity imaging for atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Ghosal, Sayan; Salapaka, Murti

    2015-01-01

    Atomic force microscopy is widely employed for imaging material at the nanoscale. However, real-time measures on image reliability are lacking in contemporary atomic force microscopy literature. In this article, we present a real-time technique that provides an image of fidelity for a high bandwidth dynamic mode imaging scheme. The fidelity images define channels that allow the user to have additional authority over the choice of decision threshold that facilitates where the emphasis is desired, on discovering most true features on the sample with the possible detection of high number of false features, or emphasizing minimizing instances of false detections. Simulation and experimental results demonstrate the effectiveness of fidelity imaging.

  8. Two-photon excitation fluorescence microscopy.

    PubMed

    So, P T; Dong, C Y; Masters, B R; Berland, K M

    2000-01-01

    Two-photon fluorescence microscopy is one of the most important recent inventions in biological imaging. This technology enables noninvasive study of biological specimens in three dimensions with submicrometer resolution. Two-photon excitation of fluorophores results from the simultaneous absorption of two photons. This excitation process has a number of unique advantages, such as reduced specimen photodamage and enhanced penetration depth. It also produces higher-contrast images and is a novel method to trigger localized photochemical reactions. Two-photon microscopy continues to find an increasing number of applications in biology and medicine.

  9. Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

    ERIC Educational Resources Information Center

    Ito, Takashi

    2008-01-01

    This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

  10. Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

    PubMed

    Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

    2015-06-11

    The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

  11. Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells

    PubMed Central

    Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

    2015-01-01

    The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results. PMID:26066680

  12. Time-Adjusted Internal Target Volume: A Novel Approach Focusing on Heterogeneity of Tumor Motion Based on 4-Dimensional Computed Tomography Imaging for Radiation Therapy Planning of Lung Cancer

    SciTech Connect

    Nishibuchi, Ikuno; Kimura, Tomoki; Nakashima, Takeo; Ochi, Yusuke; Takahashi, Ippei; Doi, Yoshiko; Kenjo, Masahiro; Kaneyasu, Yuko; Ozawa, Syuichi; Murakami, Yuji; Wadasaki, Koichi; Nagata, Yasushi

    2014-08-01

    Purpose: To consider nonuniform tumor motion within the internal target volume (ITV) by defining time-adjusted ITV (TTV), a volume designed to include heterogeneity of tumor existence on the basis of 4-dimensional computed tomography (4D-CT). Methods and Materials: We evaluated 30 lung cancer patients. Breath-hold CT (BH-CT) and free-breathing 4D-CT scans were acquired for each patient. The tumors were manually delineated using a lung CT window setting (window, 1600 HU; level, −300 HU). Tumor in BH-CT images was defined as gross tumor volume (GTV), and the sum of tumors in 4D-CT images was defined as ITV-4D. The TTV images were generated from the 4D-CT datasets, and the tumor existence probability within ITV-4D was calculated. We calculated the TTV{sub 80} value, which is the percentage of the volume with a tumor existence probability that exceeded 80% on ITV-4D. Several factors that affected the TTV{sub 80} value, such as the ITV-4D/GTV ratio or tumor centroid deviation, were evaluated. Results: Time-adjusted ITV images were acquired for all patients, and tumor respiratory motion heterogeneity was visualized. The median (range) ITV-4D/GTV ratio and median tumor centroid deviation were 1.6 (1.0-4.1) and 6.3 mm (0.1-30.3 mm), respectively. The median TTV{sub 80} value was 43.3% (2.9-98.7%). Strong correlations were observed between the TTV{sub 80} value and the ITV-4D/GTV ratio (R=−0.71) and tumor centroid deviation (R=−0.72). The TTV images revealed the tumor motion pattern features within ITV. Conclusions: The TTV images reflected nonuniform tumor motion, and they revealed the tumor motion pattern features, suggesting that the TTV concept may facilitate various aspects of radiation therapy planning of lung cancer while incorporating respiratory motion in the future.

  13. (Gene sequencing by scanning molecular exciton microscopy)

    SciTech Connect

    Not Available

    1991-01-01

    This report details progress made in setting up a laboratory for optical microscopy of genes. The apparatus including a fluorescence microscope, a scanning optical microscope, various spectrometers, and supporting computers is described. Results in developing photon and exciton tips, and in preparing samples are presented. (GHH)

  14. Value of Reflected Light Microscopy in Teaching.

    ERIC Educational Resources Information Center

    Pasteris, Jill Dill

    1983-01-01

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

  15. Scanning electron microscopy study of Trichomonas gallinae.

    PubMed

    Tasca, Tiana; De Carli, Geraldo A

    2003-12-01

    A scanning electron microscopy (SEM) study of Trichomonas gallinae (Rivolta, 1878), provided more information about the morphology of this flagellated protozoan. SEM showed the morphological features of the trophozoites; the emergence of the anterior flagella, the structure of the undulating membrane, the position and shape of the pelta, axostyle and posterior flagellum. Of special interest were the pseudocyst forms.

  16. DNA base identification by electron microscopy.

    PubMed

    Bell, David C; Thomas, W Kelley; Murtagh, Katelyn M; Dionne, Cheryl A; Graham, Adam C; Anderson, Jobriah E; Glover, William R

    2012-10-01

    Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

  17. Low voltage transmission electron microscopy of graphene.

    PubMed

    Bachmatiuk, Alicja; Zhao, Jiong; Gorantla, Sandeep Madhukar; Martinez, Ignacio Guillermo Gonzalez; Wiedermann, Jerzy; Lee, Changgu; Eckert, Juergen; Rummeli, Mark Hermann

    2015-02-04

    The initial isolation of graphene in 2004 spawned massive interest in this two-dimensional pure sp(2) carbon structure due to its incredible electrical, optical, mechanical, and thermal effects. This in turn led to the rapid development of various characterization tools for graphene. Examples include Raman spectroscopy and scanning tunneling microscopy. However, the one tool with the greatest prowess for characterizing and studying graphene is the transmission electron microscope. State-of-the-art (scanning) transmission electron microscopes enable one to image graphene with atomic resolution, and also to conduct various other characterizations simultaneously. The advent of aberration correctors was timely in that it allowed transmission electron microscopes to operate with reduced acceleration voltages, so that damage to graphene is avoided while still providing atomic resolution. In this comprehensive review, a brief introduction is provided to the technical aspects of transmission electron microscopes relevant to graphene. The reader is then introduced to different specimen preparation techniques for graphene. The different characterization approaches in both transmission electron microscopy and scanning transmission electron microscopy are then discussed, along with the different aspects of electron diffraction and electron energy loss spectroscopy. The use of graphene for other electron microscopy approaches such as in-situ investigations is also presented.

  18. Light Microscopy Module (LMM)-Emulator

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

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

  19. Atomic force microscopy of biological samples.

    PubMed

    Allison, David P; Mortensen, Ninell P; Sullivan, Claretta J; Doktycz, Mitchel J

    2010-01-01

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

  20. Atomic force microscopy of biological samples

    SciTech Connect

    Doktycz, Mitchel John

    2010-01-01

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

  1. High-aperture cryogenic light microscopy.

    PubMed

    Le Gros, M A; McDermott, G; Uchida, M; Knoechel, C G; Larabell, C A

    2009-07-01

    We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

  2. Frequency domain photoacoustic and fluorescence microscopy

    PubMed Central

    Langer, Gregor; Buchegger, Bianca; Jacak, Jaroslaw; Klar, Thomas A.; Berer, Thomas

    2016-01-01

    We report on simultaneous frequency domain optical-resolution photoacoustic and fluorescence microscopy with sub-µm lateral resolution. With the help of a blood smear, we show that photoacoustic and fluorescence images provide complementary information. Furthermore, we compare theoretically predicted signal-to-noise ratios of sinusoidal modulation in frequency domain with pulsed excitation in time domain. PMID:27446698

  3. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: AXIAL RESOLUTION

    EPA Science Inventory

    Abstract

    Confocal Microscopy System Performance: Axial resolution.
    Robert M. Zucker, PhD

    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Re...

  4. Confocal microscopy imaging of solid tissue

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

  5. Dark Field Microscopy for Analytical Laboratory Courses

    ERIC Educational Resources Information Center

    Augspurger, Ashley E.; Stender, Anthony S.; Marchuk, Kyle; Greenbowe, Thomas J.; Fang, Ning

    2014-01-01

    An innovative and inexpensive optical microscopy experiment for a quantitative analysis or an instrumental analysis chemistry course is described. The students have hands-on experience with a dark field microscope and investigate the wavelength dependence of localized surface plasmon resonance in gold and silver nanoparticles. Students also…

  6. Scanning Probe Microscopy of Organic Solar Cells

    NASA Astrophysics Data System (ADS)

    Reid, Obadiah G.

    Nanostructured composites of organic semiconductors are a promising class of materials for the manufacture of low-cost solar cells. Understanding how the nanoscale morphology of these materials affects their efficiency as solar energy harvesters is crucial to their eventual potential for large-scale deployment for primary power generation. In this thesis we describe the use of optoelectronic scanning-probe based microscopy methods to study this efficiency-structure relationship with nanoscale resolution. In particular, our objective is to make spatially resolved measurements of each step in the power conversion process from photons to an electric current, including charge generation, transport, and recombination processes, and correlate them with local device structure. We have achieved two aims in this work: first, to develop and apply novel electrically sensitive scanning probe microscopy experiments to study the optoelectronic materials and processes discussed above; and second, to deepen our understanding of the physics underpinning our experimental techniques. In the first case, we have applied conductive-, and photoconductive atomic force (cAFM & pcAFM) microscopy to measure both local photocurrent collection and dark charge transport properties in a variety of model and novel organic solar cell composites, including polymer/fullerene blends, and polymer-nanowire/fullerene blends, finding that local heterogeneity is the rule, and that improvements in the uniformity of specific beneficial nanostructures could lead to large increases in efficiency. We have used scanning Kelvin probe microscopy (SKPM) and time resolved-electrostatic force microscopy (trEFM) to characterize all-polymer blends, quantifying their sensitivity to photochemical degradation and the subsequent formation of local charge traps. We find that while trEFM provides a sensitive measure of local quantum efficiency, SKPM is generally unsuited to measurements of efficiency, less sensitive than tr

  7. Fast Ion Beam Microscopy of Whole Cells

    NASA Astrophysics Data System (ADS)

    Watt, Frank; Chen, Xiao; Chen, Ce-Belle; Udalagama, Chammika Nb; Ren, Minqin; Pastorin, G.; Bettiol, Andrew

    2013-08-01

    The way in which biological cells function is of prime importance, and the determination of such knowledge is highly dependent on probes that can extract information from within the cell. Probing deep inside the cell at high resolutions however is not easy: optical microscopy is limited by fundamental diffraction limits, electron microscopy is not able to maintain spatial resolutions inside a whole cell without slicing the cell into thin sections, and many other new and novel high resolution techniques such as atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM) are essentially surface probes. In this paper we show that microscopy using fast ions has the potential to extract information from inside whole cells in a unique way. This novel fast ion probe utilises the unique characteristic of MeV ion beams, which is the ability to pass through a whole cell while maintaining high spatial resolutions. This paper first addresses the fundamental difference between several types of charged particle probes, more specifically focused beams of electrons and fast ions, as they penetrate organic material. Simulations show that whereas electrons scatter as they penetrate the sample, ions travel in a straight path and therefore maintain spatial resolutions. Also described is a preliminary experiment in which a whole cell is scanned using a low energy (45 keV) helium ion microscope, and the results compared to images obtained using a focused beam of fast (1.2 MeV) helium ions. The results demonstrate the complementarity between imaging using low energy ions, which essentially produce a high resolution image of the cell surface, and high energy ions, which produce an image of the cell interior. The characteristics of the fast ion probe appear to be ideally suited for imaging gold nanoparticles in whole cells. Using scanning transmission ion microscopy (STIM) to image the cell interior, forward scattering transmission ion microscopy (FSTIM) to improve the

  8. Leica solution: CARS microscopy at video rates

    NASA Astrophysics Data System (ADS)

    Lurquin, V.

    2008-02-01

    Confocal and multiphoton microscopy are powerful techniques to study morphology and dynamics in cells and tissue, if fluorescent labeling is possible or autofluorescence is strong. For non-fluorescent molecules, Coherent anti-Stokes Raman scattering (CARS) microscopy provides chemical contrast based on intrinsic and highly specific vibrational properties of molecules eliminating the need for labeling. Just as other multiphoton techniques, CARS microscopy possesses three-dimensional sectioning capabilities. Leica Microsystems has combined the CARS imaging technology with its TCS SP5 confocal microscope to provide several advantages for CARS imaging. For CARS microscopy, two picosecond near-infrared lasers are overlapped spatially and temporally and sent into the scanhead of the confocal system. The software allows programmed, automatic switching between these light sources for multi-modal imaging. Furthermore the Leica TCS SP5 can be equipped with a non-descanned detector which will significantly enhance the signal. The Leica TCS SP5 scanhead combines two technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time resolution. The fast scanner allows imaging speeds as high as 25 images/per second at a resolution of 512×512 pixel. This corresponds to true video-rate allowing to follow processes at these time-scales as well as the acquisition of three-dimensional stacks in a few seconds. This time resolution is critical to study live animals or human patients for which heart beat and muscle movements lead to a blurring of the image if the acquisition time is high. Furthermore with the resonant scanhead the sectioning is truly confocal and does not suffer from spatial leakage. In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS SP5 a powerful tool for three-dimensional, label-free imaging of chemical and biological samples in vitro and in vivo.

  9. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

    SciTech Connect

    Miranda, Adelaide; De Beule, Pieter A. A.

    2015-09-15

    Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

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

    NASA Astrophysics Data System (ADS)

    Tong, Yongpeng; Li, Changming; Liang, Feng; Chen, Jianmin; Zhang, Hong; Liu, Guoqing; Sun, Huibin; Luong, John H. T.

    2008-12-01

    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 2O 3 and TiO 2) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl 2) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al 2O 3 and TiO 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 2O 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.

  11. Development and evaluation of a high-resolution reanalysis of the East Australian Current region using the Regional Ocean Modelling System (ROMS 3.4) and Incremental Strong-Constraint 4-Dimensional Variational (IS4D-Var) data assimilation

    NASA Astrophysics Data System (ADS)

    Kerry, Colette; Powell, Brian; Roughan, Moninya; Oke, Peter

    2016-10-01

    As with other Western Boundary Currents globally, the East Australian Current (EAC) is highly variable making it a challenge to model and predict. For the EAC region, we combine a high-resolution state-of-the-art numerical ocean model with a variety of traditional and newly available observations using an advanced variational data assimilation scheme. The numerical model is configured using the Regional Ocean Modelling System (ROMS 3.4) and takes boundary forcing from the BlueLink ReANalysis (BRAN3). For the data assimilation, we use an Incremental Strong-Constraint 4-Dimensional Variational (IS4D-Var) scheme, which uses the model dynamics to perturb the initial conditions, atmospheric forcing, and boundary conditions, such that the modelled ocean state better fits and is in balance with the observations. This paper describes the data assimilative model configuration that achieves a significant reduction of the difference between the modelled solution and the observations to give a dynamically consistent "best estimate" of the ocean state over a 2-year period. The reanalysis is shown to represent both assimilated and non-assimilated observations well. It achieves mean spatially averaged root mean squared (rms) residuals with the observations of 7.6 cm for sea surface height (SSH) and 0.4 °C for sea surface temperature (SST) over the assimilation period. The time-mean rms residual for subsurface temperature measured by Argo floats is a maximum of 0.9 °C between water depths of 100 and 300 m and smaller throughout the rest of the water column. Velocities at several offshore and continental shelf moorings are well represented in the reanalysis with complex correlations between 0.8 and 1 for all observations in the upper 500 m. Surface radial velocities from a high-frequency radar array are assimilated and the reanalysis provides surface velocity estimates with complex correlations with observed velocities of 0.8-1 across the radar footprint. A comparison with

  12. Enhancing Tumor Drug Delivery by Laser-Activated Vascular Barrier Disruption

    DTIC Science & Technology

    2009-12-01

    abilization, an early event commonly observed after photody- namic vascular-targeting therapy. Fig. 2. A, intravital microscopic imaging showing the...By permitting high resolution imaging of vessel structure and function in live animals, intravital microscopy offers a powerful tool to study vascular...Tumor blood vessels were continuously imaged by intravital fluorescence microscopy showing the formation of thrombi (indicated by arrows) that caused

  13. Advances in Light Microscopy for Neuroscience

    PubMed Central

    Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

    2010-01-01

    Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

  14. Stimulated Raman scattering microscopy for biomedical imaging

    NASA Astrophysics Data System (ADS)

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; He, Chengwei; Kang, Jing X.; Xie, X. Sunney

    2009-02-01

    Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is significantly greater than that of spontaneous Raman scattering, and is further enhanced by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and easily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast.

  15. Advances in light microscopy for neuroscience.

    PubMed

    Wilt, Brian A; Burns, Laurie D; Wei Ho, Eric Tatt; Ghosh, Kunal K; Mukamel, Eran A; Schnitzer, Mark J

    2009-01-01

    Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.

  16. Single molecule fluorescence and force microscopy.

    PubMed

    Schütz, G J; Hinterdorfer, P

    2002-12-01

    The investigation of biomolecules has entered a new age since the development of methodologies capable of studies at the level of single molecules. In biology, most molecules show a complex dynamical behavior, with individual motions and transitions between different states occurring highly correlated in space and time within an arrangement of various elements. Recent advances in the development of new microscopy techniques with sensitivity at the single molecule have gained access to essentially new types of information obtainable from imaging biomolecular samples. These methodologies are described here in terms of their applicability to the in vivo detection and visualization of molecular processes on surfaces, membranes, and cells. First examples of single molecule microscopy on cell membranes revealed new basic insight into the lateral organization of the plasma membrane, providing the captivating perspective of an ultra-sensitive methodology as a general tool to study local processes and heterogeneities in living cells.

  17. Scanning electron microscopy of lichen sclerosus*

    PubMed Central

    de Almeida, Hiram Larangeira; Bicca, Eduardo de Barros Coelho; Breunig, Juliano de Avelar; Rocha, Nara Moreira; Silva, Ricardo Marques e

    2013-01-01

    Lichen sclerosus is an acquired inflammatory condition characterized by whitish fibrotic plaques, with a predilection for the genital skin. We performed scanning electron microscopy of the dermis from a lesion of lichen sclerosus. Normal collagen fibers could be easily found in deeper layers of the specimen, as well as the transition to pathologic area, which seems homogenized. With higher magnifications in this transitional area collagen fibers are adherent to each other, and with very high magnifications a pearl chain aspect became evident along the collagen fibers. In the superficial dermis this homogenization is even more evident, collagen fibers are packed together and round structures are also observed. Rupture of collagen fibers and inflammatory cells were not found. These autoimmune changes of the extracellular matrix lead to the aggregation of immune complexes and/or changed matrix proteins along the collagen fibers, the reason why they seem hyalinized when examined by light microscopy. PMID:23739707

  18. Fast interferometric second harmonic generation microscopy

    PubMed Central

    Bancelin, Stéphane; Couture, Charles-André; Légaré, Katherine; Pinsard, Maxime; Rivard, Maxime; Brown, Cameron; Légaré, François

    2016-01-01

    We report the implementation of fast Interferometric Second Harmonic Generation (I-SHG) microscopy to study the polarity of non-centrosymmetric structures in biological tissues. Using a sample quartz plate, we calibrate the spatially varying phase shift introduced by the laser scanning system. Compensating this phase shift allows us to retrieve the correct phase distribution in periodically poled lithium niobate, used as a model sample. Finally, we used fast interferometric second harmonic generation microscopy to acquire phase images in tendon. Our results show that the method exposed here, using a laser scanning system, allows to recover the polarity of collagen fibrils, similarly to standard I-SHG (using a sample scanning system), but with an imaging time about 40 times shorter. PMID:26977349

  19. Circumventing photodamage in live-cell microscopy

    PubMed Central

    Magidson, Valentin; Khodjakov, Alexey

    2013-01-01

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

  20. Frontiers of in situ electron microscopy

    DOE PAGES

    Zheng, Haimei; Zhu, Yimei; Meng, Shirley Ying

    2015-01-01

    In situ transmission electron microscopy (TEM) has become an increasingly important tool for materials characterization. It provides key information on the structural dynamics of a material during transformations and the correlation between structure and properties of materials. With the recent advances in instrumentation, including aberration corrected optics, sample environment control, the sample stage, and fast and sensitive data acquisition, in situ TEM characterization has become more and more powerful. In this article, a brief review of the current status and future opportunities of in situ TEM is included. It also provides an introduction to the six articles covered by inmore » this issue of MRS Bulletin explore the frontiers of in situ electron microscopy, including liquid and gas environmental TEM, dynamic four-dimensional TEM, nanomechanics, ferroelectric domain switching studied by in situ TEM, and state-of-the-art atomic imaging of light elements (i.e., carbon atoms) and individual defects.« less

  1. Global standardization of scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Fujita, Daisuke; Itoh, Hiroshi; Ichimura, Shingo; Kurosawa, Tomizo

    2007-02-01

    Recent efforts to achieve global standardization of scanning probe microscopy (SPM) including noncontact atomic force microscopy (NC-AFM), especially through the International Organization for Standardization (ISO) and related research, are surveyed. Since the unification of terminology for SPM is a prerequisite for standardization, it should have the first priority, followed by the unification of data management and treatment, which will enable access to and processing of SPM data collected by different types of instrument. Among the various SPM analytical methods, the dimensional metrology of SPM is regarded to be the first priority for standardization. This requires solving two basic problems: calibrating the x, y, and z coordinate axes with traceability to the SI unit of length, and eliminating the morphological artefacts caused by the shape of the probe tip. Pre-standardization efforts on restoring distorted images and characterizing the tip shape during use are discussed.

  2. Reflectance Confocal Microscopy in Lentigo Maligna.

    PubMed

    Gamo, R; Pampín, A; Floristán, U

    2016-12-01

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

  3. Nuclear microscopy of sperm cell elemental structure

    NASA Astrophysics Data System (ADS)

    Bench, Graham S.; Balhorn, Rod; Friz, Alexander M.

    1995-05-01

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  4. Diffractive elements performance in chromatic confocal microscopy

    NASA Astrophysics Data System (ADS)

    Garzón, J.; Duque, D.; Alean, A.; Toledo, M.; Meneses, J.; Gharbi, T.

    2011-01-01

    The Confocal Laser Scanning Microscopy (CLSM) has been widely used in the semiconductor industry and biomedicine because of its depth discrimination capability. Subsequent to this technique has been developed in recent years Chromatic Confocal Microscopy. This method retains the same principle of confocal and offers the added advantage of removing the axial movement of the moving system. This advantage is usually accomplished with an optical element that generates a longitudinal chromatic aberration and a coding system that relates the axial position of each point of the sample with the wavelength that is focused on each. The present paper shows the performance of compact chromatic confocal microscope when some different diffractive elements are used for generation of longitudinal chromatic aberration. Diffractive elements, according to the process and manufacturing parameters, may have different diffraction efficiency and focus a specific wavelength in a specific focal position. The performance assessment is carried out with various light sources which exhibit an incoherent behaviour and a broad spectral width.

  5. Nuclear microscopy of sperm cell elemental structure

    SciTech Connect

    Bench, G.S.; Balhorn, R.; Friz, A.M.; Freeman, S.P.H.T.

    1994-09-28

    Theories suggest there is a link between protamine concentrations in individual sperm and male fertility. Previously, biochemical analyses have used pooled samples containing millions of sperm to determine protamine concentrations. These methods have not been able to determine what percentage of morphologically normal sperm are biochemically defective and potentially infertile. Nuclear microscopy has been utilized to measure elemental profiles at the single sperm level. By measuring the amount of phosphorus and sulfur, the total DNA and protamine content in individual sperm from fertile bull and mouse semen have been determined. These values agree with results obtained from other biochemical analyses. Nuclear microscopy shows promise for measuring elemental profiles in the chromatin of individual sperm. The technique may be able to resolve theories regarding the importance of protamines to male fertility and identify biochemical defects responsible for certain types of male infertility.

  6. Atomically resolved force microscopy at room temperature

    SciTech Connect

    Morita, Seizo

    2014-04-24

    Atomic force microscopy (AFM) can now not only image individual atoms but also construct atom letters using atom manipulation method even at room temperature (RT). Therefore, the AFM is the second generation atomic tool following the scanning tunneling microscopy (STM). However the AFM can image even insulating atoms, and also directly measure/map the atomic force and potential at the atomic scale. Noting these advantages, we have been developing a bottom-up nanostructuring system at RT based on the AFM. It can identify chemical species of individual atoms and then manipulate selected atom species to the predesigned site one-by-one to assemble complex nanostructures consisted of multi atom species at RT. Here we introduce our results toward atom-by-atom assembly of composite nanostructures based on the AFM at RT including the latest result on atom gating of nano-space for atom-by-atom creation of atom clusters at RT for semiconductor surfaces.

  7. Resonance response of scanning force microscopy cantilevers

    SciTech Connect

    Chen, G.Y.; Warmack, R.J.; Thundat, T.; Allison, D.P. ); Huang, A. )

    1994-08-01

    A variational method is used to calculate the deflection and the fundamental and harmonic resonance frequencies of commercial V-shaped and rectangular atomic force microscopy cantilevers. The effective mass of V-shaped cantilevers is roughly half that calculated for the equivalent rectangular cantilevers. Damping by environmental gases, including air, nitrogen, argon, and helium, affects the frequency of maximum response and to a much greater degree the quality factor [ital Q]. Helium has the lowest viscosity, resulting in the highest [ital Q], and thus provides the best sensitivity in noncontact force microscopy. Damping in liquids is dominated by an increase in effective mass of the cantilever due to an added mass of the liquid being dragged with that cantilever.

  8. Interferometric phase microscopy of red blood cells

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Sun, Nan; Tang, Xian; Wang, Yin; Wang, Shouyu

    2013-12-01

    Quantitative phase imaging of cells with high accuracy in a completely noninvasive manner is a challenging task. To provide a proper solution to this important need, interferometric phase microscopy is described which relies on the off-axis interferometry, confocal microscopy and high-speed image capture technology. Phase retrieval from the single interferogram is done by algorithms based on the fast Fourier transform, traditional Hilbert transform and two-step Hilbert transform, respectively. Furthermore, a phase aberrations compensation approach is applied to correct the phase distribution of the red blood cells obtained via the three methods mentioned before without the pre-known knowledge for removing the wave front curvature introduced by the microscope objectives, off-axis imaging, etc., which otherwise hinders the phase reconstruction. The improved results reveal the better inner structures of the red blood cells. The development of quantitative phase imaging technique is shedding light on their future directions and applications for basic and clinical research.

  9. Confocal multiview light-sheet microscopy

    PubMed Central

    Medeiros, Gustavo de; Norlin, Nils; Gunther, Stefan; Albert, Marvin; Panavaite, Laura; Fiuza, Ulla-Maj; Peri, Francesca; Hiiragi, Takashi; Krzic, Uros; Hufnagel, Lars

    2015-01-01

    Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage. PMID:26602977

  10. Speckle-field digital holographic microscopy

    PubMed Central

    Park, YongKeun; Choi, Wonshik; Yaqoob, Zahid; Dasari, Ramachandra; Badizadegan, Kamran; Feld, Michael S.

    2010-01-01

    The use of coherent light in conventional holographic phase microscopy (HPM) poses three major drawbacks: poor spatial resolution, weak depth sectioning, and fixed pattern noise due to unwanted diffraction. Here, we report a technique which can overcome these drawbacks, but maintains the advantage of phase microscopy - high contrast live cell imaging and 3D imaging. A speckle beam of a complex spatial pattern is used for illumination to reduce fixed pattern noise and to improve optical sectioning capability. By recording of the electric field of speckle, we demonstrate high contrast 3D live cell imaging without the need for axial scanning - neither objective lens nor sample stage. This technique has great potential in studying biological samples with improved sensitivity, resolution and optical sectioning capability. PMID:19654630

  11. Three-Dimensional Reflectance Traction Microscopy

    PubMed Central

    Jones, Christopher A. R.; Groves, Nicholas Scott; Sun, Bo

    2016-01-01

    Cells in three-dimensional (3D) environments exhibit very different biochemical and biophysical phenotypes compared to the behavior of cells in two-dimensional (2D) environments. As an important biomechanical measurement, 2D traction force microscopy can not be directly extended into 3D cases. In order to quantitatively characterize the contraction field, we have developed 3D reflectance traction microscopy which combines confocal reflection imaging and partial volume correlation postprocessing. We have measured the deformation field of collagen gel under controlled mechanical stress. We have also characterized the deformation field generated by invasive breast cancer cells of different morphologies in 3D collagen matrix. In contrast to employ dispersed tracing particles or fluorescently-tagged matrix proteins, our methods provide a label-free, computationally effective strategy to study the cell mechanics in native 3D extracellular matrix. PMID:27304456

  12. Reconstruction in interferometric synthetic aperture microscopy: comparison with optical coherence tomography and digital holographic microscopy.

    PubMed

    Sheppard, Colin J R; Kou, Shan Shan; Depeursinge, Christian

    2012-03-01

    It is shown that the spatial frequencies recorded in interferometric synthetic aperture microscopy do not correspond to exact backscattering [as they do in unistatic synthetic aperture radar (SAR)] and that the reconstruction process based on SAR is therefore based on an approximation. The spatial frequency response is developed based on the three-dimensional coherent transfer function approach and compared with that in optical coherence tomography and digital holographic microscopy.

  13. Application perspectives of localization microscopy in virology.

    PubMed

    Cremer, C; Kaufmann, R; Gunkel, M; Polanski, F; Müller, P; Dierkes, R; Degenhard, S; Wege, C; Hausmann, M; Birk, U

    2014-07-01

    Localization microscopy approaches allowing an optical resolution down to the single-molecule level in fluorescence-labeled biostructures have already found a variety of applications in cell biology, as well as in virology. Here, we focus on some perspectives of a special localization microscopy embodiment, spectral precision distance/position determination microscopy (SPDM). SPDM permits the use of conventional fluorophores or fluorescent proteins together with standard sample preparation conditions employing an aqueous buffered milieu and typically monochromatic excitation. This allowed superresolution imaging and studies on the aggregation state of modified tobacco mosaic virus particles on the nanoscale with a single-molecule localization accuracy of better than 8 nm, using standard fluorescent dyes in the visible spectrum. To gain a better understanding of cell entry mechanisms during influenza A virus infection, SPDM was used in conjunction with algorithms for distance and cluster analyses to study changes in the distribution of virus particles themselves or in the distribution of infection-related proteins, the hepatocyte growth factor receptors, in the cell membrane on the single-molecule level. Not requiring TIRF (total internal reflection) illumination, SPDM was also applied to study the molecular arrangement of gp36.5/m164 glycoprotein (essentially associated with murine cytomegalovirus infection) in the endoplasmic reticulum and the nuclear membrane inside cells with single-molecule resolution. On the basis of the experimental evidence so far obtained, we finally discuss additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligonucleotide fluorescence in situ hybridization, as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices.

  14. Surface Studies by Scanning Probe Microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Ho-Seob

    The scanning probe microscopy reported here includes scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS), and atomic force microscopy (AFM). The scanning tunneling microscope is a novel tool which can reveal the atomic structure and electronic properties of surfaces using a probe with a sharp tip. An additional technique, atomic force microscopy has the potential to record geometric structures for both conducting and non -conducting materials. The first AFM designs utilized short range forces between a small stylus and a sample surface to produce high resolution images of defects and structural features of the surface. The current-voltage characteristics were also investigated during dynamic changes of the tunnel current and barrier height with an additional technology, tunneling spectroscopy. An advanced design for an AFM has been developed which utilizes a dielectric tunnel junction to retain the high sensitivity of tunnel current control over force ranges between 10^{-6} and 10 ^{-11}N. This AFM has been successfully applied to physical and biological samples. Scanning probe techniques have been developed and applied to a range of sample types including conductors, semi-conductors and non-conductors. Each technique utilizes the same electronics, computers, and imaging facilities. A fundamental problem of the atomic structure of graphite has existed since the inception of STM images. The experimental and theoretical hypotheses have been considered and a resolution of the problem has been developed as reported in this dissertation. Unprecedented resolving power, greater than 1A, has confirmed our hypothesis and has been correctly correlated with the structure of graphite surface. This dissertation also presents the results from studies of the surface structure of: MoS_2 , Cu, Au, Ag, Si, CdTe, HgTe, Fe_2 O_3, mica, gypsum, purple membranes with protein chains, and an organic photoconducting material, by scanning probe microscopes.

  15. Correlative photoactivated localization and scanning electron microscopy.

    PubMed

    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.

  16. Photoacoustic microscopy of ceramic turbine blades

    NASA Technical Reports Server (NTRS)

    Khandelwal, P. K.; Kinnick, R. R.; Heitman, P. W.

    1985-01-01

    Scanning photoacoustic microscopy (SPAM) is evaluated as a nondestructive technique for the detection of both surface and subsurface flaws in polycrystalline ceramics, such as those currently under consideration for the high temperature components of small vehicular and industrial gas turbine engines; the fracture strength of these brittle materials is controlled by small, 25-200 micron flaws. Attention is given to the correlation of SPAM-detected flaws with actual, fracture-controlling flaws in ceramic turbine blades.

  17. Orbital angular momentum light in microscopy

    NASA Astrophysics Data System (ADS)

    Ritsch-Marte, Monika

    2017-02-01

    Light with a helical phase has had an impact on optical imaging, pushing the limits of resolution or sensitivity. Here, special emphasis will be given to classical light microscopy of phase samples and to Fourier filtering techniques with a helical phase profile, such as the spiral phase contrast technique in its many variants and areas of application. This article is part of the themed issue 'Optical orbital angular momentum'.

  18. Dark Field Microscopy for Analytical Laboratory Courses

    SciTech Connect

    Augspurger, Ashley E; Stender, Anthony S; Marchuk, Kyle; Greenbowe, Thomas J; Fang, Ning

    2014-06-10

    An innovative and inexpensive optical microscopy experiment for a quantitative analysis or an instrumental analysis chemistry course is described. The students have hands-on experience with a dark field microscope and investigate the wavelength dependence of localized surface plasmon resonance in gold and silver nanoparticles. Students also observe and measure individual crystal growth during a replacement reaction between copper and silver nitrate. The experiment allows for quantitative, qualitative, and image data analyses for undergraduate students.

  19. Reflection Acoustic Microscopy for Micro-NDE.

    DTIC Science & Technology

    1983-02-01

    WORDS (Coni, wu rere side. 14 It noeeeey And Idenify1 by block esife) Nondestructive Evaluation Acoustic Microscopy I Subsurface Imaging Pulsecio Cmrsin... subsurface imaging is presented and it is shown that with such lenses it is possible to obtain good focussing performance over a wide depth range...typically few millimeters at 50 MHz. A major problem in subsurface imaging derives from the large reflection obtained frnm the surface, and the small amount

  20. Biological cryo-electron microscopy in China.

    PubMed

    Wang, Hong-Wei; Lei, Jianlin; Shi, Yigong

    2017-01-01

    Cryo-electron microscopy (cryo-EM) plays an increasingly more important role in structural biology. With the construction of an arm of the Chinese National Protein Science Facility at Tsinghua University, biological cryo-EM has entered a phase of rapid development in China. This article briefly reviews the history of biological cryo-EM in China, describes its current status, comments on its impact on the various biological research fields, and presents future outlook.

  1. Biological cryo‐electron microscopy in China

    PubMed Central

    2016-01-01

    Abstract Cryo‐electron microscopy (cryo‐EM) plays an increasingly more important role in structural biology. With the construction of an arm of the Chinese National Protein Science Facility at Tsinghua University, biological cryo‐EM has entered a phase of rapid development in China. This article briefly reviews the history of biological cryo‐EM in China, describes its current status, comments on its impact on the various biological research fields, and presents future outlook. PMID:27534377

  2. A New Twist on Scanning Thermal Microscopy

    DTIC Science & Technology

    2012-01-25

    Materials and Manufacturing Directorate, Air Force Research Laboratory, WPAFB, Ohio 45433, United States ‡School of Materials Science and Engineering...bimorphs do not suffer from these setbacks. In fact, thermal bimorphs transduced with an atomic force microscopy (AFM) quadrant photodetector have a...AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Air Force Research Laboratory

  3. Quantitative imaging of bilirubin by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

    2013-03-01

    Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

  4. Image Correlation Microscopy for Uniform Illumination

    PubMed Central

    Gaborski, Thomas R.; Sealander, Michael N.; Ehrenberg, Morton; Waugh, Richard E.; McGrath, James L.

    2011-01-01

    Image cross-correlation microscopy (ICM) is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. ICM has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy (FCS). In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy (UI-ICM). Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning ICM, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function (SACF). Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function (TACF) depends strongly on particle size and not particle shape. In this report, we establish the relationships between the SACF feature size, TACF characteristic time and the diffusion coefficient for UI-ICM using analytical, Monte-Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate UI-ICM analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils. PMID:20055917

  5. Observation of Superlubricity by Scanning Tunneling Microscopy

    NASA Astrophysics Data System (ADS)

    Hirano, Motohisa; Shinjo, Kazumasa; Kaneko, Reizo; Murata, Yoshitada

    1997-02-01

    Experimental evidence of superlubricity, the state of vanishing friction, is obtained by examining systems of sliding atomically clean surfaces by using ultrahigh vacuum scanning tunneling microscopy. The experimental results agree with theoretical predictions: Friction is not observed in the superlubricity regime in measurements capable of resolving a friction force of 3×10-9 N, whereas friction of 8×10-8 N, which is comparable to theoretical values, is observed in the friction regime.

  6. Multidepth imaging by chromatic dispersion confocal microscopy

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2012-03-01

    Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

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

    PubMed

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

    2015-12-01

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

  8. Efficient illumination for microsecond tracking microscopy.

    PubMed

    Dulin, David; Barland, Stephane; Hachair, Xavier; Pedaci, Francesco

    2014-01-01

    The possibility to observe microsecond dynamics at the sub-micron scale, opened by recent technological advances in fast camera sensors, will affect many biophysical studies based on particle tracking in optical microscopy. A main limiting factor for further development of fast video microscopy remains the illumination of the sample, which must deliver sufficient light to the camera to allow microsecond exposure times. Here we systematically compare the main illumination systems employed in holographic tracking microscopy, and we show that a superluminescent diode and a modulated laser diode perform the best in terms of image quality and acquisition speed, respectively. In particular, we show that the simple and inexpensive laser illumination enables less than 1 μs camera exposure time at high magnification on a large field of view without coherence image artifacts, together with a good hologram quality that allows nm-tracking of microscopic beads to be performed. This comparison of sources can guide in choosing the most efficient illumination system with respect to the specific application.

  9. Invited Review Article: Pump-probe microscopy.

    PubMed

    Fischer, Martin C; Wilson, Jesse W; Robles, Francisco E; Warren, Warren S

    2016-03-01

    Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

  10. Invited Review Article: Pump-probe microscopy

    PubMed Central

    Wilson, Jesse W.; Robles, Francisco E.; Warren, Warren S.

    2016-01-01

    Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications. PMID:27036751

  11. Non-radiative excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Riachy, Lina; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-03-01

    Non-radiative Excitation Fluorescence Microscopy (NEFM) constitutes a new way to observe biological samples beyond the diffraction limit. Non-radiative excitation of the samples is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer. To prevent dewetting of the donor film, we have recently implemented a silanization process to covalently bond the QDs on the substrate. An homogeneous monolayer of QDs was then deposited on only one side of the coverslips. Atomic force microscopy was then used to characterize the QD layer. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed a quenching of QDs emission, together with an emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs.

  12. Mirror-enhanced super-resolution microscopy.

    PubMed

    Yang, Xusan; Xie, Hao; Alonas, Eric; Liu, Yujia; Chen, Xuanze; Santangelo, Philip J; Ren, Qiushi; Xi, Peng; Jin, Dayong

    Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.

  13. Image Restoration in Cryo-electron Microscopy

    PubMed Central

    Penczek, Pawel A.

    2011-01-01

    Image restoration techniques are used to obtain, given experimental measurements, the best possible approximation of the original object within the limits imposed by instrumental conditions and noise level in the data. In molecular electron microscopy, we are mainly interested in linear methods that preserve the respective relationships between mass densities within the restored map. Here, we describe the methodology of image restoration in structural electron microscopy, and more specifically, we will focus on the problem of the optimum recovery of Fourier amplitudes given electron microscope data collected under various defocus settings. We discuss in detail two classes of commonly used linear methods, the first of which consists of methods based on pseudoinverse restoration, and which is further subdivided into mean-square error, chi-square error, and constrained based restorations, where the methods in the latter two subclasses explicitly incorporates non-white distribution of noise in the data. The second class of methods is based on the Wiener filtration approach. We show that the Wiener filter-based methodology can be used to obtain a solution to the problem of amplitude correction (or “sharpening”) of the electron microscopy map that makes it visually comparable to maps determined by X-ray crystallography, and thus amenable to comparable interpretation. Finally, we present a semi-heuristic Wiener filter-based solution to the problem of image restoration given sets of heterogeneous solutions. We conclude the chapter with a discussion of image restoration protocols implemented in commonly used single particle software packages. PMID:20888957

  14. Mirror-enhanced super-resolution microscopy

    PubMed Central

    Yang, Xusan; Xie, Hao; Alonas, Eric; Liu, Yujia; Chen, Xuanze; Santangelo, Philip J; Ren, Qiushi; Xi, Peng; Jin, Dayong

    2016-01-01

    Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power. PMID:27398242

  15. Quantitative Aspects of Single Molecule Microscopy

    PubMed Central

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

    2015-01-01

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

  16. Single cell elemental analysis using nuclear microscopy

    NASA Astrophysics Data System (ADS)

    Ren, M. Q.; Thong, P. S. P.; Kara, U.; Watt, F.

    1999-04-01

    The use of Particle Induced X-ray Emission (PIXE), Rutherford Backscattering Spectrometry (RBS) and Scanning Transmission Ion Microscopy (STIM) to provide quantitative elemental analysis of single cells is an area which has high potential, particularly when the trace elements such as Ca, Fe, Zn and Cu can be monitored. We describe the methodology of sample preparation for two cell types, the procedures of cell imaging using STIM, and the quantitative elemental analysis of single cells using RBS and PIXE. Recent work on single cells at the Nuclear Microscopy Research Centre,National University of Singapore has centred around two research areas: (a) Apoptosis (programmed cell death), which has been recently implicated in a wide range of pathological conditions such as cancer, Parkinson's disease etc, and (b) Malaria (infection of red blood cells by the malaria parasite). Firstly we present results on the elemental analysis of human Chang liver cells (ATTCC CCL 13) where vanadium ions were used to trigger apoptosis, and demonstrate that nuclear microscopy has the capability of monitoring vanadium loading within individual cells. Secondly we present the results of elemental changes taking place in individual mouse red blood cells which have been infected with the malaria parasite and treated with the anti-malaria drug Qinghaosu (QHS).

  17. Intermittent contact hydration scanning probe microscopy.

    PubMed

    Aloisi, G; Bacci, F; Carlà, M; Dolci, D

    2010-07-01

    Hydration scanning probe microscopy is a technique similar to scanning tunneling microscopy, in which the probe current, sustained by the slight surface conduction of a thin hydration layer covering an insulating support surface, is essentially electrochemical in nature instead of electronic tunneling. Such a technique allows the imaging of a great variety of samples, including insulators, provided that they are hydrophilic, as well as the study of molecular samples of biological interest (such as DNA) fixed on a suitable supporting surface. The main problem to obtain stable and reproducible images comes from the very critical determination of the operating conditions under which the probe-hydration layer interaction does not lead to the formation of a relatively large water meniscus. It has been suggested that this issue can be removed by adding a high frequency oscillation to the probe movement, as in tapping atomic force microscopy. Meniscus formation and breakup have been investigated in order to determine the best values for the amplitude and the frequency of the oscillation. Results obtained in this mode are discussed in comparison with the usual continuous contact mode.

  18. Invited Review Article: Pump-probe microscopy

    NASA Astrophysics Data System (ADS)

    Fischer, Martin C.; Wilson, Jesse W.; Robles, Francisco E.; Warren, Warren S.

    2016-03-01

    Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.

  19. Optical microscopy beyond the diffraction limit

    PubMed Central

    Smolyaninov, Igor I.

    2008-01-01

    Over the past century the resolution of far-field optical microscopes, which rely on propagating optical modes, was widely believed to be limited because of diffraction to a value on the order of a half-wavelength λ∕2 of the light used. Although immersion microscopes had slightly improved resolution on the order of λ∕2n, the increased resolution was limited by the small range of refractive indices, n, of available transparent materials. We are experiencing quick demolition of the diffraction limit in optical microscopy. Over the past few years numerous nonlinear optical microscopy techniques based on photoswitching and saturation of fluorescence demonstrated far-field resolution of 20 to 30 nm. The latest exciting example of these techniques has been demonstrated by Huang et al. [Science 319, 810–813 (2008)]. Moreover, recent progress in metamaterials indicates that artificial optical media can be created, which do not exhibit the diffraction limit. Resolution of linear “immersion” microscopes based on such metamaterials appears limited only by losses, which can be compensated by gain media. Thus, optical microscopy is quickly moving towards the 10 nm resolution scale, which should bring about numerous revolutionary advances in biomedical imaging. PMID:19404465

  20. Shaping field for deep tissue microscopy

    NASA Astrophysics Data System (ADS)

    Colon, J.; Lim, H.

    2015-05-01

    Information capacity of a lossless image-forming system is a conserved property determined by two imaging parameters - the resolution and the field of view (FOV). Adaptive optics improves the former by manipulating the phase, or wavefront, in the pupil plane. Here we describe a homologous approach, namely adaptive field microscopy, which aims to enhance the FOV by controlling the phase, or defocus, in the focal plane. In deep tissue imaging, the useful FOV can be severely limited if the region of interest is buried in a thick sample and not perpendicular to the optic axis. One must acquire many z-scans and reconstruct by post-processing, which exposes tissue to excessive radiation and is also time consuming. We demonstrate the effective FOV can be substantially enhanced by dynamic control of the image plane. Specifically, the tilt of the image plane is continuously adjusted in situ to match the oblique orientation of the sample plane within tissue. The utility of adaptive field microscopy is tested for imaging tissue with non-planar morphology. Ocular tissue of small animals was imaged by two-photon excited fluorescence. Our results show that adaptive field microscopy can utilize the full FOV. The freedom to adjust the image plane to account for the geometrical variations of sample could be extremely useful for 3D biological imaging. Furthermore, it could facilitate rapid surveillance of cellular features within deep tissue while avoiding photo damages, making it suitable for in vivo imaging.

  1. Electron microscopy of Crotalaria pulmonary hypertension

    PubMed Central

    Kay, J. M.; Smith, Paul; Heath, Donald

    1969-01-01

    The lungs of 11 rats fed on Crotalaria spectabilis seeds for periods ranging from 12 to 61 days were examined by both light and electron microscopy. The findings were compared with those obtained from nine control rats given a normal diet. Eight of the 11 test rats showed morphological evidence of pulmonary arterial hypertension in the form of right ventricular hypertrophy; the exceptions were rats killed after receiving the Crotalaria diet for 12, 22, and 29 days respectively. On light microscopy, all the test rats showed exudative lesions in the lungs consisting of eosinophilic alveolar coagulum, intra-alveolar haemorrhage, interstitial fibrosis, and a proliferation of mast cells. Enlarged and proliferated cells were seen to line the alveolar walls or lie free within the alveolar spaces. Electron microscopy showed these cells to be enlarged granular pneumocytes containing enlarged, electron-dense, lamellar secretory inclusions. Scanty macrophages were also seen in the alveolar spaces, in which excessive numbers of myelin figures and lattices were seen: these structures resembled phospholipid membranes and were probably related to pulmonary surfactant. We think that proliferation of granular pneumocytes is a non-specific reaction of the alveolar walls to injury. The alveolar-capillary wall showed interstitial oedema with the formation of intraluminal endothelial vesicles, probably representing the early ultrastructural phase of pulmonary oedema, and more likely to be an effect of the pulmonary hypertension than its cause. Images PMID:5348317

  2. Laser beam shaping for biomedical microscopy techniques

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  3. Advances in fiber lasers for nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Wise, F. W.; Ouzounov, D.; Kieu, K.; Renninger, W.; Chong, A.; Liu, H.

    2008-02-01

    In the past 30 years major advances in medical imaging have been made in areas such as magnetic resonance imaging, computed tomography, and ultrasound. These techniques have become quite effective for structural imaging at the organ or tissue level, but do not address the clear need for imaging technologies that exploit existing knowledge of the genetic and molecular bases of disease. Techniques that can provide similar information on the cellular and molecular scale would be very powerful, and ultimately the extension of such techniques to in vivo measurements will be desired. The availability of these imaging capabilities would allow monitoring of the early stages of disease or therapy, for example. Optical techniques provide excellent imaging capabilities, with sub-micron spatial resolution, and are noninvasive. An overall goal of biomedical imaging is to obtain diagnostic or functional information about biological structures. The difficulty of acquiring high-resolution images of structures deep in tissue presents a major challenge, however, owing to strong scattering of light. As a consequence, optical imaging has been limited to thin (typically ~0.5 mm) samples or superficial tissue. In contrast, techniques such as ultrasound and magnetic resonance provide images of structures centimeters deep in tissue, with ~100-micron resolution. It is desirable to develop techniques that offer the resolution of optics with the depth-penetration of other techniques. Since 1990, a variety of nonlinear microscopies have been demonstrated. These include 2- and 3-photon fluorescence microscopy, and 2nd- and 3rd-harmonic generation microscopies. These typically employ femtosecond-pulse excitation, for maximum peak power (and thus nonlinear excitation) for a given pulse energy. A relative newcomer to the group is CARS microscopy [1], which exploits resonant vibrational excitation of molecules or bonds. The CARS signal contrast arises from intrinsic elements of cells, and thus

  4. Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity

    NASA Astrophysics Data System (ADS)

    De Luca, Giulia; Breedijk, Ronald; Hoebe, Ron; Stallinga, Sjoerd; Manders, Erik

    2017-03-01

    Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.49 NA. As the theory predicts, this improved lateral resolution is independent of the pinhole diameter. In standard confocal microscopy, the same lateral resolution can only be achieved with an almost closed pinhole and, consequently, with a major loss of signal. We show that the sectioning capabilities of the standard confocal microscope are preserved in RCM and that the axial resolution of RCM is slightly better (~15%) than the standard confocal microscope. Furthermore, the signal-to-noise ratio in RCM is a factor of 2 higher than in standard confocal microscopy, also due to the use of highly sensitive modern cameras. In case the pinhole of a confocal microscope is adjusted in such way that the lateral resolution is comparable to that of RCM, the signal-to-noise ratio in RCM is 4 times higher than standard confocal microscopy. Therefore, RCM offers a good alternative to standard confocal microscopy for higher lateral resolution with the main advantage of strongly improved sensitivity.

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

    SciTech Connect

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

    2015-12-15

    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.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. A Correlative Optical Microscopy and Scanning Electron Microscopy Approach to Locating Nanoparticles in Brain Tumors

    PubMed Central

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

    2014-01-01

    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. PMID:25464144

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

    PubMed

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

    2015-01-01

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

  10. Resolution doubling using confocal microscopy via analogy with structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Hayashi, Shinichi

    2016-08-01

    Structured illumination microscopy (SIM) is a super-resolution fluorescence microscopy with a 2-fold higher lateral resolution than conventional wide-field fluorescence (WF) microscopy. Confocal fluorescence (CF) microscopy has approximately the same optical cutoff frequency as SIM; however, the maximum theoretical increase in lateral resolution over that of WF is 1.4-fold with an infinitesimal pinhole diameter. Quantitative comparisons based on an analytical imaging formula revealed that modulation transfer functions (MTFs) of SIM reconstructed images before postprocessing are nearly identical to those of CF images recorded with an infinitesimal pinhole diameter. Here, we propose a new method using an adequate pinhole diameter combined with the use of an apodized Fourier inverse filter to increase the lateral resolution of CF images to as much as that SIM images without significant noise degradation in practice. Furthermore, the proposed method does not require a posteriori parameterization and has reproducibility. This approach can be easily applied to conventional laser scanning CF, spinning disk CF, and multiphoton microscopies.

  11. Complete and rapid switch from light microscopy to virtual microscopy for teaching medical histology.

    PubMed

    Krippendorf, Beth B; Lough, John

    2005-07-01

    During the interim between the 2003 and 2004 academic years, the cell and tissue biology and integrated medical neuroscience courses at the Medical College of Wisconsin made a complete and rapid switch from light microscopy- to virtual microscopy-based histology laboratories. This switch was prompted by the difficulties in maintaining and the cost of replacing the college's microscopes and microscope slides, and primarily by the desire to promote and streamline learning for our large classes (n > 200) of first-year medical students. A group of students who used the virtual microscope, another group of students who used the light microscope, and faculty with experience using both tools rated the effectiveness of the virtual microscope for learning and teaching. Also, to determine whether virtual microscopy affected student learning, laboratory examination scores for the 2004 class (n = 209) were compared with those of four previous classes that used light microscopes exclusively (n = 811). The switch from light microscopy to virtual microscopy was very favorably received by both students and faculty. More importantly, data from examination scores and course evaluation surveys indicated that use of the virtual microscope may significantly improve student performance and learning efficiency. Procedures for successfully implementing this change are described.

  12. Scanning Ion Conductance Microscopy of Live Keratinocytes

    NASA Astrophysics Data System (ADS)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

  13. Atomic force microscopy study of tooth surfaces.

    PubMed

    Farina, M; Schemmel, A; Weissmüller, G; Cruz, R; Kachar, B; Bisch, P M

    1999-03-01

    Atomic force microscopy (AFM) was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces. Human teeth and teeth from a rodent (Golden hamster), from a fish (piranha), and from a grazing mollusk (chiton) with distinct feeding habits were analyzed in terms of particle arrangement, packing, and size distribution. Scanning electron microscopy and transmission electron microscopy were used for comparison. It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of complementary and/or new structural information. All teeth were cleaned and some were etched with acidic solutions before analysis. It was observed that human enamel (permanent teeth) presents particles tightly packed in the outer surface, whereas enamel from the hamster (continuously growing teeth) shows particles of less dense packing. The piranha teeth have a thin cuticle covering the long apatite crystals of the underlying enameloid. This cuticle has a rough surface of particles that have a globular appearance after the brief acidic treatment. The similar appearance of the in vivo naturally etched tooth surface suggests that the pattern of globule distribution may be due to the presence of an organic material. Elemental analysis of this cuticle indicated that calcium, phosphorus, and iron are the main components of the structure while electron microdiffraction of pulverized cuticle particles showed a pattern consistent with hydroxyapatite. The chiton mineralized tooth cusp had a smooth surface in an unabraded region and a very rough structure with the magnetite crystals (already known to make part of the structure) protruding from the surface. It was concluded that the structures analyzed are optimized for efficiency in feeding mechanism and life span of the teeth.

  14. Light Microscopy Module Imaging Tested and Demonstrated

    NASA Technical Reports Server (NTRS)

    Gati, Frank

    2004-01-01

    The Fluids Integrated Rack (FIR), a facility-class payload, and the Light Microscopy Module (LMM), a subrack payload, are integrated research facilities that will fly in the U.S. Laboratory module, Destiny, aboard the International Space Station. Both facilities are being engineered, designed, and developed at the NASA Glenn Research Center by Northrop Grumman Information Technology. The FIR is a modular, multiuser scientific research facility that is one of two racks that make up the Fluids and Combustion Facility (the other being the Combustion Integrated Rack). The FIR has a large volume dedicated for experimental hardware; easily reconfigurable diagnostics, power, and data systems that allow for unique experiment configurations; and customizable software. The FIR will also provide imagers, light sources, power management and control, command and data handling for facility and experiment hardware, and data processing and storage. The first payload in the FIR will be the LMM. The LMM integrated with the FIR is a remotely controllable, automated, on-orbit microscope subrack facility, with key diagnostic capabilities for meeting science requirements--including video microscopy to observe microscopic phenonema and dynamic interactions, interferometry to make thin-film measurements with nanometer resolution, laser tweezers to manipulate micrometer-sized particles, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure the photonic properties of materials. Vibration disturbances were identified early in the LMM development phase as a high risk for contaminating the science microgravity environment. An integrated FIR-LMM test was conducted in Glenn's Acoustics Test Laboratory to assess mechanical sources of vibration and their impact to microscopic imaging. The primary purpose of the test was to characterize the LMM response at the sample location, the x-y stage within the microscope, to vibration

  15. Super-resolved spatial light interference microscopy.

    PubMed

    Chu, Kaiqin; Smith, Zachary J; Wachsmann-Hogiu, Sebastian; Lane, Stephen

    2012-03-01

    We report a scheme to achieve resolution beyond the diffraction limit in spatial light interference microscopy (SLIM). By adding a grating to the optical path, the structured illumination technique can be used to improve the resolution by a factor of 2. We show that a direct application of the structured illumination technique, however, has proved to be unsuccessful. Through two crucial modifications, namely, one to the pupil plane of the objective and the other to the demodulation procedure, faithful phase information of the object is recovered and the resolution is improved by a factor of 2.

  16. Cell surface fluctuations studied with defocusing microscopy

    NASA Astrophysics Data System (ADS)

    Agero, U.; Monken, C. H.; Ropert, C.; Gazzinelli, R. T.; Mesquita, O. N.

    2003-05-01

    Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the

  17. Nonlinear Optical Microscopy of Single Nanostructures

    NASA Astrophysics Data System (ADS)

    Huang, Libai; Cheng, Ji-Xin

    2013-07-01

    We review recent advances in nonlinear optical (NLO) microscopy studies of single nanostructures. NLO signals are intrinsically sensitive to the electronic, vibrational, and structural properties of such nanostructures. Ultrafast excitation allows for mapping of energy relaxation pathways at the single-particle level. The strong nonlinear response of nanostructures makes them highly attractive for applications as novel NLO imaging agents in biological and biomedical research. NLO modalities based on harmonic generation, multiphoton photoluminescence, four-wave mixing, and pump-probe processes are discussed in detail.

  18. Improvement of image quality in holographic microscopy.

    PubMed

    Budhiraja, C J; Som, S C

    1981-05-15

    A novel technique of noise reduction in holographic microscopy has been experimentally studied. It has been shown that significant improvement in the holomicroscopic images of actual low-contrast continuous tone biological objects can be achieved without trade off in image resolution. The technique makes use of holographically produced multidirectional phase gratings used as diffusers and the continuous addition of subchannel holograms. It has been shown that the self-imaging property of this type of diffuser makes the use of these diffusers ideal for microscopic objects. Experimental results have also been presented to demonstrate real-time image processing capability of this technique.

  19. Scanning electron microscopy study of Tritrichomonas augusta.

    PubMed

    Borges, Fernanda P; Wiltuschnig, Renata C M; Tasca, Tiana; De Carli, Geraldo A

    2004-09-01

    Tritrichomonas augusta is a flagellated protozoan that parasitizes amphibians and reptiles. According to scanning electron microscopy (SEM), the cell shape of T. augusta varies from slender pyriform to ovoidal. Our data show the morphological features of the trophozoites: the emergence of the anterior flagella, the structure of the undulating membrane and the position and shape of the pelta, axostyle and posterior flagellum. In addition, herein we describe spherical forms which are probably pseudocysts. The description of the external structure of T. augusta, as demonstrated by SEM, contributes to the understanding of the biology of this parasite.

  20. System analysis of force feedback microscopy

    SciTech Connect

    Rodrigues, Mario S.; Chevrier, Joël; Comin, Fabio

    2014-02-07

    It was shown recently that the Force Feedback Microscope (FFM) can avoid the jump-to-contact in Atomic force Microscopy even when the cantilevers used are very soft, thus increasing force resolution. In this letter, we explore theoretical aspects of the associated real time control of the tip position. We take into account lever parameters such as the lever characteristics in its environment, spring constant, mass, dissipation coefficient, and the operating conditions such as controller gains and interaction force. We show how the controller parameters are determined so that the FFM functions at its best and estimate the bandwidth of the system under these conditions.