WorldWideScience

Sample records for two-photon microscopic imaging

  1. Adapting a compact confocal microscope system to a two-photon excitation fluorescence imaging architecture.

    Science.gov (United States)

    Diaspro, A; Corosu, M; Ramoino, P; Robello, M

    1999-11-01

    Within the framework of a national National Institute of Physics of Matter (INFM) project, we have realised a two-photon excitation (TPE) fluorescence microscope based on a new generation commercial confocal scanning head. The core of the architecture is a mode-locked Ti:Sapphire laser (Tsunami 3960, Spectra Physics Inc., Mountain View, CA) pumped by a high-power (5 W, 532 nm) laser (Millennia V, Spectra Physics Inc.) and an ultracompact confocal scanning head, Nikon PCM2000 (Nikon Instruments, Florence, Italy) using a single-pinhole design. Three-dimensional point-spread function has been measured to define spatial resolution performances. The TPE microscope has been used with a wide range of excitable fluorescent molecules (DAPI, Fura-2, Indo-1, DiOC(6)(3), fluoresceine, Texas red) covering a single photon spectral range from UV to green. An example is reported on 3D imaging of the helical structure of the sperm head of the Octopus Eledone cirrhosa labelled with an UV excitable dye, i.e., DAPI. The system can be easily switched for operating both in conventional and two-photon mode. Copyright 1999 Wiley-Liss, Inc.

  2. Automatic quantification of mitochondrial fragmentation from two-photon microscope images of mouse brain tissue.

    Science.gov (United States)

    Lihavainen, E; Kislin, M; Toptunov, D; Khiroug, L; Ribeiro, A S

    2015-12-01

    The morphology of mitochondria can inform about their functional state and, thus, about cell vitality. For example, fragmentation of the mitochondrial network is associated with many diseases. Recent advances in neuronal imaging have enabled the observation of mitochondria in live brains for long periods of time, enabling the study of their dynamics in animal models of diseases. To aid these studies, we developed an automatic method, based on supervised learning, for quantifying the degree of mitochondrial fragmentation in tissue images acquired via two-photon microscopy from transgenic mice, which exclusively express Enhanced cyan fluorescent protein (ECFP) under Thy1 promoter, targeted to the mitochondrial matrix in subpopulations of neurons. We tested the method on images prior to and after cardiac arrest, and found it to be sensitive to significant changes in mitochondrial morphology because of the arrest. We conclude that the method is useful in detecting morphological abnormalities in mitochondria and, likely, in other subcellular structures as well. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  3. Deep two-photon microscopic imaging through brain tissue using the second singlet state from fluorescent agent chlorophyll α in spinach leaf.

    Science.gov (United States)

    Shi, Lingyan; Rodríguez-Contreras, Adrián; Budansky, Yury; Pu, Yang; Nguyen, Thien An; Alfano, Robert R

    2014-06-01

    Two-photon (2P) excitation of the second singlet (S₂) state was studied to achieve deep optical microscopic imaging in brain tissue when both the excitation (800 nm) and emission (685 nm) wavelengths lie in the "tissue optical window" (650 to 950 nm). S₂ state technique was used to investigate chlorophyll α (Chl α) fluorescence inside a spinach leaf under a thick layer of freshly sliced rat brain tissue in combination with 2P microscopic imaging. Strong emission at the peak wavelength of 685 nm under the 2P S₂ state of Chl α enabled the imaging depth up to 450 μm through rat brain tissue.

  4. Apertureless near-field/far-field CW two-photon microscope for biological and material imaging and spectroscopic applications.

    Science.gov (United States)

    Nowak, Derek B; Lawrence, A J; Sánchez, Erik J

    2010-12-10

    We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.

  5. Two-Photon Fluorescence Microscope for Microgravity Research

    Science.gov (United States)

    Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

    2005-01-01

    A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the

  6. Development of a two photon microscope for tracking Drosophila larvae

    Science.gov (United States)

    Karagyozov, Doycho; Mihovilovic Skanata, Mirna; Gershow, Marc

    Current in vivo methods for measuring neural activity in Drosophila larva require immobilization of the animal. Although we can record neural signals while stimulating the sensory organs, we cannot read the behavioral output because we have prevented the animal from moving. Many research questions cannot be answered without observation of neural activity in behaving (freely-moving) animals. We incorporated a Tunable Acoustic Gradient (TAG) lens into a two-photon microscope to achieve a 70kHz axial scan rate, enabling volumetric imaging at tens of hertz. We then implemented a tracking algorithm based on a Kalman filter to maintain the neurons of interest in the field of view and in focus during the rapid three dimensional motion of a free larva. Preliminary results show successful tracking of a neuron moving at speeds reaching 500 μm/s. NIH Grant 1DP2EB022359 and NSF Grant PHY-1455015.

  7. Microsphere imaging with confocal microscopy and two photon microscopy

    International Nuclear Information System (INIS)

    Chun, Hyung Su; An, Kyung Won; Lee, Jai Hyung

    2002-01-01

    We have acquired images of polystyrene and fused-silica microsphere by using conventional optical microscopy, confocal microscopy and two-photon microscopy, and performed comparative analysis of these images. Different from conventional optical microscopy, confocal and two-photon microscopy had good optical sectioning capability. In addition, confocal microscopy and two-photon microscopy had better lateral resolution than conventional optical microscopy. These results are attributed to confocality and nonlinearity of confocal microscopy and two photon microscopy, respectively.

  8. An integrated single- and two-photon non-diffracting light-sheet microscope

    Science.gov (United States)

    Lau, Sze Cheung; Chiu, Hoi Chun; Zhao, Luwei; Zhao, Teng; Loy, M. M. T.; Du, Shengwang

    2018-04-01

    We describe a fluorescence optical microscope with both single-photon and two-photon non-diffracting light-sheet excitations for large volume imaging. With a special design to accommodate two different wavelength ranges (visible: 400-700 nm and near infrared: 800-1200 nm), we combine the line-Bessel sheet (LBS, for single-photon excitation) and the scanning Bessel beam (SBB, for two-photon excitation) light sheet together in a single microscope setup. For a transparent thin sample where the scattering can be ignored, the LBS single-photon excitation is the optimal imaging solution. When the light scattering becomes significant for a deep-cell or deep-tissue imaging, we use SBB light-sheet two-photon excitation with a longer wavelength. We achieved nearly identical lateral/axial resolution of about 350/270 nm for both imagings. This integrated light-sheet microscope may have a wide application for live-cell and live-tissue three-dimensional high-speed imaging.

  9. Two-photon imaging and analysis of neural network dynamics

    International Nuclear Information System (INIS)

    Luetcke, Henry; Helmchen, Fritjof

    2011-01-01

    The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

  10. Two-photon imaging and analysis of neural network dynamics

    Science.gov (United States)

    Lütcke, Henry; Helmchen, Fritjof

    2011-08-01

    The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

  11. Two-photon imaging and analysis of neural network dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Luetcke, Henry; Helmchen, Fritjof [Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland)

    2011-08-15

    The glow of a starry night sky, the smell of a freshly brewed cup of coffee or the sound of ocean waves breaking on the beach are representations of the physical world that have been created by the dynamic interactions of thousands of neurons in our brains. How the brain mediates perceptions, creates thoughts, stores memories and initiates actions remains one of the most profound puzzles in biology, if not all of science. A key to a mechanistic understanding of how the nervous system works is the ability to measure and analyze the dynamics of neuronal networks in the living organism in the context of sensory stimulation and behavior. Dynamic brain properties have been fairly well characterized on the microscopic level of individual neurons and on the macroscopic level of whole brain areas largely with the help of various electrophysiological techniques. However, our understanding of the mesoscopic level comprising local populations of hundreds to thousands of neurons (so-called 'microcircuits') remains comparably poor. Predominantly, this has been due to the technical difficulties involved in recording from large networks of neurons with single-cell spatial resolution and near-millisecond temporal resolution in the brain of living animals. In recent years, two-photon microscopy has emerged as a technique which meets many of these requirements and thus has become the method of choice for the interrogation of local neural circuits. Here, we review the state-of-research in the field of two-photon imaging of neuronal populations, covering the topics of microscope technology, suitable fluorescent indicator dyes, staining techniques, and in particular analysis techniques for extracting relevant information from the fluorescence data. We expect that functional analysis of neural networks using two-photon imaging will help to decipher fundamental operational principles of neural microcircuits.

  12. Imaging Live Drosophila Brain with Two-Photon Fluorescence Microscopy

    Science.gov (United States)

    Ahmed, Syeed Ehsan

    Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance change in nanometer scale. In this study we have investigated the effect of dopamine in cAMP dynamics in vivo. In our study two-photon fluorescence microscope was used for imaging mushroom bodies inside live Drosophila melanogaster brain and we developed a method for studying the change in cyclic AMP level.

  13. Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

    NARCIS (Netherlands)

    Uzunbajakava, N.; Otto, Cornelis

    2003-01-01

    We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous

  14. Wide-band acousto-optic deflectors for large field of view two-photon microscope.

    Science.gov (United States)

    Jiang, Runhua; Zhou, Zhenqiao; Lv, Xiaohua; Zeng, Shaoqun

    2012-04-01

    Acousto-optic deflector (AOD) is an attractive scanner for two-photon microscopy because it can provide fast and versatile laser scanning and does not involve any mechanical movements. However, due to the small scan range of available AOD, the field of view (FOV) of the AOD-based microscope is typically smaller than that of the conventional galvanometer-based microscope. Here, we developed a novel wide-band AOD to enlarge the scan angle. Considering the maximum acceptable acoustic attenuation in the acousto-optic crystal, relatively lower operating frequencies and moderate aperture were adopted. The custom AOD was able to provide 60 MHz 3-dB bandwidth and 80% peak diffraction efficiency at 840 nm wavelength. Based on a pair of such AOD, a large FOV two-photon microscope was built with a FOV up to 418.5 μm (40× objective). The spatiotemporal dispersion was compensated simultaneously with a single custom-made prism. By means of dynamic power modulation, the variation of laser intensity within the FOV was reduced below 5%. The lateral and axial resolution of the system were 0.58-2.12 μm and 2.17-3.07 μm, respectively. Pollen grain images acquired by this system were presented to demonstrate the imaging capability at different positions across the entire FOV. © 2012 American Institute of Physics

  15. Design and performance of an ultra-flexible two-photon microscope for in vivo research

    Science.gov (United States)

    Mayrhofer, Johannes M.; Haiss, Florent; Haenni, Dominik; Weber, Stefan; Zuend, Marc; Barrett, Matthew J. P.; Ferrari, Kim David; Maechler, Philipp; Saab, Aiman S.; Stobart, Jillian L.; Wyss, Matthias T.; Johannssen, Helge; Osswald, Harald; Palmer, Lucy M.; Revol, Vincent; Schuh, Claus-Dieter; Urban, Claus; Hall, Andrew; Larkum, Matthew E.; Rutz-Innerhofer, Edith; Zeilhofer, Hanns Ulrich; Ziegler, Urs; Weber, Bruno

    2015-01-01

    We present a cost-effective in vivo two-photon microscope with a highly flexible frontend for in vivo research. Our design ensures fast and reproducible access to the area of interest, including rotation of imaging plane, and maximizes space for auxiliary experimental equipment in the vicinity of the animal. Mechanical flexibility is achieved with large motorized linear stages that move the objective in the X, Y, and Z directions up to 130 mm. 360° rotation of the frontend (rotational freedom for one axis) is achieved with the combination of a motorized high precision bearing and gearing. Additionally, the modular design of the frontend, based on commercially available optomechanical parts, allows straightforward updates to future scanning technologies. The design exceeds the mobility of previous movable microscope designs while maintaining high optical performance. PMID:26600989

  16. Designing a large field-of-view two-photon microscope using optical invariant analysis.

    Science.gov (United States)

    Bumstead, Jonathan R; Park, Jasmine J; Rosen, Isaac A; Kraft, Andrew W; Wright, Patrick W; Reisman, Matthew D; Côté, Daniel C; Culver, Joseph P

    2018-04-01

    Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter [Formula: see text]. Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image [Formula: see text] lateral and [Formula: see text] axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.

  17. Two-photon calcium imaging in mice navigating a virtual reality environment.

    Science.gov (United States)

    Leinweber, Marcus; Zmarz, Pawel; Buchmann, Peter; Argast, Paul; Hübener, Mark; Bonhoeffer, Tobias; Keller, Georg B

    2014-02-20

    In recent years, two-photon imaging has become an invaluable tool in neuroscience, as it allows for chronic measurement of the activity of genetically identified cells during behavior(1-6). Here we describe methods to perform two-photon imaging in mouse cortex while the animal navigates a virtual reality environment. We focus on the aspects of the experimental procedures that are key to imaging in a behaving animal in a brightly lit virtual environment. The key problems that arise in this experimental setup that we here address are: minimizing brain motion related artifacts, minimizing light leak from the virtual reality projection system, and minimizing laser induced tissue damage. We also provide sample software to control the virtual reality environment and to do pupil tracking. With these procedures and resources it should be possible to convert a conventional two-photon microscope for use in behaving mice.

  18. Development and design of advanced two-photon microscope used in neuroscience

    International Nuclear Information System (INIS)

    Doronin, M S; Popov, A V

    2016-01-01

    This work represents the real steps to development and design advanced two-photon microscope by efforts of laboratory staff. Self-developed microscopy system provides possibility to service it and modify the structure of microscope depending on highly specialized experimental design and scientific goals. We are presenting here module-based microscopy system which provides an opportunity to looking for new applications of this setup depending on laboratories needs using with galvo and resonant scanners. (paper)

  19. Scanless functional imaging of hippocampal networks using patterned two-photon illumination through GRIN lenses

    KAUST Repository

    Moretti, Claudio

    2016-09-12

    Patterned illumination through the phase modulation of light is increasingly recognized as a powerful tool to investigate biological tissues in combination with two-photon excitation and light-sensitive molecules. However, to date two-photon patterned illumination has only been coupled to traditional microscope objectives, thus limiting the applicability of these methods to superficial biological structures. Here, we show that phase modulation can be used to efficiently project complex two-photon light patterns, including arrays of points and large shapes, in the focal plane of graded index (GRIN) lenses. Moreover, using this approach in combination with the genetically encoded calcium indicator GCaMP6, we validate our system performing scanless functional imaging in rodent hippocampal networks in vivo ~1.2 mm below the brain surface. Our results open the way to the application of patterned illumination approaches to deep regions of highly scattering biological tissues, such as the mammalian brain.

  20. High contrast two-photon imaging of fingermarks

    Science.gov (United States)

    Stoltzfus, Caleb R.; Rebane, Aleksander

    2016-04-01

    Optically-acquired fingermarks are widely used as evidence across law enforcement agencies as well as in the courts of law. A common technique for visualizing latent fingermarks on nonporous surfaces consists of cyanoacrylate fuming of the fingerprint material, followed by impregnation with a fluorescent dye, which under ultra violet (UV) illumination makes the fingermarks visible and thus accessible for digital recording. However, there exist critical circumstances, when the image quality is compromised due to high background scattering, high auto-fluorescence of the substrate material, or other detrimental photo-physical and photo-chemical effects such as light-induced damage to the sample. Here we present a novel near-infrared (NIR), two-photon induced fluorescence imaging modality, which significantly enhances the quality of the fingermark images, especially when obtained from highly reflective and/or scattering surfaces, while at the same time reducing photo-damage to sensitive forensic samples.

  1. Whole brain imaging with Serial Two-Photon Tomography

    Directory of Open Access Journals (Sweden)

    Stephen P Amato

    2016-03-01

    Full Text Available Imaging entire mouse brains at submicron resolution has historically been a challenging undertaking and largely confined to the province of dedicated atlasing initiatives. The has limited systematic investigations into important areas of neuroscience, such as neural circuits, brain mapping and neurodegeneration. In this paper, we describe in detail Serial Two-Photon (STP tomography, a robust, reliable method for imaging entire brains with histological detail. We provide examples of how the basic methodology can be extended to other imaging modalities, such as optical coherence tomography, in order to provide unique contrast mechanisms. Furthermore we provide a survey of the research that STP tomography has enabled in the field of neuroscience, provide examples of how this technology enables quantitative whole brain studies, and discuss the current limitations of STP tomography-based approaches

  2. Fluorescent Pluronic nanodots for in vivo two-photon imaging

    International Nuclear Information System (INIS)

    Maurin, Mathieu; Vurth, Laeticia; Vial, Jean-Claude; Baldeck, Patrice; Stephan, Olivier; Marder, Seth R; Sanden, Boudewijn Van der

    2009-01-01

    We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 μm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.

  3. Time gated fluorescence lifetime imaging and micro-volume spectroscopy using two-photon excitation

    NARCIS (Netherlands)

    Sytsma, J.; Vroom, J.M.; de Grauw, C.J.; Gerritsen, H.C.

    A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes.

  4. An ultrafast electron microscope gun driven by two-photon photoemission from a nanotip cathode

    International Nuclear Information System (INIS)

    Bormann, Reiner; Strauch, Stefanie; Schäfer, Sascha; Ropers, Claus

    2015-01-01

    We experimentally and numerically investigate the performance of an advanced ultrafast electron source, based on two-photon photoemission from a tungsten needle cathode incorporated in an electron microscope gun geometry. Emission properties are characterized as a function of the electrostatic gun settings, and operating conditions leading to laser-triggered electron beams of very low emittance (below 20 nm mrad) are identified. The results highlight the excellent suitability of optically driven nano-cathodes for the further development of ultrafast transmission electron microscopy

  5. Two-Photon Ghost Image and Interference-Diffraction

    Science.gov (United States)

    Shih, Y. H.; Sergienko, A. V.; Pittman, T. B.; Strekalov, D. V.; Klyshko, D. N.

    1996-01-01

    One of the most surprising consequences of quantum mechanics is entanglement of two or more distance particles. The two-particle entangled state was mathematically formulated by Schrodinger. Based on this unusual quantum behavior, EPR defined their 'physical reality' and then asked the question: 'Can Quantum-Mechanical Description of Physical Reality Be Considered Complete?' One may not appreciate EPR's criterion of physical reality and insist that 'no elementary quantum phenomenon is a phenomenon until it is a recorded phenomenon'. Optical spontaneous parametric down conversion (SPDC) is the most effective mechanism to generate an EPR type entangled two-photon state. In SPDC, an optical beam, called the pump, is incident on a birefringent crystal. The pump is intense enough so that nonlinear effects lead to the conversion of pump photons into pairs of photons, historically called signal and idler. Technically, the SPDC is said to be type-1 or type-2, depending on whether the signal and idler beams have parallel or orthogonal polarization. The SPDC conversion efficiency is typically on the order of 10(exp -9) to 10(exp -11), depending on the SPDC nonlinear material. The signal and idler intensities are extremely low, only single photon detection devices can register them. The quantum entanglement nature of SPDC has been demonstrated in EPR-Bohm experiments and Bell's inequality measurements. The following two experiments were recently performed in our laboratory, which are more closely related to the original 1935 EPR gedankenezperiment. The first experiment is a two-photon optical imaging type experiment, which has been named 'ghost image' by the physics community. The signal and idler beams of SPDC are sent in different directions, so that the detection of the signal and idler photons can be performed by two distant photon counting detectors. An aperture object (mask) is placed in front of the signal photon detector and illuminated by the signal beam through a

  6. Hybrid of two-photon microscopy and optical multimodality imaging for multi-scale imaging of small animals

    Science.gov (United States)

    Li, Tianmeng; Hui, Hui; Ma, He; Yang, Xin; Tian, Jie

    2018-02-01

    Non-invasive imaging technologies, such as magnetic resonance imaging (MRI) and optical multimodality imaging methods, are commonly used for diagnosing and supervising the development of inflammatory bowel disease (IBD). These in vivo imaging methods can provide morphology changes information of IBD in macro-scale. However, it is difficult to investigate the intestinal wall in molecular and cellular level. State-of-art light-sheet and two-photon microscopy have the ability to acquire the changes for IBD in micro-scale. The aim of this work is to evaluate the size of the enterocoel and the thickness of colon wall using both MRI for in vivo imaging, and light-sheet and two-photon microscope for in vitro imaging. C57BL/6 mice were received 3.5% Dextran sodium sulfate (DSS) in the drinking water for 5 days to build IBD model. Mice were imaged with MRI on days 0, 6 to observe colitis progression. After MRI imaging, the mice were sacrificed to take colons for tissue clearing. Then, light-sheet and two-photon microscopies are used for in vitro imaging of the cleared samples. The experimental group showed symptoms of bloody stools, sluggishness and weight loss. It showed that the colon wall was thicker while the enterocoel was narrower compare to control group. The more details are observed using light-sheet and two-photon microscope. It is demonstrated that hybrid of MRI in macro-scale and light-sheet and two-photon microscopy in micro-scale imaging is feasible for colon inflammation diagnosing and supervising.

  7. Multisite two-photon imaging of neurons on multielectrode arrays

    Science.gov (United States)

    Potter, Steve M.; Lukina, Natalia; Longmuir, Kenneth J.; Wu, Yan

    2001-04-01

    We wish to understand how neural systems store, recall, and process information. We are using cultured networks of cortical neurons grown on microelectrode arrays as a model system for studying the emergent properties of ensembles of living neurons. We have developed a 2-way communication interface between the cultured network and a computer- generated animal, the Neurally Controlled Animat. Neural activity is used to control the behavior of the Animat, and 2- photon time-lapse imaging is carried out in order to observe the morphological changes that might underlie changes in neural processing. The 2-photon microscope is ideal for repeated imaging over hours or days, with submicron resolution and little photodamage. We have designed a computer-controlled microscope stage that allows imaging several locations in sequence, in order to collect more image data. For the latest progress, see: http://www.caltech.edu/~pinelab/PotterGroup.htm.

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

    Directory of Open Access Journals (Sweden)

    David G Rosenegger

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Doronin, Maxim; Popov, Alexander

    2017-02-01

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

  11. Simultaneous morphological and functional imaging of the honeybee's brain by two-photon microscopy

    International Nuclear Information System (INIS)

    Haase, A.

    2011-01-01

    Thanks to its rather simply structured but highly performing brain, the honeybee (Apis mellifera) is an important model for neurobiological studies. Therefore there is a great need for new functional imaging modalities adapted to this species. Herein we give a detailed report on the development and performance of a platform for in vivo functional and morphological imaging of the honeybee's brain, focusing on its primary olfactory centres, the antennal lobes (ALs). The experimental setup consists of a two-photon microscope combined with a synchronized odour stimulus generator. Our imaging platform allows to simultaneously obtain both morphological measurements of the ALs functional units, the glomeruli, and in vivo calcium recording of their neural activity. We were able to record the characteristic glomerular response maps to odour stimuli applied to the bee's antennae. Our approach offers several advantages over the commonly used conventional fluorescence microscopy. Two-photon microscopy provides substantial enhancement in both spatial and temporal resolutions, while minimizing photo damage. Calcium recordings show a more than fourfold improvement in the functional signal with respect to the techniques available up to now. Finally, the extended penetration depth, thanks to the infrared excitation, allows the functional imaging of profound glomeruli which have not been optically accessible up to now.

  12. Two-photon excited autofluorescence imaging of freshly isolated frog retinas.

    Science.gov (United States)

    Lu, Rong-Wen; Li, Yi-Chao; Ye, Tong; Strang, Christianne; Keyser, Kent; Curcio, Christine A; Yao, Xin-Cheng

    2011-06-01

    The purpose of this study was to investigate cellular sources of autofluorescence signals in freshly isolated frog (Rana pipiens) retinas. Equipped with an ultrafast laser, a laser scanning two-photon excitation fluorescence microscope was employed for sub-cellular resolution examination of both sliced and flat-mounted retinas. Two-photon imaging of retinal slices revealed autofluorescence signals over multiple functional layers, including the photoreceptor layer (PRL), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Using flat-mounted retinas, depth-resolved imaging of individual retinal layers further confirmed multiple sources of autofluorescence signals. Cellular structures were clearly observed at the PRL, ONL, INL, and GCL. At the PRL, the autofluorescence was dominantly recorded from the intracellular compartment of the photoreceptors; while mixed intracellular and extracellular autofluorescence signals were observed at the ONL, INL, and GCL. High resolution autofluorescence imaging clearly revealed mosaic organization of rod and cone photoreceptors; and sub-cellular bright autofluorescence spots, which might relate to connecting cilium, was observed in the cone photoreceptors only. Moreover, single-cone and double-cone outer segments could be directly differentiated.

  13. A compact two photon light sheet microscope for applications in neuroscience

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2016-01-01

    We present a compact setup for two photon light sheet microscopy. By using pulsed Airy beam illumination we demonstrate eight-fold increase of the FOV compared to Gaussian light sheet with the same axial resolution....

  14. Measurement of drug-target engagement in live cells by two-photon fluorescence anisotropy imaging.

    Science.gov (United States)

    Vinegoni, Claudio; Fumene Feruglio, Paolo; Brand, Christian; Lee, Sungon; Nibbs, Antoinette E; Stapleton, Shawn; Shah, Sunil; Gryczynski, Ignacy; Reiner, Thomas; Mazitschek, Ralph; Weissleder, Ralph

    2017-07-01

    The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules. Our technique allows the direct characterization of target engagement of fluorescently labeled drugs, using fluorophores with a fluorescence lifetime larger than the rotational correlation of the bound complex. Here we describe an optimized protocol for simultaneous dual-channel two-photon fluorescence anisotropy microscopy acquisition to perform drug-target measurements. We also provide the necessary software to implement stream processing to visualize images and to calculate quantitative parameters. The assembly and characterization part of the protocol can be implemented in 1 d. Sample preparation, characterization and imaging of drug binding can be completed in 2 d. Although currently adapted to an Olympus FV1000MPE microscope, the protocol can be extended to other commercial or custom-built microscopes.

  15. In vivo two-photon imaging of retina in rabbits and rats.

    Science.gov (United States)

    Jayabalan, Gopal Swamy; Wu, Yi-Kai; Bille, Josef F; Kim, Samuel; Mao, Xiao Wen; Gimbel, Howard V; Rauser, Michael E; Fan, Joseph T

    2018-01-01

    The purpose of this study was to evaluate the retina using near-infrared (NIR) two-photon scanning laser ophthalmoscopy. New Zealand white rabbits, albino rats, and brown Norway rats were used in this study. An autofluorescence image of the retina, including the retinal cells and its associated vasculatures was obtained by a real-time scan using the ophthalmoscope. Furthermore, the retinal vessels, nerve fiber layers and the non-pigmented retina were recorded with two-photon fluorescein angiography (FA); and the choroidal vasculatures were recorded using two-photon indocyanine green angiography (ICGA). Two-photon ICGA was achieved by exciting a second singlet state at ∼398 nm. Simultaneous two-photon FA and two-photon ICGA were performed to characterize the retinal and choroidal vessels with a single injection. The minimum laser power threshold required to elicit two-photon fluorescence was determined. The two-photon ophthalmoscope could serve as a promising tool to detect and monitor the disease progression in animal models. Moreover, these high-resolution images of retinal and choroidal vessels can be acquired in a real-time scan with a single light source, requiring no additional filters for FA or ICGA. The combination of FA and ICGA using the two-photon ophthalmoscope will help researchers to characterize the retinal diseases in animal models, and also to classify the types (classic, occult or mixed) of choroidal neovascularization (CNV) in macular degeneration. Furthermore, the prototype can be adapted to image the retina of rodents and rabbits. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy

    Science.gov (United States)

    Hunter, Jennifer J.; Masella, Benjamin; Dubra, Alfredo; Sharma, Robin; Yin, Lu; Merigan, William H.; Palczewska, Grazyna; Palczewski, Krzysztof; Williams, David R.

    2011-01-01

    In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors. PMID:21326644

  17. Two-Photon Autofluorescence Imaging Reveals Cellular Structures Throughout the Retina of the Living Primate Eye.

    Science.gov (United States)

    Sharma, Robin; Williams, David R; Palczewska, Grazyna; Palczewski, Krzysztof; Hunter, Jennifer J

    2016-02-01

    Although extrinsic fluorophores can be introduced to label specific cell types in the retina, endogenous fluorophores, such as NAD(P)H, FAD, collagen, and others, are present in all retinal layers. These molecules are a potential source of optical contrast and can enable noninvasive visualization of all cellular layers. We used a two-photon fluorescence adaptive optics scanning light ophthalmoscope (TPF-AOSLO) to explore the native autofluorescence of various cell classes spanning several layers in the unlabeled retina of a living primate eye. Three macaques were imaged on separate occasions using a custom TPF-AOSLO. Two-photon fluorescence was evoked by pulsed light at 730 and 920 nm excitation wavelengths, while fluorescence emission was collected in the visible range from several retinal layers and different locations. Backscattered light was recorded simultaneously in confocal modality and images were postprocessed to remove eye motion. All retinal layers yielded two-photon signals and the heterogeneous distribution of fluorophores provided optical contrast. Several structural features were observed, such as autofluorescence from vessel walls, Müller cell processes in the nerve fibers, mosaics of cells in the ganglion cell and other nuclear layers of the inner retina, as well as photoreceptor and RPE layers in the outer retina. This in vivo survey of two-photon autofluorescence throughout the primate retina demonstrates a wider variety of structural detail in the living eye than is available through conventional imaging methods, and broadens the use of two-photon imaging of normal and diseased eyes.

  18. High-resolution, label-free two-photon imaging of diseased human corneas

    Science.gov (United States)

    Batista, Ana; Breunig, Hans Georg; König, Aisada; Schindele, Andreas; Hager, Tobias; Seitz, Berthold; König, Karsten

    2018-03-01

    The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells' metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells' morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells' metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.

  19. Applications of two-photon fluorescence microscopy in deep-tissue imaging

    Science.gov (United States)

    Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

    2000-07-01

    Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

  20. Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Breunig, Hans Georg; König, Karsten; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Christopher

    2011-07-01

    Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential.

  1. Near-IR Two-Photon Fluorescent Sensor for K(+) Imaging in Live Cells.

    Science.gov (United States)

    Sui, Binglin; Yue, Xiling; Kim, Bosung; Belfield, Kevin D

    2015-08-19

    A new two-photon excited fluorescent K(+) sensor is reported. The sensor comprises three moieties, a highly selective K(+) chelator as the K(+) recognition unit, a boron-dipyrromethene (BODIPY) derivative modified with phenylethynyl groups as the fluorophore, and two polyethylene glycol chains to afford water solubility. The sensor displays very high selectivity (>52-fold) in detecting K(+) over other physiological metal cations. Upon binding K(+), the sensor switches from nonfluorescent to highly fluorescent, emitting red to near-IR (NIR) fluorescence. The sensor exhibited a good two-photon absorption cross section, 500 GM at 940 nm. Moreover, it is not sensitive to pH in the physiological pH range. Time-dependent cell imaging studies via both one- and two-photon fluorescence microscopy demonstrate that the sensor is suitable for dynamic K(+) sensing in living cells.

  2. Gold Core Mesoporous Organosilica Shell Degradable Nanoparticles for Two-Photon Imaging and Gemcitabine Monophosphate Delivery

    KAUST Repository

    Rhamani, Saher; Chaix, Arnaud; Aggad, Dina; Hoang, Phuong Mai; Moosa, Basem; Garcia, Marcel; Gary-Bobo, Magali; Charnay, Clarence; Almalik, Abdulaziz; Durand, Jean-Olivier; Khashab, Niveen M.

    2017-01-01

    The synthesis of gold core degradable mesoporous organosilica shell nanoparticles is described. The nanopaticles were very efficient for two-photon luminescence imaging of cancer cells and for in vitro gemcitabine monophosphate delivery, allowing promising theranostic applications in the nanomedicine field.

  3. Gold Core Mesoporous Organosilica Shell Degradable Nanoparticles for Two-Photon Imaging and Gemcitabine Monophosphate Delivery

    KAUST Repository

    Rhamani, Saher

    2017-09-12

    The synthesis of gold core degradable mesoporous organosilica shell nanoparticles is described. The nanopaticles were very efficient for two-photon luminescence imaging of cancer cells and for in vitro gemcitabine monophosphate delivery, allowing promising theranostic applications in the nanomedicine field.

  4. Volumetric Two-photon Imaging of Neurons Using Stereoscopy (vTwINS)

    Science.gov (United States)

    Song, Alexander; Charles, Adam S.; Koay, Sue Ann; Gauthier, Jeff L.; Thiberge, Stephan Y.; Pillow, Jonathan W.; Tank, David W.

    2017-01-01

    Two-photon laser scanning microscopy of calcium dynamics using fluorescent indicators is a widely used imaging method for large scale recording of neural activity in vivo. Here we introduce volumetric Two-photon Imaging of Neurons using Stereoscopy (vTwINS), a volumetric calcium imaging method that employs an elongated, V-shaped point spread function to image a 3D brain volume. Single neurons project to spatially displaced “image pairs” in the resulting 2D image, and the separation distance between images is proportional to depth in the volume. To demix the fluorescence time series of individual neurons, we introduce a novel orthogonal matching pursuit algorithm that also infers source locations within the 3D volume. We illustrate vTwINS by imaging neural population activity in mouse primary visual cortex and hippocampus. Our results demonstrate that vTwINS provides an effective method for volumetric two-photon calcium imaging that increases the number of neurons recorded while maintaining a high frame-rate. PMID:28319111

  5. Multiscale vision model for event detection and reconstruction in two-photon imaging data

    DEFF Research Database (Denmark)

    Brazhe, Alexey; Mathiesen, Claus; Lind, Barbara Lykke

    2014-01-01

    on a modified multiscale vision model, an object detection framework based on the thresholding of wavelet coefficients and hierarchical trees of significant coefficients followed by nonlinear iterative partial object reconstruction, for the analysis of two-photon calcium imaging data. The framework is discussed...... of the multiscale vision model is similar in the denoising, but provides a better segmenation of the image into meaningful objects, whereas other methods need to be combined with dedicated thresholding and segmentation utilities....

  6. Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

    Science.gov (United States)

    Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

    2006-01-01

    Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

  7. Two-photon-induced hot-electron transfer to a single molecule in a scanning tunneling microscope

    International Nuclear Information System (INIS)

    Wu, S. W.; Ho, W.

    2010-01-01

    The junction of a scanning tunneling microscope (STM) operating in the tunneling regime was irradiated with femtosecond laser pulses. A photoexcited hot electron in the STM tip resonantly tunnels into an excited state of a single molecule on the surface, converting it from the neutral to the anion. The electron-transfer rate depends quadratically on the incident laser power, suggesting a two-photon excitation process. This nonlinear optical process is further confirmed by the polarization measurement. Spatial dependence of the electron-transfer rate exhibits atomic-scale variations. A two-pulse correlation experiment reveals the ultrafast dynamic nature of photoinduced charging process in the STM junction. Results from these experiments are important for understanding photoinduced interfacial charge transfer in many nanoscale inorganic-organic structures.

  8. Diagnosis of basal cell carcinoma by two photon excited fluorescence combined with lifetime imaging

    Science.gov (United States)

    Fan, Shunping; Peng, Xiao; Liu, Lixin; Liu, Shaoxiong; Lu, Yuan; Qu, Junle

    2014-02-01

    Basal cell carcinoma (BCC) is the most common type of human skin cancer. The traditional diagnostic procedure of BCC is histological examination with haematoxylin and eosin staining of the tissue biopsy. In order to reduce complexity of the diagnosis procedure, a number of noninvasive optical methods have been applied in skin examination, for example, multiphoton tomography (MPT) and fluorescence lifetime imaging microscopy (FLIM). In this study, we explored two-photon optical tomography of human skin specimens using two-photon excited autofluorescence imaging and FLIM. There are a number of naturally endogenous fluorophores in skin sample, such as keratin, melanin, collagen, elastin, flavin and porphyrin. Confocal microscopy was used to obtain structures of the sample. Properties of epidermic and cancer cells were characterized by fluorescence emission spectra, as well as fluorescence lifetime imaging. Our results show that two-photon autofluorescence lifetime imaging can provide accurate optical biopsies with subcellular resolution and is potentially a quantitative optical diagnostic method in skin cancer diagnosis.

  9. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

    Science.gov (United States)

    Chamberland, Simon; Yang, Helen H; Pan, Michael M; Evans, Stephen W; Guan, Sihui; Chavarha, Mariya; Yang, Ying; Salesse, Charleen; Wu, Haodi; Wu, Joseph C; Clandinin, Thomas R; Toth, Katalin; Lin, Michael Z; St-Pierre, François

    2017-07-27

    Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila . These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

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

    Science.gov (United States)

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

    2014-01-01

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

  11. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions

    OpenAIRE

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2010-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p ...

  12. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

    Science.gov (United States)

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  13. Additive controlled synthesis of gold nanorods (GNRs) for two-photon luminescence imaging of cancer cells

    International Nuclear Information System (INIS)

    Zhu Jing; Roy, Indrajit; Hu Rui; Ding Hong; Zhao Lingling; He, Guang S; Prasad, Paras N; Yong, Ken-Tye; Swihart, Mark T; Cui Yiping

    2010-01-01

    Gold nanorods (GNRs) with a longitudinal surface plasmon resonance peak that is tunable from 600 to 1100 nm have been fabricated in a cetyl trimethylammoniumbromide (CTAB) micellar medium using hydrochloric acid and silver nitrate as additives to control their shape and size. By manipulating the concentrations of silver nitrate and hydrochloric acid, the aspect ratio of the GNRs was reliably and reproducibly tuned from 2.5 to 8. The GNRs were first coated with polyelectrolyte multilayers and then bioconjugated to transferrin (Tf) to target pancreatic cancer cells. Two-photon imaging excited from the bioconjugated GNRs demonstrated receptor-mediated uptake of the bioconjugates into Panc-1 cells, overexpressing the transferrin receptor (TfR). The bioconjugated GNR formulation exhibited very low toxicity, suggesting that it is biocompatible and potentially suitable for targeted two-photon bioimaging.

  14. Additive controlled synthesis of gold nanorods (GNRs) for two-photon luminescence imaging of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu Jing; Roy, Indrajit; Hu Rui; Ding Hong; Zhao Lingling; He, Guang S; Prasad, Paras N [Institute for Lasers, Photonics and Biophotonics, University at Buffalo, State University of New York, Buffalo, NY 14260-4200 (United States); Yong, Ken-Tye [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Swihart, Mark T [Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, NY 14260-4200 (United States); Cui Yiping, E-mail: ktyong@ntu.edu.sg, E-mail: cyp@seu.edu.cn, E-mail: pnprasad@buffalo.edu [Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096 (China)

    2010-07-16

    Gold nanorods (GNRs) with a longitudinal surface plasmon resonance peak that is tunable from 600 to 1100 nm have been fabricated in a cetyl trimethylammoniumbromide (CTAB) micellar medium using hydrochloric acid and silver nitrate as additives to control their shape and size. By manipulating the concentrations of silver nitrate and hydrochloric acid, the aspect ratio of the GNRs was reliably and reproducibly tuned from 2.5 to 8. The GNRs were first coated with polyelectrolyte multilayers and then bioconjugated to transferrin (Tf) to target pancreatic cancer cells. Two-photon imaging excited from the bioconjugated GNRs demonstrated receptor-mediated uptake of the bioconjugates into Panc-1 cells, overexpressing the transferrin receptor (TfR). The bioconjugated GNR formulation exhibited very low toxicity, suggesting that it is biocompatible and potentially suitable for targeted two-photon bioimaging.

  15. Additive controlled synthesis of gold nanorods (GNRs) for two-photon luminescence imaging of cancer cells

    Science.gov (United States)

    Zhu, Jing; Yong, Ken-Tye; Roy, Indrajit; Hu, Rui; Ding, Hong; Zhao, Lingling; Swihart, Mark T.; He, Guang S.; Cui, Yiping; Prasad, Paras N.

    2010-07-01

    Gold nanorods (GNRs) with a longitudinal surface plasmon resonance peak that is tunable from 600 to 1100 nm have been fabricated in a cetyl trimethylammoniumbromide (CTAB) micellar medium using hydrochloric acid and silver nitrate as additives to control their shape and size. By manipulating the concentrations of silver nitrate and hydrochloric acid, the aspect ratio of the GNRs was reliably and reproducibly tuned from 2.5 to 8. The GNRs were first coated with polyelectrolyte multilayers and then bioconjugated to transferrin (Tf) to target pancreatic cancer cells. Two-photon imaging excited from the bioconjugated GNRs demonstrated receptor-mediated uptake of the bioconjugates into Panc-1 cells, overexpressing the transferrin receptor (TfR). The bioconjugated GNR formulation exhibited very low toxicity, suggesting that it is biocompatible and potentially suitable for targeted two-photon bioimaging.

  16. Efficient multi-site two-photon functional imaging of neuronal circuits.

    Science.gov (United States)

    Castanares, Michael Lawrence; Gautam, Vini; Drury, Jack; Bachor, Hans; Daria, Vincent R

    2016-12-01

    Two-photon imaging using high-speed multi-channel detectors is a promising approach for optical recording of cellular membrane dynamics at multiple sites. A main bottleneck of this technique is the limited number of photons captured within a short exposure time (~1ms). Here, we implement temporal gating to improve the two-photon fluorescence yield from holographically projected multiple foci whilst maintaining a biologically safe incident average power. We observed up to 6x improvement in the signal-to-noise ratio (SNR) in Fluorescein and cultured hippocampal neurons showing evoked calcium transients. With improved SNR, we could pave the way to achieving multi-site optical recording of fluorogenic probes with response times in the order of ~1ms.

  17. Deep brain two-photon NIR fluorescence imaging for study of Alzheimer's disease

    Science.gov (United States)

    Chen, Congping; Liang, Zhuoyi; Zhou, Biao; Ip, Nancy Y.; Qu, Jianan Y.

    2018-02-01

    Amyloid depositions in the brain represent the characteristic hallmarks of Alzheimer's disease (AD) pathology. The abnormal accumulation of extracellular amyloid-beta (Aβ) and resulting toxic amyloid plaques are considered to be responsible for the clinical deficits including cognitive decline and memory loss. In vivo two-photon fluorescence imaging of amyloid plaques in live AD mouse model through a chronic imaging window (thinned skull or craniotomy) provides a mean to greatly facilitate the study of the pathological mechanism of AD owing to its high spatial resolution and long-term continuous monitoring. However, the imaging depth for amyloid plaques is largely limited to upper cortical layers due to the short-wavelength fluorescence emission of commonly used amyloid probes. In this work, we reported that CRANAD-3, a near-infrared (NIR) probe for amyloid species with excitation wavelength at 900 nm and emission wavelength around 650 nm, has great advantages over conventionally used probes and is well suited for twophoton deep imaging of amyloid plaques in AD mouse brain. Compared with a commonly used MeO-X04 probe, the imaging depth of CRANAD-3 is largely extended for open skull cranial window. Furthermore, by using two-photon excited fluorescence spectroscopic imaging, we characterized the intrinsic fluorescence of the "aging pigment" lipofuscin in vivo, which has distinct spectra from CRANAD-3 labeled plaques. This study reveals the unique potential of NIR probes for in vivo, high-resolution and deep imaging of brain amyloid in Alzheimer's disease.

  18. Two-photon fluorescence and fluorescence imaging of two styryl heterocyclic dyes combined with DNA.

    Science.gov (United States)

    Gao, Chao; Liu, Shu-yao; Zhang, Xian; Liu, Ying-kai; Qiao, Cong-de; Liu, Zhao-e

    2016-03-05

    Two new styryl heterocyclic two-photon (TP) materials, 4-[4-(N-methyl)styrene]-imidazo [4,5-f][1,10] phenanthroline-benzene iodated salt (probe-1) and 4,4-[4-(N-methyl)styrene]-benzene iodated salt (probe-2) were successfully synthesized and studied as potential fluorescent probes of DNA detection. The linear and nonlinear photophysical properties of two compounds in different solvents were investigated. The absorption, one- and two-photon fluorescent spectra of the free dye and dye-DNA complex were also examined to evaluate their photophysical properties. The binding constants of dye-DNA were obtained according to Scatchard equation with good values. The results showed that two probes could be used as fluorescent DNA probes by two-photon excitation, and TP fluorescent properties of probe-1 are superior to that of probe-2. The fluorescent method date indicated that the mechanisms of dye-DNA complex interaction may be groove binding for probe-1 and electrostatic interaction for probe-2, respectively. The MTT assay experiments showed two probes are low toxicity. Moreover, the TP fluorescence imaging of DNA detection in living cells at 800 nm indicated that the ability to locate in cell nuclei of probe-1 is better than that of probe-2. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  20. An automated detection for axonal boutons in vivo two-photon imaging of mouse

    Science.gov (United States)

    Li, Weifu; Zhang, Dandan; Xie, Qiwei; Chen, Xi; Han, Hua

    2017-02-01

    Activity-dependent changes in the synaptic connections of the brain are tightly related to learning and memory. Previous studies have shown that essentially all new synaptic contacts were made by adding new partners to existing synaptic elements. To further explore synaptic dynamics in specific pathways, concurrent imaging of pre and postsynaptic structures in identified connections is required. Consequently, considerable attention has been paid for the automated detection of axonal boutons. Different from most previous methods proposed in vitro data, this paper considers a more practical case in vivo neuron images which can provide real time information and direct observation of the dynamics of a disease process in mouse. Additionally, we present an automated approach for detecting axonal boutons by starting with deconvolving the original images, then thresholding the enhanced images, and reserving the regions fulfilling a series of criteria. Experimental result in vivo two-photon imaging of mouse demonstrates the effectiveness of our proposed method.

  1. Microscopic theory of cavity-enhanced single-photon emission from optical two-photon Raman processes

    Science.gov (United States)

    Breddermann, Dominik; Praschan, Tom; Heinze, Dirk; Binder, Rolf; Schumacher, Stefan

    2018-03-01

    We consider cavity-enhanced single-photon generation from stimulated two-photon Raman processes in three-level systems. We compare four fundamental system configurations, one Λ -, one V-, and two ladder (Ξ -) configurations. These can be realized as subsystems of a single quantum dot or of quantum-dot molecules. For a new microscopic understanding of the Raman process, we analyze the Heisenberg equation of motion applying the cluster-expansion scheme. Within this formalism an exact and rigorous definition of a cavity-enhanced Raman photon via its corresponding Raman correlation is possible. This definition for example enables us to systematically investigate the on-demand potential of Raman-transition-based single-photon sources. The four system arrangements can be divided into two subclasses, Λ -type and V-type, which exhibit strongly different Raman-emission characteristics and Raman-emission probabilities. Moreover, our approach reveals whether the Raman path generates a single photon or just induces destructive quantum interference with other excitation paths. Based on our findings and as a first application, we gain a more detailed understanding of experimental data from the literature. Our analysis and results are also transferable to the case of atomic three-level-resonator systems and can be extended to more complicated multilevel schemes.

  2. In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH.

    Science.gov (United States)

    Yaseen, Mohammad A; Sakadžić, Sava; Wu, Weicheng; Becker, Wolfgang; Kasischke, Karl A; Boas, David A

    2013-02-01

    Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

  3. Simple fibre based dispersion management for two-photon excited fluorescence imaging through an endoscope

    DEFF Research Database (Denmark)

    Dimopoulos, Konstantinos; Marti, Dominik; Andersen, Peter E.

    2018-01-01

    We want to implement two-photon excitation fluorescence microscopy (TPEFM) into endoscopes, since TPEFM can provide relevant biomarkers for cancer staging and grading in hollow organs, endoscopically accessible through natural orifices. However, many obstacles must be overcome, among others...... the delivery of short laser pulses to the distal end of the endoscope. To this avail, we present imaging results using an all-fibre dispersion management scheme in a TPEFM setup. The scheme has been conceived by Jespersen et al. in 20101 and relies on the combination of a single mode fibre with normal...

  4. Imaging hydrogen flames by two-photon, laser-induced fluorescence

    Science.gov (United States)

    Miles, R.; Lempert, W.; Kumar, V.; Diskin, G.

    1991-01-01

    A nonintrusive multicomponent imaging system is developed which can image hydrogen, hot oxygen, and air simultaneously. An Ar-F excimer laser is injection-locked to cover the Q1 two-photon transition in molecular hydrogen which allows the observation of both hot oxygen and cold hydrogen. Rayleigh scattering from the water molecules occurs at the same frequency as the illuminating laser allowing analysis of the air density. Images of ignited and nonignited hydrogen jets are recorded with a high-sensitivity gated video camera. The images permit the analysis of turbulent hydrogen-core jet, the combustion zone, and the surrounding air, and two-dimensional spatial correlations can be made to study the turbulent structure and couplings between different regions of the flow field. The method is of interest to the study of practical combustion systems which employ hydrogen-air diffusion flames.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-07

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

  6. Thermally activated delayed fluorescence organic dots for two-photon fluorescence lifetime imaging

    Science.gov (United States)

    He, Tingchao; Ren, Can; Li, Zhuohua; Xiao, Shuyu; Li, Junzi; Lin, Xiaodong; Ye, Chuanxiang; Zhang, Junmin; Guo, Lihong; Hu, Wenbo; Chen, Rui

    2018-05-01

    Autofluorescence is a major challenge in complex tissue imaging when molecules present in the biological tissue compete with the fluorophore. This issue may be resolved by designing organic molecules with long fluorescence lifetimes. The present work reports the two-photon absorption (TPA) properties of a thermally activated delayed fluorescence (TADF) molecule with carbazole as the electron donor and dicyanobenzene as the electron acceptor (i.e., 4CzIPN). The results indicate that 4CzIPN exhibits a moderate TPA cross-section (˜9 × 10-50 cm4 s photon-1), high fluorescence quantum yield, and a long fluorescence lifetime (˜1.47 μs). 4CzIPN was compactly encapsulated into an amphiphilic copolymer via nanoprecipitation to achieve water-soluble organic dots. Interestingly, 4CzIPN organic dots have been utilized in applications involving two-photon fluorescence lifetime imaging (FLIM). Our work aptly demonstrates that TADF molecules are promising candidates of nonlinear optical probes for developing next-generation multiphoton FLIM applications.

  7. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions.

    Science.gov (United States)

    Kasischke, Karl A; Lambert, Elton M; Panepento, Ben; Sun, Anita; Gelbard, Harris A; Burgess, Robert W; Foster, Thomas H; Nedergaard, Maiken

    2011-01-01

    Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO(2) range with a p(50) of 3.4 ± 0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution.

  8. Single pulse two photon fluorescence lifetime imaging (SP-FLIM) with MHz pixel rate.

    Science.gov (United States)

    Eibl, Matthias; Karpf, Sebastian; Weng, Daniel; Hakert, Hubertus; Pfeiffer, Tom; Kolb, Jan Philip; Huber, Robert

    2017-07-01

    Two-photon-excited fluorescence lifetime imaging microscopy (FLIM) is a chemically specific 3-D sensing modality providing valuable information about the microstructure, composition and function of a sample. However, a more widespread application of this technique is hindered by the need for a sophisticated ultra-short pulse laser source and by speed limitations of current FLIM detection systems. To overcome these limitations, we combined a robust sub-nanosecond fiber laser as the excitation source with high analog bandwidth detection. Due to the long pulse length in our configuration, more fluorescence photons are generated per pulse, which allows us to derive the lifetime with a single excitation pulse only. In this paper, we show high quality FLIM images acquired at a pixel rate of 1 MHz. This approach is a promising candidate for an easy-to-use and benchtop FLIM system to make this technique available to a wider research community.

  9. Active learning of cortical connectivity from two-photon imaging data

    Science.gov (United States)

    Wang, Ye; Dunson, David; Sapiro, Guillermo; Ringach, Dario

    2018-01-01

    Understanding how groups of neurons interact within a network is a fundamental question in system neuroscience. Instead of passively observing the ongoing activity of a network, we can typically perturb its activity, either by external sensory stimulation or directly via techniques such as two-photon optogenetics. A natural question is how to use such perturbations to identify the connectivity of the network efficiently. Here we introduce a method to infer sparse connectivity graphs from in-vivo, two-photon imaging of population activity in response to external stimuli. A novel aspect of the work is the introduction of a recommended distribution, incrementally learned from the data, to optimally refine the inferred network. Unlike existing system identification techniques, this “active learning” method automatically focuses its attention on key undiscovered areas of the network, instead of targeting global uncertainty indicators like parameter variance. We show how active learning leads to faster inference while, at the same time, provides confidence intervals for the network parameters. We present simulations on artificial small-world networks to validate the methods and apply the method to real data. Analysis of frequency of motifs recovered show that cortical networks are consistent with a small-world topology model. PMID:29718955

  10. Active learning of cortical connectivity from two-photon imaging data.

    Directory of Open Access Journals (Sweden)

    Martín A Bertrán

    Full Text Available Understanding how groups of neurons interact within a network is a fundamental question in system neuroscience. Instead of passively observing the ongoing activity of a network, we can typically perturb its activity, either by external sensory stimulation or directly via techniques such as two-photon optogenetics. A natural question is how to use such perturbations to identify the connectivity of the network efficiently. Here we introduce a method to infer sparse connectivity graphs from in-vivo, two-photon imaging of population activity in response to external stimuli. A novel aspect of the work is the introduction of a recommended distribution, incrementally learned from the data, to optimally refine the inferred network. Unlike existing system identification techniques, this "active learning" method automatically focuses its attention on key undiscovered areas of the network, instead of targeting global uncertainty indicators like parameter variance. We show how active learning leads to faster inference while, at the same time, provides confidence intervals for the network parameters. We present simulations on artificial small-world networks to validate the methods and apply the method to real data. Analysis of frequency of motifs recovered show that cortical networks are consistent with a small-world topology model.

  11. Two-photon calcium imaging during fictive navigation in virtual environments

    Directory of Open Access Journals (Sweden)

    Misha Benjamin Ahrens

    2013-06-01

    Full Text Available A full understanding of nervous system function requires recording from large populations of neurons during naturalistic behaviors. Here we enable paralyzed larval zebrafish to fictively navigate two-dimensional virtual environments while we record optically from many neurons with two-photon imaging. Electrical recordings from motor nerves in the tail are decoded into intended forward swims and turns, which are used to update a virtual environment displayed underneath the fish. Several behavioral features - such as turning responses to whole-field motion and dark avoidance - are well-replicated in this virtual setting. We readily observed neuronal populations in the hindbrain with laterally selective responses that correlated with right or left optomotor behavior. We also observed neurons in the habenula, pallium, and midbrain with response properties specific to environmental features. Beyond single-cell correlations, the classification of network activity in such virtual settings promises to reveal principles of brainwide neural dynamics during behavior.

  12. Femtosecond two-photon laser-induced fluorescence of krypton for high-speed flow imaging.

    Science.gov (United States)

    Wang, Yejun; Capps, Cade; Kulatilaka, Waruna D

    2017-02-15

    Ultrashort-pulse (femtosecond-duration) two-photon laser-induced fluorescence (fs-TPLIF) of an inert gas tracer krypton (Kr) is investigated. A detailed spectroscopic study of fluorescence channels followed by the 5p'←←4p excitation of Kr at 204.1 nm is reported. The experimental line positions in the 750-840 nm emission region agree well with the NIST Atomic Spectra Database. The present work provides an accurate listing of relative line strengths in this spectral region. In the range of laser pulse energies investigated, a quadratic dependence was observed between the Kr-TPLIF signal and the laser pulse energy. The single-laser-shot 2D TPLIF images recorded in an unsteady jet demonstrate the potential of using fs excitation at 204.1 nm for mixing and flow diagnostic studies using Kr as an inert gas tracer.

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

    Science.gov (United States)

    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

  14. A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging.

    Science.gov (United States)

    Koo, Chi-Kin; Wong, Ka-Leung; Man, Cornelia Wing-Yin; Lam, Yun-Wah; So, Leo King-Yan; Tam, Hoi-Lam; Tsao, Sai-Wah; Cheah, Kok-Wai; Lau, Kai-Chung; Yang, Yang-Yi; Chen, Jin-Can; Lam, Michael Hon-Wah

    2009-02-02

    The cyclometalated platinum(II) complex [Pt(L)Cl], where HL is a new cyclometalating ligand 2-phenyl-6-(1H-pyrazol-3-yl)pyridine containing C(phenyl), N(pyridyl), and N(pyrazolyl) donor moieties, was found to possess two-photon-induced luminescent properties. The two-photon-absorption cross section of the complex in N,N-dimethylformamide at room temperature was measured to be 20.8 GM. Upon two-photon excitation at 730 nm from a Ti:sapphire laser, bright-green emission was observed. Besides its two-photon-induced luminescent properties, [Pt(L)Cl] was able to be rapidly accumulated in live HeLa and NIH3T3 cells. The two-photon-induced luminescence of the complex was retained after live cell internalization and can be observed by two-photon confocal microscopy. Its bioaccumulation properties enabled time-lapse imaging of the internalization process of the dye into living cells. Cytotoxicity of [Pt(L)Cl] to both tested cell lines was low, according to MTT assays, even at loadings as high as 20 times the dose concentration for imaging for 6 h.

  15. Imaging-guided two-photon excitation-emission-matrix measurements of human skin tissues

    Science.gov (United States)

    Yu, Yingqiu; Lee, Anthony M. D.; Wang, Hequn; Tang, Shuo; Zhao, Jianhua; Lui, Harvey; Zeng, Haishan

    2012-07-01

    There are increased interests on using multiphoton imaging and spectroscopy for skin tissue characterization and diagnosis. However, most studies have been done with just a few excitation wavelengths. Our objective is to perform a systematic study of the two-photon fluorescence (TPF) properties of skin fluorophores, normal skin, and diseased skin tissues. A nonlinear excitation-emission-matrix (EEM) spectroscopy system with multiphoton imaging guidance was constructed. A tunable femtosecond laser was used to vary excitation wavelengths from 730 to 920 nm for EEM data acquisition. EEM measurements were performed on excised fresh normal skin tissues, seborrheic keratosis tissue samples, and skin fluorophores including: NADH, FAD, keratin, melanin, collagen, and elastin. We found that in the stratum corneum and upper epidermis of normal skin, the cells have large sizes and the TPF originates from keratin. In the lower epidermis, cells are smaller and TPF is dominated by NADH contributions. In the dermis, TPF is dominated by elastin components. The depth resolved EEM measurements also demonstrated that keratin structure has intruded into the middle sublayers of the epidermal part of the seborrheic keratosis lesion. These results suggest that the imaging guided TPF EEM spectroscopy provides useful information for the development of multiphoton clinical devices for skin disease diagnosis.

  16. Fluorenyl benzothiadiazole and benzoselenadiazole near-IR fluorescent probes for two-photon fluorescence imaging (Conference Presentation)

    Science.gov (United States)

    Belfield, Kevin D.; Yao, Sheng; Kim, Bosung; Yue, Xiling

    2016-03-01

    Imaging biological samples with two-photon fluorescence (2PF) microscopy has the unique advantage of resulting high contrast 3D resolution subcellular image that can reach up to several millimeters depth. 2PF probes that absorb and emit at near IR region need to be developed. Two-photon excitation (2PE) wavelengths are less concerned as 2PE uses wavelengths doubles the absorption wavelength of the probe, which means 2PE wavelengths for probes even with absorption at visible wavelength will fall into NIR region. Therefore, probes that fluoresce at near IR region with high quantum yields are needed. A series of dyes based on 5-thienyl-2, 1, 3-benzothiadiazole and 5-thienyl-2, 1, 3-benzoselenadiazole core were synthesized as near infrared two-photon fluorophores. Fluorescence maxima wavelengths as long as 714 nm and fluorescence quantum yields as high as 0.67 were achieved. The fluorescence quantum yields of the dyes were nearly constant, regardless of solvents polarity. These diazoles exhibited large Stokes shift (GM), and high two-photon fluorescence figure of merit (FM , 1.04×10-2 GM). Cells incubated on a 3D scaffold with one of the new probes (encapsulated in Pluronic micelles) exhibited bright fluorescence, enabling 3D two-photon fluorescence imaging to a depth of 100 µm.

  17. Spatiotemporal dynamics of rhythmic spinal interneurons measured with two-photon calcium imaging and coherence analysis.

    Science.gov (United States)

    Kwan, Alex C; Dietz, Shelby B; Zhong, Guisheng; Harris-Warrick, Ronald M; Webb, Watt W

    2010-12-01

    In rhythmic neural circuits, a neuron often fires action potentials with a constant phase to the rhythm, a timing relationship that can be functionally significant. To characterize these phase preferences in a large-scale, cell type-specific manner, we adapted multitaper coherence analysis for two-photon calcium imaging. Analysis of simulated data showed that coherence is a simple and robust measure of rhythmicity for calcium imaging data. When applied to the neonatal mouse hindlimb spinal locomotor network, the phase relationships between peak activity of >1,000 ventral spinal interneurons and motor output were characterized. Most interneurons showed rhythmic activity that was coherent and in phase with the ipsilateral motor output during fictive locomotion. The phase distributions of two genetically identified classes of interneurons were distinct from the ensemble population and from each other. There was no obvious spatial clustering of interneurons with similar phase preferences. Together, these results suggest that cell type, not neighboring neuron activity, is a better indicator of an interneuron's response during fictive locomotion. The ability to measure the phase preferences of many neurons with cell type and spatial information should be widely applicable for studying other rhythmic neural circuits.

  18. Linking Neurons to Network Function and Behavior by Two-Photon Holographic Optogenetics and Volumetric Imaging.

    Science.gov (United States)

    Dal Maschio, Marco; Donovan, Joseph C; Helmbrecht, Thomas O; Baier, Herwig

    2017-05-17

    We introduce a flexible method for high-resolution interrogation of circuit function, which combines simultaneous 3D two-photon stimulation of multiple targeted neurons, volumetric functional imaging, and quantitative behavioral tracking. This integrated approach was applied to dissect how an ensemble of premotor neurons in the larval zebrafish brain drives a basic motor program, the bending of the tail. We developed an iterative photostimulation strategy to identify minimal subsets of channelrhodopsin (ChR2)-expressing neurons that are sufficient to initiate tail movements. At the same time, the induced network activity was recorded by multiplane GCaMP6 imaging across the brain. From this dataset, we computationally identified activity patterns associated with distinct components of the elicited behavior and characterized the contributions of individual neurons. Using photoactivatable GFP (paGFP), we extended our protocol to visualize single functionally identified neurons and reconstruct their morphologies. Together, this toolkit enables linking behavior to circuit activity with unprecedented resolution. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Plasmon-resonant nanorods as multimodal agents for two-photon luminescent imaging and photothermal therapy

    Science.gov (United States)

    Huff, Terry B.; Hansen, Matthew N.; Tong, Ling; Zhao, Yan; Wang, Haifeng; Zweifel, Daniel A.; Cheng, Ji-Xin; Wei, Alexander

    2007-02-01

    Plasmon-resonant gold nanorods have outstanding potential as multifunctional agents for image-guided therapies. Nanorods have large absorption cross sections at near-infrared (NIR) frequencies, and produce two-photon luminescence (TPL) when excited by fs-pulsed laser irradiation. The TPL signals can be detected with single-particle sensitivity, enabling nanorods to be imaged in vivo while passing through blood vessels at subpicomolar concentrations. Furthermore, cells labeled with nanorods become highly susceptible to photothermal damage when irradiated at plasmon resonance, often resulting in a dramatic blebbing of the cell membrane. However, the straightforward application of gold nanorods for cell-specific labeling is obstructed by the presence of CTAB, a cationic surfactant carried over from nanorod synthesis which also promotes their nonspecific uptake into cells. Careful exchange and replacement of CTAB can be achieved by introducing oligoethyleneglycol (OEG) units capable of chemisorption onto nanorod surfaces by in situ dithiocarbamate formation, a novel method of surface functionalization. Nanorods with a dense coating of methyl-terminated OEG chains are shielded from nonspecific cell uptake, whereas nanorods functionalized with folate-terminated OEG chains accumulate on the surface of tumor cells overexpressing their cognate receptor, with subsequent delivery of photoinduced cell damage at low laser fluence.

  20. Identification and two-photon imaging of oligodendrocyte in CA1 region of hippocampal slices

    International Nuclear Information System (INIS)

    Zhou Wei; Ge Wooping; Zeng Shaoqun; Duan Shumin; Luo Qingming

    2007-01-01

    Oligodendrocyte (OL) plays a critical role in myelination and axon maintenance in central nervous system. Recent studies show that OL can also express NMDA receptors in development and pathological situations in white matter. There is still lack of studies about OL properties and function in gray matter of brain. Here we reported that some glial cells in CA1 region of rat hippocampal slices (P15-23) had distinct electrophysiological characteristics from the other glia cells in this region, while they displayed uniform properties with OL from white matter in previous report; therefore, they were considered as OL in hippocampus. By loading dye in recording pipette and imaging with two-photon laser scanning microscopy, we acquired the high spatial resolution, three-dimension images of these special cells in live slices. The OL in hippocampus shows a complex process-bearing shape and the distribution of several processes is parallel to Schaffer fiber in CA1 region. When stimulating Schaffer fiber, OL displays a long duration depolarization mediated by inward rectifier potassium channel. This suggested that the OL in CA1 region could sense the neuronal activity and contribute to potassium clearance

  1. Mannose-functionalized porous silica-coated magnetic nanoparticles for two-photon imaging or PDT of cancer cells

    International Nuclear Information System (INIS)

    Perrier, Marine; Gary-Bobo, Magali; Lartigue, Lenaïc; Brevet, David; Morère, Alain; Garcia, Marcel; Maillard, Philippe; Raehm, Laurence; Guari, Yannick; Larionova, Joulia; Durand, Jean-Olivier; Mongin, Olivier; Blanchard-Desce, Mireille

    2013-01-01

    An original fluorophore engineered for two-photon excitation or a porphyrin derivative were entrapped in the silica shell of magnetic porous silica nanoparticles during the synthesis of the silica moiety without damaging the structure of the organic part. The mild conditions involved allowed obtaining microporous or mesoporous silica magnetic nanoparticles, respectively. Mannose was grafted on the surface of the nanoparticles to target MCF-7 breast cancer cells. The studies of magnetic properties of these hybrid nanoparticles show that they present a blocking temperature at 190 K. The nano-objects designed with the two-photon fluorophore were efficient for two-photon imaging of MCF-7 cancer cells, whereas the nano-objects with the photosensitizer efficiently killed cancer cells. The presence of the mannose moiety was demonstrated to improve both imaging and therapy properties.

  2. Mannose-functionalized porous silica-coated magnetic nanoparticles for two-photon imaging or PDT of cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Perrier, Marine [UMR 5253 CNRS-UM2-ENSCM-UM1, Institut Charles Gerhardt Montpellier (France); Gary-Bobo, Magali [Faculte de Pharmacie, Universite Montpellier 1, Universite Montpellier 2, Institut des Biomolecules Max Mousseron UMR 5247 CNRS (France); Lartigue, Lenaiec; Brevet, David [UMR 5253 CNRS-UM2-ENSCM-UM1, Institut Charles Gerhardt Montpellier (France); Morere, Alain; Garcia, Marcel [Faculte de Pharmacie, Universite Montpellier 1, Universite Montpellier 2, Institut des Biomolecules Max Mousseron UMR 5247 CNRS (France); Maillard, Philippe [Universite Paris-Sud, UMR 176 CNRS, Institut Curie (France); Raehm, Laurence; Guari, Yannick, E-mail: yannick.guari@um2.fr; Larionova, Joulia; Durand, Jean-Olivier, E-mail: durand@univ-montp2.fr [UMR 5253 CNRS-UM2-ENSCM-UM1, Institut Charles Gerhardt Montpellier (France); Mongin, Olivier [Universite de Rennes 1, Institut des Sciences Chimiques de Rennes, CNRS UMR 6226 (France); Blanchard-Desce, Mireille [Universite Bordeaux, Institut des Sciences Moleculaires, UMR CNRS 5255 (France)

    2013-05-15

    An original fluorophore engineered for two-photon excitation or a porphyrin derivative were entrapped in the silica shell of magnetic porous silica nanoparticles during the synthesis of the silica moiety without damaging the structure of the organic part. The mild conditions involved allowed obtaining microporous or mesoporous silica magnetic nanoparticles, respectively. Mannose was grafted on the surface of the nanoparticles to target MCF-7 breast cancer cells. The studies of magnetic properties of these hybrid nanoparticles show that they present a blocking temperature at 190 K. The nano-objects designed with the two-photon fluorophore were efficient for two-photon imaging of MCF-7 cancer cells, whereas the nano-objects with the photosensitizer efficiently killed cancer cells. The presence of the mannose moiety was demonstrated to improve both imaging and therapy properties.

  3. Two-Photon Probes for Lysosomes and Mitochondria: Simultaneous Detection of Lysosomes and Mitochondria in Live Tissues by Dual-Color Two-Photon Microscopy Imaging.

    Science.gov (United States)

    Lim, Chang Su; Hong, Seung Taek; Ryu, Seong Shick; Kang, Dong Eun; Cho, Bong Rae

    2015-10-01

    Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Review: two-photon scanning systems for clinical high resolution in vivo tissue imaging

    Science.gov (United States)

    König, K.; Müller, J.; Höfer, M.; Müller, C.; Weinigel, M.; Bückle, R.; Elsner, P.; Kaatz, M.; Messerschmidt, B.

    2008-02-01

    The femtosecond laser multiphoton tomograph DermaInspect as well as high NA two-photon GRIN microendoscopes for in vivo tomography of human skin have been used to detect malignant melanoma as well as to study the diffusion and intradermal accumulation of topically applied cosmetical and pharmaceutical components. So far, more than 500 patients and volunteers in Europe, Australia, and Asia have been investigated with this unique tomograph. Near infrared 80 MHz picojoule femtosecond laser pulses were employed to excite endogenous fluorophores such as NAD(P)H, flavoproteins, melanin, and elastin as well as fluorescent components of a variety of ointments via a twophoton excitation process. In addition, collagen has been imaged by second harmonic generation. Using a two-PMT detection system, the ratio of elastin to collagen was determined during optical sectioning. A high submicron spatial resolution and 50 picosecond temporal resolution was achieved using galvoscan mirrors and piezodriven focusing optics as well as a time-correlated single photon counting module with a fast microchannel plate detector and fast photomultipliers. Individual intratissue cells, mitochondria, melanosomes, and the morphology of the nuclei as well as extracellular matrix elements could be clearly visualized due to molecular imaging and the calculation of fluorescence lifetime images. Nanoparticles and intratissue drugs have been detected non-invasively, in situ and over a period of up to 3 months. In addition, hydration effects and UV effects were studied by monitoring modifications of cellular morphology and autofluorescence. The system was used to observe the diffusion through the stratum corneum and the accumulation and release of functionalized nanoparticles along hair shafts and epidermal ridges. The DermaInspect been also employed to gain information on skin age and wound healing in patients with ulcers. Novel developments include a galvo/piezo-scan driven flexible articulated arm as

  5. Two-Photon Functional Imaging of the Auditory Cortex in Behaving Mice: From Neural Networks to Single Spines

    Directory of Open Access Journals (Sweden)

    Ruijie Li

    2018-04-01

    Full Text Available In vivo two-photon Ca2+ imaging is a powerful tool for recording neuronal activities during perceptual tasks and has been increasingly applied to behaving animals for acute or chronic experiments. However, the auditory cortex is not easily accessible to imaging because of the abundant temporal muscles, arteries around the ears and their lateral locations. Here, we report a protocol for two-photon Ca2+ imaging in the auditory cortex of head-fixed behaving mice. By using a custom-made head fixation apparatus and a head-rotated fixation procedure, we achieved two-photon imaging and in combination with targeted cell-attached recordings of auditory cortical neurons in behaving mice. Using synthetic Ca2+ indicators, we recorded the Ca2+ transients at multiple scales, including neuronal populations, single neurons, dendrites and single spines, in auditory cortex during behavior. Furthermore, using genetically encoded Ca2+ indicators (GECIs, we monitored the neuronal dynamics over days throughout the process of associative learning. Therefore, we achieved two-photon functional imaging at multiple scales in auditory cortex of behaving mice, which extends the tool box for investigating the neural basis of audition-related behaviors.

  6. Detection of axonal synapses in 3D two-photon images.

    Directory of Open Access Journals (Sweden)

    Cher Bass

    Full Text Available Studies of structural plasticity in the brain often require the detection and analysis of axonal synapses (boutons. To date, bouton detection has been largely manual or semi-automated, relying on a step that traces the axons before detection the boutons. If tracing the axon fails, the accuracy of bouton detection is compromised. In this paper, we propose a new algorithm that does not require tracing the axon to detect axonal boutons in 3D two-photon images taken from the mouse cortex. To find the most appropriate techniques for this task, we compared several well-known algorithms for interest point detection and feature descriptor generation. The final algorithm proposed has the following main steps: (1 a Laplacian of Gaussian (LoG based feature enhancement module to accentuate the appearance of boutons; (2 a Speeded Up Robust Features (SURF interest point detector to find candidate locations for feature extraction; (3 non-maximum suppression to eliminate candidates that were detected more than once in the same local region; (4 generation of feature descriptors based on Gabor filters; (5 a Support Vector Machine (SVM classifier, trained on features from labelled data, and was used to distinguish between bouton and non-bouton candidates. We found that our method achieved a Recall of 95%, Precision of 76%, and F1 score of 84% within a new dataset that we make available for accessing bouton detection. On average, Recall and F1 score were significantly better than the current state-of-the-art method, while Precision was not significantly different. In conclusion, in this article we demonstrate that our approach, which is independent of axon tracing, can detect boutons to a high level of accuracy, and improves on the detection performance of existing approaches. The data and code (with an easy to use GUI used in this article are available from open source repositories.

  7. Phosphorus ligand imaging with two-photon fluorescence spectroscopy: towards rational catalyst immobilization

    NARCIS (Netherlands)

    Marras, F.; Kluwer, A.M.; Siekierzycka, J.R.; Vozza, A.; Brouwer, A.M.; Reek, J.N.H.

    2010-01-01

    Spotless catalysts: Ligand immobilization was studied by two-photon fluorescence microscopy with a fluorescent nixantphos ligand as probe (see picture). In the immobilization process ligand aggregates form in solution and are deposited on the support, where they appear as bright spots in

  8. Two-photon imaging of field enhancement by groups of gold nanostrip antennas

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Sondergaard, Thomas

    2009-01-01

    Resonant field enhancement by groups of 16 nm thin gold nanostrip antennas consisting of four strips (widths of 70, 100, and 130 nm) and fixed gap (50, 100, 150, or 200 nm) between them and positioned on a quartz substrate is investigated by reflection spectroscopy and two-photon photoluminescenc...

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

    Directory of Open Access Journals (Sweden)

    Chunqiang Li

    2016-01-01

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

  10. Imaging arrangement and microscope

    Science.gov (United States)

    Pertsinidis, Alexandros; Chu, Steven

    2015-12-15

    An embodiment of the present invention is an imaging arrangement that includes imaging optics, a fiducial light source, and a control system. In operation, the imaging optics separate light into first and second tight by wavelength and project the first and second light onto first and second areas within first and second detector regions, respectively. The imaging optics separate fiducial light from the fiducial light source into first and second fiducial light and project the first and second fiducial light onto third and fourth areas within the first and second detector regions, respectively. The control system adjusts alignment of the imaging optics so that the first and second fiducial light projected onto the first and second detector regions maintain relatively constant positions within the first and second detector regions, respectively. Another embodiment of the present invention is a microscope that includes the imaging arrangement.

  11. Water-Soluble Triarylborane Chromophores for One- and Two-Photon Excited Fluorescence Imaging of Mitochondria in Cells.

    Science.gov (United States)

    Griesbeck, Stefanie; Zhang, Zuolun; Gutmann, Marcus; Lühmann, Tessa; Edkins, Robert M; Clermont, Guillaume; Lazar, Adina N; Haehnel, Martin; Edkins, Katharina; Eichhorn, Antonius; Blanchard-Desce, Mireille; Meinel, Lorenz; Marder, Todd B

    2016-10-04

    Three water-soluble tetracationic quadrupolar chromophores comprising two three-coordinate boron π-acceptor groups bridged by thiophene-containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5-(3,5-Me2 C6 H2 )-2,2'-(C4 H2 S)2 -5'-(3,5-Me2 C6 H2 ) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one- and two-photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one- and two-photon-excited fluorescence imaging of mitochondrial function in cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Imaging of Fluoride Ion in Living Cells and Tissues with a Two-Photon Ratiometric Fluorescence Probe

    Directory of Open Access Journals (Sweden)

    Xinyue Zhu

    2015-01-01

    Full Text Available A reaction-based two-photon (TP ratiometric fluorescence probe Z2 has been developed and successfully applied to detect and image fluoride ion in living cells and tissues. The Z2 probe was designed designed to utilize an ICT mechanism between n-butylnaphthalimide as a fluorophore and tert-butyldiphenylsilane (TBDPS as a response group. Upon addition of fluoride ion, the Si-O bond in the Z2 would be cleaved, and then a stronger electron-donating group was released. The fluorescent changes at 450 and 540 nm, respectively, made it possible to achieve ratiometric fluorescence detection. The results indicated that the Z2 could ratiometrically detect and image fluoride ion in living cells and tissues in a depth of 250 μm by two-photon microscopy (TPM.

  13. 3D imaging of intrinsic crystalline defects in zinc oxide by spectrally resolved two-photon fluorescence microscopy

    Science.gov (United States)

    Al-Tabich, A.; Inami, W.; Kawata, Y.; Jablonski, R.; Worasawat, S.; Mimura, H.

    2017-05-01

    We present a method for three-dimensional intrinsic defect imaging in zinc oxide (ZnO) by spectrally resolved two-photon fluorescence microscopy, based on the previously presented method of observing a photoluminescence distribution in wide-gap semiconductor crystals [Noor et al., Appl. Phys. Lett. 92(16), 161106 (2008)]. A tightly focused light beam radiated by a titanium-sapphire laser is used to obtain a two-photon excitation of selected area of the ZnO sample. Photoluminescence intensity of a specific spectral range is then selected by optical band pass filters and measured by a photomultiplier tube. Reconstruction of the specimen image is done by scanning the volume of interest by a piezoelectric positioning stage and measuring the spectrally resolved photoluminescence intensity at each point. The method has been proved to be effective at locating intrinsic defects of the ZnO crystalline structure in the volume of the crystal. The method was compared with other defect imaging and 3D imaging techniques like scanning tunneling microscopy and confocal microscopy. In both cases, our method shows superior penetration abilities and, as the only method, allows location of the defects of the chosen type in 3D. In this paper, we present the results of oxygen vacancies and zinc antisites imaging in ZnO nanorods.

  14. Construction of an efficient two-photon fluorescent probe for imaging nitroreductase in live cells and tissues

    Science.gov (United States)

    Zhou, Liyi; Gong, Liang; Hu, Shunqin

    2018-06-01

    Compared with traditional confocal microscopy, two-photon fluorescence microscopy (TPFM), which excites a two-photon (TP) fluorophore by near-infrared light, provides improved three-dimensional image resolution with increased tissue-image depth (>500 μm) and an extended observation time. Therefore, the development of novel functional TP fluorophores has attracted great attention in recent years. Herein, a novel TP fluorophore CM-NH2, which have the donor-π-acceptor (D-π-A)-structure, was designed and synthesized. We further used this dye developed a new type of TP fluorescent probe CM-NO2 for detecting nitroreductase (NTR). Upon incubated with NTR for 15 min, CM-NO2 displayed a 90-fold fluorescence enhancement at 505 nm and the maximal TP action cross-section value after reaction was detected and calculated to be 200 GM at 760 nm. The probe exhibited excellent properties such as high sensitivity, high selectivity, low cytotoxicity, and high photostability. Moreover, the probe was utilized to image the tumor hypoxia in live HeLa cells. Finally, using the CM-NO2 to image NTR in tissues was demonstrated.

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

    Science.gov (United States)

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

    2017-10-01

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

  16. Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

    Energy Technology Data Exchange (ETDEWEB)

    Mueller-Harvey, Irene, E-mail: i.mueller-harvey@reading.ac.uk [Chemistry and Biochemistry Laboratory, Food Production and Quality Research Division, School of Agriculture, Policy and Development, University of Reading, P O Box 236, Reading RG6 6AT (United Kingdom); Feucht, Walter, E-mail: walter.feucht@gmail.com [Department of Plant Sciences, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Polster, Juergen, E-mail: j.polster@wzw.tum.de [Department of Physical Biochemistry, Technical University of Munich (TUM), Wissenschaftszentrum Weihenstephan (WZW), D-85354 Freising (Germany); Trnkova, Lucie, E-mail: lucie.trnkova@uhk.cz [University of Hradec Kralove, Faculty of Science, Department of Chemistry, Rokitanskeho 62, 50003 Hradec Kralove (Czech Republic); Burgos, Pierre, E-mail: pierre.burgos@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Parker, Anthony W., E-mail: tony.parker@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom); Botchway, Stanley W., E-mail: stan.botchway@stfc.ac.uk [Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell-Oxford, Didcot, Oxfordshire, OX11 0QX (United Kingdom)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer This fluorescence lifetime imaging microscopy (FLIM) technique for flavanols overcomes autofluorescence interference in cells. Black-Right-Pointing-Pointer Plant flavanols differed in their lifetimes. Black-Right-Pointing-Pointer Dissolved and bound flavanols revealed contrasting lifetime changes. Black-Right-Pointing-Pointer This technique will allow studying of flavanol trafficking in live cells. - Abstract: Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were {approx}1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018). In vivo experiments with onion cells demonstrated that tryptophan and quercetin, which tend to be major contributors of background fluorescence in plant cells, have sufficiently low cross sections for two-photon excitation at 630 nm and therefore do not interfere with detection of externally added or endogenous flavanols in Allium cepa or Taxus baccata cells. Applying two-photon excitation to flavanols enabled 3-D fluorescence lifetime imaging microscopy and showed that added EGCG penetrated the whole nucleus of onion cells. Interestingly, EGCG and catechin showed different lifetime behaviour when bound to the nucleus: EGCG lifetime increased from <45 to 200 ps, whilst catechin lifetime decreased from 1.0 ns to 500 ps. Semi-quantitative measurements revealed that the relative ratios of EGCG concentrations in nucleoli associated vesicles: nucleus: cytoplasm were ca. 100:10:1. Solution experiments with catechin, epicatechin and histone proteins provided preliminary evidence, via the appearance of a second lifetime ({tau}{sub 2} = 1.9-3.1 ns), that both flavanols may be interacting with histone proteins. We conclude that there

  17. FocusStack and StimServer: a new open source MATLAB toolchain for visual stimulation and analysis of two-photon calcium neuronal imaging data.

    Science.gov (United States)

    Muir, Dylan R; Kampa, Björn M

    2014-01-01

    Two-photon calcium imaging of neuronal responses is an increasingly accessible technology for probing population responses in cortex at single cell resolution, and with reasonable and improving temporal resolution. However, analysis of two-photon data is usually performed using ad-hoc solutions. To date, no publicly available software exists for straightforward analysis of stimulus-triggered two-photon imaging experiments. In addition, the increasing data rates of two-photon acquisition systems imply increasing cost of computing hardware required for in-memory analysis. Here we present a Matlab toolbox, FocusStack, for simple and efficient analysis of two-photon calcium imaging stacks on consumer-level hardware, with minimal memory footprint. We also present a Matlab toolbox, StimServer, for generation and sequencing of visual stimuli, designed to be triggered over a network link from a two-photon acquisition system. FocusStack is compatible out of the box with several existing two-photon acquisition systems, and is simple to adapt to arbitrary binary file formats. Analysis tools such as stack alignment for movement correction, automated cell detection and peri-stimulus time histograms are already provided, and further tools can be easily incorporated. Both packages are available as publicly-accessible source-code repositories.

  18. Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

    Science.gov (United States)

    Krasieva, Tatiana B.; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Tromberg, Bruce J.

    2012-03-01

    The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6+/-0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55+/-0.05 and 0.17+/-0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

  19. Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment

    Directory of Open Access Journals (Sweden)

    Clément eRicard

    2014-02-01

    Full Text Available The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate of each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

  20. Six-color intravital two-photon imaging of brain tumors and their dynamic microenvironment.

    Science.gov (United States)

    Ricard, Clément; Debarbieux, Franck Christian

    2014-01-01

    The majority of intravital studies on brain tumor in living animal so far rely on dual color imaging. We describe here a multiphoton imaging protocol to dynamically characterize the interactions between six cellular components in a living mouse. We applied this methodology to a clinically relevant glioblastoma multiforme (GBM) model designed in reporter mice with targeted cell populations labeled by fluorescent proteins of different colors. This model permitted us to make non-invasive longitudinal and multi-scale observations of cell-to-cell interactions. We provide examples of such 5D (x,y,z,t,color) images acquired on a daily basis from volumes of interest, covering most of the mouse parietal cortex at subcellular resolution. Spectral deconvolution allowed us to accurately separate each cell population as well as some components of the extracellular matrix. The technique represents a powerful tool for investigating how tumor progression is influenced by the interactions of tumor cells with host cells and the extracellular matrix micro-environment. It will be especially valuable for evaluating neuro-oncological drug efficacy and target specificity. The imaging protocol provided here can be easily translated to other mouse models of neuropathologies, and should also be of fundamental interest for investigations in other areas of systems biology.

  1. Two-photon microscopy for imaging of the (atherosclerotic) vascular wall: a proof of concept study

    NARCIS (Netherlands)

    van Zandvoort, Marc; Engels, Wim; Douma, Kim; Beckers, Linda; Oude Egbrink, Mirjam; Daemen, Mat; Slaaf, Dick W.

    2004-01-01

    Understanding atherogenesis will benefit significantly from simultaneous imaging, both ex vivo and in vivo, of structural and functional information at the (sub)cellular level within intact arteries. Due to limited penetration depth and loss of resolution with depth, intravital and confocal

  2. Scanless functional imaging of hippocampal networks using patterned two-photon illumination through GRIN lenses

    KAUST Repository

    Moretti, Claudio; Antonini, Andrea; Bovetti, Serena; Liberale, Carlo; Fellin, Tommaso

    2016-01-01

    functional imaging in rodent hippocampal networks in vivo ~1.2 mm below the brain surface. Our results open the way to the application of patterned illumination approaches to deep regions of highly scattering biological tissues, such as the mammalian brain.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-12

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

  5. Femtosecond, two-photon laser-induced-fluorescence imaging of atomic oxygen in an atmospheric-pressure plasma jet

    Science.gov (United States)

    Schmidt, Jacob B.; Sands, Brian L.; Kulatilaka, Waruna D.; Roy, Sukesh; Scofield, James; Gord, James R.

    2015-06-01

    Femtosecond, two-photon-absorption laser-induced-fluorescence (fs-TALIF) spectroscopy is employed to measure space- and time-resolved atomic-oxygen distributions in a nanosecond, repetitively pulsed, externally grounded, atmospheric-pressure plasma jet flowing helium with a variable oxygen admixture. The high-peak-intensity, low-average-energy femtosecond pulses result in increased TALIF signal with reduced photolytic inferences. This allows 2D imaging of absolute atomic-oxygen number densities ranging from 5.8   ×   1015 to 2.0   ×   1012cm-3 using a cooled CCD with an external intensifier. Xenon is used for signal and imaging-system calibrations to quantify the atomic-oxygen fluorescence signal. Initial results highlight a transition in discharge morphology from annular to filamentary, corresponding with a change in plasma chemistry from ozone to atomic oxygen production, as the concentration of oxygen in the feed gas is changed at a fixed voltage-pulse-repetition rate. In this configuration, significant concentrations of reactive oxygen species may be remotely generated by sustaining an active discharge beyond the confines of the dielectric capillary, which may benefit applications that require large concentrations of reactive oxygen species such as material processing or biomedical devices.

  6. Mercury effects on Thalassiosira weissflogii: Applications of two-photon excitation chlorophyll fluorescence lifetime imaging and flow cytometry

    International Nuclear Information System (INIS)

    Wu Yun; Zeng Yan; Qu, Jianan Y.; Wang Wenxiong

    2012-01-01

    The toxic effects of inorganic mercury [Hg(II)] and methylmercury (MeHg) on the photosynthesis and population growth in a marine diatom Thalassiosira weissflogii were investigated using two methods: two-photon excitation fluorescence lifetime imaging (FLIM) and flow cytometry (FCM). For photosynthesis, Hg(II) exposure increased the average chlorophyll fluorescence lifetime, whereas such increment was not found under MeHg stress. This may be caused by the inhibitory effect of Hg(II) instead of MeHg on the electron transport chain. For population growth, modeled specific growth rate data showed that the reduction in population growth by Hg(II) mainly resulted from an increased number of injured cells, while the live cells divided at the normal rates. However, MeHg inhibitory effects on population growth were contributed by the reduced division rates of all cells. Furthermore, the cell images and the FCM data reflected the morphological changes of diatom cells under Hg(II)/MeHg exposure vividly and quantitatively. Our results demonstrated that the toxigenicity mechanisms between Hg(II) and MeHg were different in the algal cells.

  7. Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

    Science.gov (United States)

    Collot, Mayeul; Loukou, Christina; Yakovlev, Aleksey V; Wilms, Christian D; Li, Dongdong; Evrard, Alexis; Zamaleeva, Alsu; Bourdieu, Laurent; Léger, Jean-François; Ropert, Nicole; Eilers, Jens; Oheim, Martin; Feltz, Anne; Mallet, Jean-Maurice

    2012-09-12

    We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

  8. Two-photon physics

    International Nuclear Information System (INIS)

    Bardeen, W.A.

    1981-10-01

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

  9. Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging.

    Science.gov (United States)

    Chen, Bin; Feng, Guangxue; He, Bairong; Goh, Chiching; Xu, Shidang; Ramos-Ortiz, Gabriel; Aparicio-Ixta, Laura; Zhou, Jian; Ng, Laiguan; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong

    2016-02-10

    Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Differential magnetic force microscope imaging.

    Science.gov (United States)

    Wang, Ying; Wang, Zuobin; Liu, Jinyun; Hou, Liwei

    2015-01-01

    This paper presents a method for differential magnetic force microscope imaging based on a two-pass scanning procedure to extract differential magnetic forces and eliminate or significantly reduce background forces with reversed tip magnetization. In the work, the difference of two scanned images with reversed tip magnetization was used to express the local magnetic forces. The magnetic sample was first scanned with a low lift distance between the MFM tip and the sample surface, and the magnetization direction of the probe was then changed after the first scan to perform the second scan. The differential magnetic force image was obtained through the subtraction of the two images from the two scans. The theoretical and experimental results have shown that the proposed method for differential magnetic force microscope imaging is able to reduce the effect of background or environment interference forces, and offers an improved image contrast and signal to noise ratio (SNR). © Wiley Periodicals, Inc.

  11. Interface-Targeting Strategy Enables Two-Photon Fluorescent Lipid Droplet Probes for High-Fidelity Imaging of Turbid Tissues and Detecting Fatty Liver.

    Science.gov (United States)

    Guo, Lifang; Tian, Minggang; Feng, Ruiqing; Zhang, Ge; Zhang, Ruoyao; Li, Xuechen; Liu, Zhiqiang; He, Xiuquan; Sun, Jing Zhi; Yu, Xiaoqiang

    2018-04-04

    Lipid droplets (LDs) with unique interfacial architecture not only play crucial roles in protecting a cell from lipotoxicity and lipoapoptosis but also closely relate with many diseases such as fatty liver and diabetes. Thus, as one of the important applied biomaterials, fluorescent probes with ultrahigh selectivity for in situ and high-fidelity imaging of LDs in living cells and tissues are critical to elucidate relevant physiological and pathological events as well as detect related diseases. However, available probes only utilizing LDs' waterless neutral cores but ignoring the unique phospholipid monolayer interfaces exhibit low selectivity. They cannot differentiate neutral cores of LDs from intracellular other lipophilic microenvironments, which results in extensively cloud-like background noise and severely limited their bioapplications. Herein, to design LD probes with ultrahigh selectivity, the exceptional interfacial architecture of LDs is considered adequately and thus an interface-targeting strategy is proposed for the first time. According to the novel strategy, we have developed two amphipathic fluorescent probes (N-Cy and N-Py) by introducing different cations into a lipophilic fluorophore (nitrobenzoxadiazole (NBD)). Consequently, their cationic moiety precisely locates the interfaces through electrostatic interaction and simultaneously NBD entirely embeds into the waterless core via hydrophobic interaction. Thus, high-fidelity and background-free fluorescence imaging of LDs are expectably realized in living cells in situ. Moreover, LDs in turbid tissues like skeletal muscle slices have been clearly imaged (up to 82 μm depth) by a two-photon microscope. Importantly, using N-Cy, we not only intuitively monitored the variations of LDs in number, size, and morphology but also clearly revealed their abnormity in hepatic tissues resulting from fatty liver. Therefore, these unique probes provide excellent imaging tools for elucidating LD

  12. Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

    Science.gov (United States)

    Xie, Xilei; Tang, Fuyan; Shangguan, Xiaoyan; Che, Shiyi; Niu, Jinye; Xiao, Yongsheng; Wang, Xu; Tang, Bo

    2017-06-13

    Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

  13. Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging

    Directory of Open Access Journals (Sweden)

    Mély Yves

    2008-09-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

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

    Directory of Open Access Journals (Sweden)

    Rebane Aleks

    2010-02-01

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

  15. Wide-field two-photon microscopy with temporal focusing and HiLo background rejection

    Science.gov (United States)

    Yew, Elijah Y. S.; Choi, Heejin; Kim, Daekeun; So, Peter T. C.

    2011-03-01

    Scanningless depth-resolved microscopy is achieved through spatial-temporal focusing and has been demonstrated previously. The advantage of this method is that a large area may be imaged without scanning resulting in higher throughput of the imaging system. Because it is a widefield technique, the optical sectioning effect is considerably poorer than with conventional spatial focusing two-photon microscopy. Here we propose wide-field two-photon microscopy based on spatio-temporal focusing and employing background rejection based on the HiLo microscope principle. We demonstrate the effects of applying HiLo microscopy to widefield temporally focused two-photon microscopy.

  16. Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy.

    Science.gov (United States)

    Appleton, P L; Quyn, A J; Swift, S; Näthke, I

    2009-05-01

    Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 mum within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of image at high-resolution deep within tissue. We show that manipulating the refractive index of the mounting media and decreasing sample opacity greatly improves image quality such that the limiting factor for a standard, inverted multi-photon microscope is determined by the working distance of the objective as opposed to detectable fluorescence. This method negates the need for mechanical sectioning of tissue and enables the routine generation of high-quality, quantitative image data that can significantly advance our understanding of tissue architecture and physiology.

  17. N-doped carbon dots derived from bovine serum albumin and formic acid with one- and two-photon fluorescence for live cell nuclear imaging.

    Science.gov (United States)

    Tan, Mingqian; Li, Xintong; Wu, Hao; Wang, Beibei; Wu, Jing

    2015-12-01

    Carbon dots with both one- and two-photon fluorescence have drawn great attention for biomedical imaging. Herein, nitrogen-doped carbon dots were facilely developed by one-pot hydrothermal method using bovine serum albumin and formic acid as carbon sources. They are highly water-soluble with strong fluorescence when excited with ultraviolet or near infrared light. The carbon dots have a diameter of ~8.32 nm and can emit strong two-photon induced fluorescence upon excitation at 750 nm with a femtosecond laser. X-ray photoelectron spectrometer analysis revealed that the carbon dots contained three components, C, N and O, corresponding to the peak at 285, 398 and 532 eV, respectively. The Fourier-transform infrared spectroscopy analysis revealed that there are carboxyl and carboxylic groups on the surface, which allowed further linking of functional molecules. pH stability study demonstrated that the carbon dots are able to be used in a wide range of pH values. The fluorescence mechanism is also discussed in this study. Importantly, these carbon dots are biocompatible and highly photostable, which can be directly applied for both one- and two-photon living cell imaging. After proper surface functionalization with TAT peptide, they can be used as fluorescent probes for live cell nuclear-targeted imaging. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Tale of two photons

    International Nuclear Information System (INIS)

    Anon.

    1994-01-01

    A very profitable spinoff from electron- positron collisions is two-photon physics. Rather than the electron and positron interacting directly via an exchanged photon, two virtual (transient) photons, one from each particle, get tangled up. With new electron-positron colliders appearing on the scene, a topical meeting on two-photon physics - 'From DAPHNE to LEP 200 and beyond' - held from 2-4 February in Paris, in the premises of the Ministry of Higher Education and Research, was particularly timely. Some 60 physicists, both experimentalists and theorists, participated, with some thirty speakers

  19. Multifunctional biocompatible graphene oxide quantum dots decorated magnetic nanoplatform for efficient capture and two-photon imaging of rare tumor cells.

    Science.gov (United States)

    Shi, Yongliang; Pramanik, Avijit; Tchounwou, Christine; Pedraza, Francisco; Crouch, Rebecca A; Chavva, Suhash Reddy; Vangara, Aruna; Sinha, Sudarson Sekhar; Jones, Stacy; Sardar, Dhiraj; Hawker, Craig; Ray, Paresh Chandra

    2015-05-27

    Circulating tumor cells (CTCs) are extremely rare cells in blood containing billions of other cells. The selective capture and identification of rare cells with sufficient sensitivity is a real challenge. Driven by this need, this manuscript reports the development of a multifunctional biocompatible graphene oxide quantum dots (GOQDs) coated, high-luminescence magnetic nanoplatform for the selective separation and diagnosis of Glypican-3 (GPC3)-expressed Hep G2 liver cancer tumor CTCs from infected blood. Experimental data show that an anti-GPC3-antibody-attached multifunctional nanoplatform can be used for selective Hep G2 hepatocellular carcinoma tumor cell separation from infected blood containing 10 tumor cells/mL of blood in a 15 mL sample. Reported data indicate that, because of an extremely high two-photon absorption cross section (40530 GM), an anti-GPC3-antibody-attached GOQDs-coated magnetic nanoplatform can be used as a two-photon luminescence platform for selective and very bright imaging of a Hep G2 tumor cell in a biological transparency window using 960 nm light. Experimental results with nontargeted GPC3(-) and SK-BR-3 breast cancer cells show that multifunctional-nanoplatform-based cell separation, followed by two-photon imaging, is highly selective for Hep G2 hepatocellular carcinoma tumor cells.

  20. A fast-response two-photon fluorescent probe for imaging endogenous H2O2 in living cells and tissues

    Science.gov (United States)

    Lu, Yanan; Shi, Xiaomin; Fan, Wenlong; Black, Cory A.; Lu, Zhengliang; Fan, Chunhua

    2018-02-01

    As a second messenger, hydrogen peroxide plays significant roles in numerous physiological and pathological processes and is related to various diseases including inflammatory disease, diabetes, neurodegenerative disorders, cardiovascular disease and Alzheimer's disease. Two-photon (TP) fluorescent probes reported for the detection of endogenous H2O2 are rare and most have drawbacks such as slow response and low sensitivity. In this report, we demonstrate a simple H2O2-specific TP fluorescent probe (TX-HP) containing a two-photon dye 6-hydroxy-2,3,4,4a-tetrahydro-1H-xanthen-1-one (TX) on the modulation of the ICT process. The probe exhibits a rapid fluorescent response to H2O2 in 9 min with both high sensitivity and selectivity. The probe can detect exogenous H2O2 in living cells. Furthermore, the probe is successfully utilized for imaging H2O2 in liver tissues.

  1. Confocal multispot microscope for fast and deep imaging in semicleared tissues

    Science.gov (United States)

    Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

    2018-02-01

    Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

  2. Two Photon Distribution Amplitudes

    International Nuclear Information System (INIS)

    El Beiyad, M.; Pire, B.; Szymanowski, L.; Wallon, S.

    2008-01-01

    The factorization of the amplitude of the process γ*γ→γγ in the low energy and high photon virtuality region is demonstrated at the Born order and in the leading logarithmic approximation. The leading order two photon (generalized) distribution amplitudes exhibit a characteristic ln Q 2 behaviour and obey new inhomogeneous evolution equations

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

    Science.gov (United States)

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

    2015-10-01

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

  4. Quantitative evaluation of redox ratio and collagen characteristics during breast cancer chemotherapy using two-photon intrinsic imaging.

    Science.gov (United States)

    Wu, Shulian; Huang, Yudian; Tang, Qinggong; Li, Zhifang; Horng, Hannah; Li, Jiatian; Wu, Zaihua; Chen, Yu; Li, Hui

    2018-03-01

    Preoperative neoadjuvant treatment in locally advanced breast cancer is recognized as an effective adjuvant therapy, as it improves treatment outcomes. However, the potential complications remain a threat, so there is an urgent clinical need to assess both the tumor response and changes in its microenvironment using non-invasive and precise identification techniques. Here, two-photon microscopy was employed to detect morphological alterations in breast cancer progression and recession throughout chemotherapy. The changes in structure were analyzed based on the autofluorescence and collagen of differing statuses. Parameters, including optical redox ratio, the ratio of second harmonic generation and auto-fluorescence signal, collagen density, and collagen shape orientation, were studied. Results indicate that these parameters are potential indicators for evaluating breast tumors and their microenvironment changes during progression and chemotherapy. Combined analyses of these parameters could provide a quantitative, novel method for monitoring tumor therapy.

  5. The Use of Intravital Two-Photon and Thick Section Confocal Imaging to Analyze B Lymphocyte Trafficking in Lymph Nodes and Spleen.

    Science.gov (United States)

    Park, Chung; Hwang, Il-Young; Kehrl, John H

    2018-01-01

    Intravital two-photon laser scanning microscopy (TP-LSM) has allowed the direct observation of immune cells in intact organs of living animals. In the B cell biology field TP-LSM has detailed the movement of B cells in high endothelial venules and during their transmigration into lymph organs; described the movement and positioning of B cells within lymphoid organs; outlined the mechanisms by which antigen is delivered to B cells; observed B cell interacting with T cells, other cell types, and even with pathogens; and delineated the egress of B cells from the lymph node (LN) parenchyma into the efferent lymphatics. As the quality of TP-LSM improves and as new fluorescent probes become available additional insights into B cell behavior and function await new investigations. Yet intravital TP-LSM has some disadvantages including a lower resolution than standard confocal microscopy, a narrow imaging window, and a shallow depth of imaging. We have found that supplementing intravital TP-LSM with conventional confocal microscopy using thick LN sections helps to overcome some of these shortcomings. Here, we describe procedures for visualizing the behavior and trafficking of fluorescently labeled, adoptively transferred antigen-activated B cells within the inguinal LN of live mice using two-photon microscopy. Also, we introduce procedures for fixed thick section imaging using standard confocal microscopy, which allows imaging of fluorescently labeled cells deep in the LN cortex and in the spleen with high resolution.

  6. Water soluble two-photon fluorescent organic probes for long-term imaging of lysosomes in live cells and tumor spheroids.

    Science.gov (United States)

    Kumari, Pratibha; Verma, Sanjay K; Mobin, Shaikh M

    2018-01-11

    The morphological alteration of lysosomes is a powerful indicator of various pathological disorders. In this regard, we have designed and synthesized a new water soluble fluorescent Schiff-base ligand (L-lyso) containing two hydroxyl groups. L-lyso exhibits excellent two-photon properties with tracking of lysosomes in live cells as well as in 3D tumor spheroids. Furthermore, it can label lysosomes for more than 3 days. Thus, L-lyso has an edge over the commercially available expensive LysoTracker probes and also over other reported probes in terms of its long-term imaging, water solubility and facile synthesis.

  7. Two-photon imaging during prolonged middle cerebral artery occlusion in mice reveals recovery of dendritic structure after reperfusion.

    Science.gov (United States)

    Li, Ping; Murphy, Timothy H

    2008-11-12

    Filament occlusion of the middle cerebral artery (MCA) is a well accepted animal model of focal ischemia. Advantages of the model are relatively long occlusion times and a large penumbra region that simulates aspects of human stroke. Here, we use two-photon and confocal microscopy in combination with regional measurement of blood flow using laser speckle to assess the spatial relationship between the borders of the MCA ischemic territory and loss of dendrite structure, as well as the effect of reperfusion on dendritic damage in adult YFP (yellow fluorescent protein) and GFP (green fluorescent protein) C57BL/6 transgenic mice with fluorescent (predominantly layer 5) neurons. By examining the spatial extent of dendritic damage, we determined that 60 min of MCA occlusion produced a core with severe structural damage that did not recover after reperfusion (begins approximately 3.8 mm lateral to midline), a reversibly damaged area up to 0.6 mm medial to the core that recovered after reperfusion (penumbra), and a relatively structurally intact area ( approximately 1 mm wide; medial penumbra) with hypoperfusion. Loss of structure was preceded by a single ischemic depolarization 122.1 +/- 10.2 s after occlusion onset. Reperfusion of animals after 60 min of ischemia was not associated with exacerbation of damage (reperfusion injury) and resulted in a significant restoration of blebbed dendritic structure, but only within approximately 0.6 mm lateral of the dendritic damage structural border. In summary, we find that recovery of dendritic structure can occur after reperfusion after even 60 min of ischemia, but is likely restricted to a relatively small penumbra region with partial blood flow or oxygenation.

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

    DEFF Research Database (Denmark)

    Beermann, Jonas; Bozhevolnyi, Sergey I.

    2005-01-01

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

  9. Real-time two-photon lithography in controlled flow to create a single-microparticle array and particle-cluster array for optofluidic imaging.

    Science.gov (United States)

    Xu, Bing; Shi, Yang; Lao, Zhaoxin; Ni, Jincheng; Li, Guoqiang; Hu, Yanlei; Li, Jiawen; Chu, Jiaru; Wu, Dong; Sugioka, Koji

    2018-01-30

    Microarray technology provides an excellent platform for biomedical and biochemical research including basic scientific studies, drug discovery, and diagnostics. Here, we develop a novel method referred to as real-time two-photon lithography in a controlled flow in which femtosecond laser two-photon lithography is performed in situ in the sequential mode stopping and flowing the flow of liquid resin containing microparticles to achieve 100% trapping on a one-bead-to-one-trap basis. Polydisperse particles can be all trapped to form a desired array by freely designing trap structures, resulting in an unprecedentedly high capture efficiency of ∼100%. No persistent pressure is needed after trapping which reduces the complexity of the system. In addition, trapping of particle-cluster arrays with a controlled number of particles is also achieved via this method. The trapped particles inside the microchip are successfully applied as microlenses for high quality imaging. The present technology marks an essential step towards a versatile platform for the integration of bead-based assays and paves the way for developing innovative microfluidics, optofluidics, micro-optics and single-cell analysis devices.

  10. Quantitative 3-dimensional imaging of auxin and cytokinin levels in transgenic soybean and medicago truncatula roots via two-photon induced fluorescence imaging

    Science.gov (United States)

    Fisher, Jon; Gaillard, Paul; Nurmalasari, Ni Putu Dewi; Fellbaum, Carl; Subramaniam, Sen; Smith, Steve

    2018-02-01

    Industrial nitrogen fertilizers account for nearly 50% of the fossil fuel costs in modern agriculture and contribute to soil and water pollution. Therefore, significant interest exists in understanding and characterizing the efficiency of nitrogen fixation, and the biochemical signaling pathways which orchestrate the plant-microbial symbiosis through which plants fix nitrogen. Legume plant species exhibit a particularly efficient nitrogen uptake mechanism, using root nodules which house nitrogen-fixing rhizobial bacteria. While nodule development has been widely studied, there remain significant gaps in understanding the regulatory hormones' role in plant development. In this work, we produce 3-dimensional maps of auxin (AX) and cytokinin (CK) hormone concentrations within model plant root tips and nodules with respect to root architecture and cell type. Soybean and Medicago plants were transfected with a two-color fluorescent vector with AXsensitive green fluorescent protein (GFP) and CK-sensitive TdTomato (TdT). 3D images of soybean root nodules were captured using two-photon induced fluorescence microscopy. The resulting images were computationally analyzed using the localization code first developed by Weeks and later adapted by Kilfoil, and analyzed in the context of the root architecture. Statistical analysis of the resulting 3D hormone level maps reproduce-well the known roles of AX and CK in developing plant roots, and are the first quantitative description of these regulatory hormones tied to specific plant architecture. The analytical methods used, and the spatial distribution of these key regulatory hormones in plant roots, nodule primordia and root nodules, and their statistical interpretation are presented.

  11. Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

    Science.gov (United States)

    Sivaguru, Mayandi; Fried, Glenn; Sivaguru, Barghav S; Sivaguru, Vignesh A; Lu, Xiaochen; Choi, Kyung Hwa; Saif, M Taher A; Lin, Brian; Sadayappan, Sakthivel

    2015-11-01

    The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

  12. Nonlinear spectral imaging of human normal skin, basal cell carcinoma and squamous cell carcinoma based on two-photon excited fluorescence and second-harmonic generation

    Science.gov (United States)

    Xiong, S. Y.; Yang, J. G.; Zhuang, J.

    2011-10-01

    In this work, we use nonlinear spectral imaging based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) for analyzing the morphology of collagen and elastin and their biochemical variations in basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and normal skin tissue. It was found in this work that there existed apparent differences among BCC, SCC and normal skin in terms of their thickness of the keratin and epithelial layers, their size of elastic fibers, as well as their distribution and spectral characteristics of collagen. These differences can potentially be used to distinguish BCC and SCC from normal skin, and to discriminate between BCC and SCC, as well as to evaluate treatment responses.

  13. Dipolar versus octupolar triphenylamine-based fluorescent organic nanoparticles as brilliant one- and two-photon emitters for (bio)imaging.

    Science.gov (United States)

    Parthasarathy, Venkatakrishnan; Fery-Forgues, Suzanne; Campioli, Elisa; Recher, Gaëlle; Terenziani, Francesca; Blanchard-Desce, Mireille

    2011-11-18

    Two related triphenylamine-based dipolar and octupolar fluorophores are used to prepare aqueous suspensions of fluorescent organic nanoparticles (FONs) via the reprecipitation method. The obtained spherical nanoparticles (30-40 nm in diameter) are fluorescent in aqueous solution (up to 15% fluorescence quantum yield) and exhibit extremely high one- and two-photon brightness, superior to those obtained for quantum dots. Despite the two chromophores showing similar fluorescence in solution, the fluorescence of FONs made from the octupolar derivative is significantly red-shifted compared to that generated by the dipolar FONs. In addition, the maximum two-photon absorption cross section of the FONs made from the octupolar derivative is 55% larger than that of the dipolar derivative FONs. The experimental observations provide evidence that the different molecular shape (rodlike versus three-branched) and charge distribution (dipolar versus octupolar) of the two chromophores strongly affect the packing inside the nanoparticles as well as their spectroscopic properties and colloidal stability in pure water. The use of these FONs as probes for biphotonic in-vivo imaging is investigated on Xenopus laevis tadpoles to test their utilization for angiography. When using FONs made from the octupolar dye, the formation of microagglomerates (2-5 μm scale) is observed in vivo, with subsequent lethal occlusion of the blood vessels. Conversely, the nanoparticles of the dipolar dye allow acute imaging of blood vessels thanks to their suitable size and brightness, while no toxic effect is observed. Such a goal cannot be achieved with the dissolved dye, which permeates the vessel walls. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Two-photon excited fluorescence imaging of the pancreatic solid pseudopapillary tumor without hematoxylin and eosin stains.

    Science.gov (United States)

    Xu, Yahao; Liao, Chenxi; Chen, Jing; Chen, Youting; Zhu, Xiaoqin; Chen, Jianxin

    2016-05-01

    Solid pseudopapillary tumor (SPT) of the pancreas is an epithelial tumor with low-grade malignant potential and present more common in females. At present, the gold standard for accurate diagnosis of pancreatic tumor was mostly depending on the pathological and/or cytological evaluation. In this work, TPEF microscopy was applied to obtain the images of human normal pancreas and SPT of the pancreas without hematoxylin and eosin (H&E) staining, for the purpose of identifying the organization structural, cell morphological, and cytoplasm changing, which were then compared to their corresponding H&E stained histopathological results. Our results showed that high-resolution TPEF imaging of the pancreatic SPT can clearly distinguish the pathological features from normal pancreas in unstained histological sections, and the results are consistent with the histological results. Moreover, we measured the nuclear-cytoplasmic ratios of the pancreatic SPT and normal pancreas to characterize their difference in the cytomorphological feature. It indicated that this technique can achieve the consistent information of pathological diagnosis, and has the potential to substantially improve the optical diagnosis and treatment of the pancreatic SPT without H&E staining in the future. SCANNING 38:245-250, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  15. Correlating two-photon excited fluorescence imaging of breast cancer cellular redox state with seahorse flux analysis of normalized cellular oxygen consumption

    Science.gov (United States)

    Hou, Jue; Wright, Heather J.; Chan, Nicole; Tran, Richard; Razorenova, Olga V.; Potma, Eric O.; Tromberg, Bruce J.

    2016-06-01

    Two-photon excited fluorescence (TPEF) imaging of the cellular cofactors nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide is widely used to measure cellular metabolism, both in normal and pathological cells and tissues. When dual-wavelength excitation is used, ratiometric TPEF imaging of the intrinsic cofactor fluorescence provides a metabolic index of cells-the "optical redox ratio" (ORR). With increased interest in understanding and controlling cellular metabolism in cancer, there is a need to evaluate the performance of ORR in malignant cells. We compare TPEF metabolic imaging with seahorse flux analysis of cellular oxygen consumption in two different breast cancer cell lines (MCF-7 and MDA-MB-231). We monitor metabolic index in living cells under both normal culture conditions and, for MCF-7, in response to cell respiration inhibitors and uncouplers. We observe a significant correlation between the TPEF-derived ORR and the flux analyzer measurements (R=0.7901, p<0.001). Our results confirm that the ORR is a valid dynamic index of cell metabolism under a range of oxygen consumption conditions relevant for cancer imaging.

  16. Opposite reactivity of meningeal versus cortical microvessels to the nitric oxide donor glyceryl trinitrate evaluated in vivo with two-photon imaging.

    Directory of Open Access Journals (Sweden)

    Evgeny Pryazhnikov

    Full Text Available Vascular changes underlying headache in migraine patients induced by Glyceryl trinitrate (GTN were previously studied with various imaging techniques. Despite the long history of medical and experimental use of GTN, its effects on the brain vasculature are still poorly understood presumably due to low spatial resolution of the imaging modalities used so far. We took advantage of the micrometer-scale vertical resolution of two-photon microscopy to differentiate between the vasodynamic effects of GTN on meningeal versus cortical vessels imaged simultaneously in anesthetized rats through either thinned skull or glass-sealed cranial window. Intermediate and small calibre vessels were visualized in vivo by imaging intravascular fluorescent dextran, and detection of blood flow direction allowed identification of individual arterioles and venules. We found that i.p.-injected GTN induced a transient constriction of meningeal arterioles, while their cortical counterparts were, in contrast, dilated. These opposing effects of GTN were restricted to arterioles, whereas the effects on venules were insignificant. Interestingly, the NO synthase inhibitor L-NAME did not affect the diameter of meningeal vessels but induced a constriction of cortical vessels. The different cellular environment in cortex versus meninges as well as distinct vessel wall anatomical features probably play crucial role in the observed phenomena. These findings highlight differential region- and vessel-type-specific effects of GTN on cranial vessels, and may implicate new vascular mechanisms of NO-mediated primary headaches.

  17. Towards vortex imaging with scanning tunneling microscope

    International Nuclear Information System (INIS)

    Fuchs, Dan T.

    1994-02-01

    A low temperature, Besocke beetle type scanning tunneling microscope, with a scan range of 10 by 10 microns was built. The scanning tunneling microscope was calibrates for various temperatures and tested on several samples. Gold monolayers evaporated at 400 deg C were resolved and their dynamic behavior observed. Atomic resolution images of graphite were obtained. The scanning tunneling microscope was designed for future applications of vortex imaging in superconductors. The special design considerations for this application are discussed and the physics underlying it reviewed. (author)

  18. Image processing for HTS SQUID probe microscope

    International Nuclear Information System (INIS)

    Hayashi, T.; Koetitz, R.; Itozaki, H.; Ishikawa, T.; Kawabe, U.

    2005-01-01

    An HTS SQUID probe microscope has been developed using a high-permeability needle to enable high spatial resolution measurement of samples in air even at room temperature. Image processing techniques have also been developed to improve the magnetic field images obtained from the microscope. Artifacts in the data occur due to electromagnetic interference from electric power lines, line drift and flux trapping. The electromagnetic interference could successfully be removed by eliminating the noise peaks from the power spectrum of fast Fourier transforms of line scans of the image. The drift between lines was removed by interpolating the mean field value of each scan line. Artifacts in line scans occurring due to flux trapping or unexpected noise were removed by the detection of a sharp drift and interpolation using the line data of neighboring lines. Highly detailed magnetic field images were obtained from the HTS SQUID probe microscope by the application of these image processing techniques

  19. Imaging differential polarization microscope with electronic readout

    International Nuclear Information System (INIS)

    Mickols, W.; Tinoco, I.; Katz, J.E.; Maestre, M.F.; Bustamante, C.

    1985-01-01

    A differential polarization microscope forms two images: one of the transmitted intensity and the other due to the change in intensity between images formed when different polarizations of light are used. The interpretation of these images for linear dichroism and circular dichroism are described. The design constraints on the data acquisition systems and the polarization modulation are described. The advantage of imaging several biological systems which contain optically anisotropic structures are described

  20. Multi-compartment microscopic diffusion imaging

    OpenAIRE

    Kaden, Enrico; Kelm, Nathaniel D.; Carson, Robert P.; Does, Mark D.; Alexander, Daniel C.

    2016-01-01

    This paper introduces a multi-compartment model for microscopic diffusion anisotropy imaging. The aim is to estimate microscopic features specific to the intra- and extra-neurite compartments in nervous tissue unconfounded by the effects of fibre crossings and orientation dispersion, which are ubiquitous in the brain. The proposed MRI method is based on the Spherical Mean Technique (SMT), which factors out the neurite orientation distribution and thus provides direct estimates of the microsco...

  1. Seamless stitching of tile scan microscope images.

    Science.gov (United States)

    Legesse, F B; Chernavskaia, O; Heuke, S; Bocklitz, T; Meyer, T; Popp, J; Heintzmann, R

    2015-06-01

    For diagnostic purposes, optical imaging techniques need to obtain high-resolution images of extended biological specimens in reasonable time. The field of view of an objective lens, however, is often smaller than the sample size. To image the whole sample, laser scanning microscopes acquire tile scans that are stitched into larger mosaics. The appearance of such image mosaics is affected by visible edge artefacts that arise from various optical aberrations which manifest in grey level jumps across tile boundaries. In this contribution, a technique for stitching tiles into a seamless mosaic is presented. The stitching algorithm operates by equilibrating neighbouring edges and forcing the brightness at corners to a common value. The corrected image mosaics appear to be free from stitching artefacts and are, therefore, suited for further image analysis procedures. The contribution presents a novel method to seamlessly stitch tiles captured by a laser scanning microscope into a large mosaic. The motivation for the work is the failure of currently existing methods for stitching nonlinear, multimodal images captured by our microscopic setups. Our method eliminates the visible edge artefacts that appear between neighbouring tiles by taking into account the overall illumination differences among tiles in such mosaics. The algorithm first corrects the nonuniform brightness that exists within each of the tiles. It then compensates for grey level differences across tile boundaries by equilibrating neighbouring edges and forcing the brightness at the corners to a common value. After these artefacts have been removed further image analysis procedures can be applied on the microscopic images. Even though the solution presented here is tailored for the aforementioned specific case, it could be easily adapted to other contexts where image tiles are assembled into mosaics such as in astronomical or satellite photos. © 2015 The Authors Journal of Microscopy © 2015 Royal

  2. Scanning laser microscope for imaging nanostructured superconductors

    International Nuclear Information System (INIS)

    Ishida, Takekazu; Arai, Kohei; Akita, Yukio; Miyanari, Mitsunori; Minami, Yusuke; Yotsuya, Tsutomu; Kato, Masaru; Satoh, Kazuo; Uno, Mayumi; Shimakage, Hisashi; Miki, Shigehito; Wang, Zhen

    2010-01-01

    The nanofabrication of superconductors yields various interesting features in superconducting properties. A variety of different imaging techniques have been developed for probing the local superconducting profiles. A scanning pulsed laser microscope has been developed by the combination of the XYZ piezo-driven stages and an optical fiber with an aspheric focusing lens. The scanning laser microscope is used to understand the position-dependent properties of a superconducting MgB 2 stripline of length 100 μm and width of 3 μm under constant bias current. Our results show that the superconducting stripline can clearly be seen in the contour image of the scanning laser microscope on the signal voltage. It is suggested from the observed image that the inhomogeneity is relevant in specifying the operating conditions such as detection efficiency of the sensor.

  3. Scanning laser microscope for imaging nanostructured superconductors

    Science.gov (United States)

    Ishida, Takekazu; Arai, Kohei; Akita, Yukio; Miyanari, Mitsunori; Minami, Yusuke; Yotsuya, Tsutomu; Kato, Masaru; Satoh, Kazuo; Uno, Mayumi; Shimakage, Hisashi; Miki, Shigehito; Wang, Zhen

    2010-10-01

    The nanofabrication of superconductors yields various interesting features in superconducting properties. A variety of different imaging techniques have been developed for probing the local superconducting profiles. A scanning pulsed laser microscope has been developed by the combination of the XYZ piezo-driven stages and an optical fiber with an aspheric focusing lens. The scanning laser microscope is used to understand the position-dependent properties of a superconducting MgB 2 stripline of length 100 μm and width of 3 μm under constant bias current. Our results show that the superconducting stripline can clearly be seen in the contour image of the scanning laser microscope on the signal voltage. It is suggested from the observed image that the inhomogeneity is relevant in specifying the operating conditions such as detection efficiency of the sensor.

  4. Three-dimensional cell organization leads to almost immediate HRE activity as demonstrated by molecular imaging of MG-63 spheroids using two-photon excitation microscopy.

    Science.gov (United States)

    Indovina, Paola; Collini, Maddalena; Chirico, Giuseppe; Santini, Maria Teresa

    2007-02-20

    Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stably transfected with a vector constructed with multiple copies of the HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1alpha (hypoxia-inducible factor-1alpha) is stabilized, the binding of HIF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfected monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities.

  5. Two-Photon Processor and SeNeCA: a freely available software package to process data from two-photon calcium imaging at speeds down to several milliseconds per frame

    Czech Academy of Sciences Publication Activity Database

    Tomek, J.; Novák, Ondřej; Syka, Josef

    2013-01-01

    Roč. 110, č. 1 (2013), s. 243-256 ISSN 0022-3077 R&D Projects: GA ČR(CZ) GAP303/12/1347; GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:68378041 Keywords : algorithm * segmentation * calcium imaging Subject RIV: FH - Neurology Impact factor: 3.041, year: 2013

  6. Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

    Science.gov (United States)

    Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

    2004-01-01

    Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

  7. IMIS: An intelligence microscope imaging system

    Science.gov (United States)

    Caputo, Michael; Hunter, Norwood; Taylor, Gerald

    1994-01-01

    Until recently microscope users in space relied on traditional microscopy techniques that required manual operation of the microscope and recording of observations in the form of written notes, drawings, or photographs. This method was time consuming and required the return of film and drawings from space for analysis. No real-time data analysis was possible. Advances in digital and video technologies along with recent developments in article intelligence will allow future space microscopists to have a choice of three additional modes of microscopy: remote coaching, remote control, and automation. Remote coaching requires manual operations of the microscope with instructions given by two-way audio/video transmission during critical phases of the experiment. When using the remote mode of microscopy, the Principal Investigator controls the microscope from the ground. The automated mode employs artificial intelligence to control microscope functions and is the only mode that can be operated in the other three modes as well. The purpose of this presentation is to discuss the advantages and disadvantages of the four modes of of microscopy and how the IMIS, a proposed intelligent microscope imaging system, can be used as a model for developing and testing concepts, operating procedures, and equipment design of specifications required to provide a comprehensive microscopy/imaging capability onboard Space Station Freedom.

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

    Directory of Open Access Journals (Sweden)

    Claudia Laperchia

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

  9. Quantitative Imaging with a Mobile Phone Microscope

    Science.gov (United States)

    Skandarajah, Arunan; Reber, Clay D.; Switz, Neil A.; Fletcher, Daniel A.

    2014-01-01

    Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone–based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications. PMID:24824072

  10. Quantitative imaging with a mobile phone microscope.

    Directory of Open Access Journals (Sweden)

    Arunan Skandarajah

    Full Text Available Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone-based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications.

  11. Preparation of a Two-Photon Fluorescent Probe for Imaging H2O2 in Lysosomes in Living Cells and Tissues.

    Science.gov (United States)

    Ren, Mingguang; Deng, Beibei; Kong, Xiuqi; Tang, Yonghe; Lin, Weiying

    2017-01-01

    Hydrogen peroxide (H 2 O 2 ) plays important roles in many physiological and pathological processes. At the cellular organelle level, the abnormal concentrations of H 2 O 2 in the lysosomes may cause redox imbalance and the loss of the critical functions of the lysosomes. Herein, we describe the preparation of a potent lysosome-targeted two-photon fluorescent probe (Lyso-HP) for the detection of H 2 O 2 in the lysosomes in the living cells. This unique fluorescent probe can also be employed to effectively detect H 2 O 2 in the living tissues using two-photon fluorescence microscopy.

  12. DHM (Digital Holography Microscope) for imaging cells

    International Nuclear Information System (INIS)

    Emery, Yves; Cuche, Etienne; Colomb, Tristan; Depeursinge, Christian; Rappaz, Benjamin; Marquet, Pierre; Magistretti, Pierre

    2007-01-01

    Light interaction with a sample modifies both intensity and phase of the illuminating wave. Any available supports for image recording are only sensitive to intensity, but Denis Gabor [P. Marquet, B. Rappaz, P. Magistretti, et. al. Digital Holography for quantitative phase-contrast imaging, Optics Letters, 30, 5, pp 291-93 (2005)] invented in 1948 a way to encode the phase as an intensity variation: the h ologram . Digital Holographic Microscopy (DHM) [D. Gabor, A new microscopic principle, Nature, 1948] implements digitally this powerful hologram. Characterization of various pollen grains and of morphology changes of neurones associated with hypotonic shock demonstrates the potential of DHM for imaging cells

  13. Improved Scanners for Microscopic Hyperspectral Imaging

    Science.gov (United States)

    Mao, Chengye

    2009-01-01

    Improved scanners to be incorporated into hyperspectral microscope-based imaging systems have been invented. Heretofore, in microscopic imaging, including spectral imaging, it has been customary to either move the specimen relative to the optical assembly that includes the microscope or else move the entire assembly relative to the specimen. It becomes extremely difficult to control such scanning when submicron translation increments are required, because the high magnification of the microscope enlarges all movements in the specimen image on the focal plane. To overcome this difficulty, in a system based on this invention, no attempt would be made to move either the specimen or the optical assembly. Instead, an objective lens would be moved within the assembly so as to cause translation of the image at the focal plane: the effect would be equivalent to scanning in the focal plane. The upper part of the figure depicts a generic proposed microscope-based hyperspectral imaging system incorporating the invention. The optical assembly of this system would include an objective lens (normally, a microscope objective lens) and a charge-coupled-device (CCD) camera. The objective lens would be mounted on a servomotor-driven translation stage, which would be capable of moving the lens in precisely controlled increments, relative to the camera, parallel to the focal-plane scan axis. The output of the CCD camera would be digitized and fed to a frame grabber in a computer. The computer would store the frame-grabber output for subsequent viewing and/or processing of images. The computer would contain a position-control interface board, through which it would control the servomotor. There are several versions of the invention. An essential feature common to all versions is that the stationary optical subassembly containing the camera would also contain a spatial window, at the focal plane of the objective lens, that would pass only a selected portion of the image. In one version

  14. Active Mask Segmentation of Fluorescence Microscope Images

    OpenAIRE

    Srinivasa, Gowri; Fickus, Matthew C.; Guo, Yusong; Linstedt, Adam D.; Kovačević, Jelena

    2009-01-01

    We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the “contour” to that of “inside and outside”, or, masks, allowing for easy mul...

  15. Atomic Force Microscope for Imaging and Spectroscopy

    Science.gov (United States)

    Pike, W. T.; Hecht, M. H.; Anderson, M. S.; Akiyama, T.; Gautsch, S.; deRooij, N. F.; Staufer, U.; Niedermann, Ph.; Howald, L.; Mueller, D.

    2000-01-01

    We have developed, built, and tested an atomic force microscope (AFM) for extraterrestrial applications incorporating a micromachined tip array to allow for probe replacement. It is part of a microscopy station originally intended for NASA's 2001 Mars lander to identify the size, distribution, and shape of Martian dust and soil particles. As well as imaging topographically down to nanometer resolution, this instrument can be used to reveal chemical information and perform infrared and Raman spectroscopy at unprecedented resolution.

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

    Science.gov (United States)

    Vinogradov, Sergei A.; Esipova, Tatiana V.

    2016-03-01

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

  17. Ultra-large field-of-view two-photon microscopy

    OpenAIRE

    Tsai, Philbert S.; Mateo, Celine; Field, Jeffrey J.; Schaffer, Chris B.; Anderson, Matthew E.; Kleinfeld, David

    2015-01-01

    We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain thro...

  18. Comparative study of image contrast in scanning electron microscope and helium ion microscope.

    Science.gov (United States)

    O'Connell, R; Chen, Y; Zhang, H; Zhou, Y; Fox, D; Maguire, P; Wang, J J; Rodenburg, C

    2017-12-01

    Images of Ga + -implanted amorphous silicon layers in a 110 n-type silicon substrate have been collected by a range of detectors in a scanning electron microscope and a helium ion microscope. The effects of the implantation dose and imaging parameters (beam energy, dwell time, etc.) on the image contrast were investigated. We demonstrate a similar relationship for both the helium ion microscope Everhart-Thornley and scanning electron microscope Inlens detectors between the contrast of the images and the Ga + density and imaging parameters. These results also show that dynamic charging effects have a significant impact on the quantification of the helium ion microscope and scanning electron microscope contrast. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  19. Active mask segmentation of fluorescence microscope images.

    Science.gov (United States)

    Srinivasa, Gowri; Fickus, Matthew C; Guo, Yusong; Linstedt, Adam D; Kovacević, Jelena

    2009-08-01

    We propose a new active mask algorithm for the segmentation of fluorescence microscope images of punctate patterns. It combines the (a) flexibility offered by active-contour methods, (b) speed offered by multiresolution methods, (c) smoothing offered by multiscale methods, and (d) statistical modeling offered by region-growing methods into a fast and accurate segmentation tool. The framework moves from the idea of the "contour" to that of "inside and outside," or masks, allowing for easy multidimensional segmentation. It adapts to the topology of the image through the use of multiple masks. The algorithm is almost invariant under initialization, allowing for random initialization, and uses a few easily tunable parameters. Experiments show that the active mask algorithm matches the ground truth well and outperforms the algorithm widely used in fluorescence microscopy, seeded watershed, both qualitatively, as well as quantitatively.

  20. The Digital Microscope and Its Image Processing Utility

    Directory of Open Access Journals (Sweden)

    Tri Wahyu Supardi

    2011-12-01

    Full Text Available Many institutions, including high schools, own a large number of analog or ordinary microscopes. These microscopes are used to observe small objects. Unfortunately, object observations on the ordinary microscope require precision and visual acuity of the user. This paper discusses the development of a high-resolution digital microscope from an analog microscope, including the image processing utility, which allows the digital microscope users to capture, store and process the digital images of the object being observed. The proposed microscope is constructed from hardware components that can be easily found in Indonesia. The image processing software is capable of performing brightness adjustment, contrast enhancement, histogram equalization, scaling and cropping. The proposed digital microscope has a maximum magnification of 1600x, and image resolution can be varied from 320x240 pixels up to 2592x1944 pixels. The microscope was tested with various objects with a variety of magnification, and image processing was carried out on the image of the object. The results showed that the digital microscope and its image processing system were capable of enhancing the observed object and other operations in accordance with the user need. The digital microscope has eliminated the need for direct observation by human eye as with the traditional microscope.

  1. Performance evaluation of image segmentation algorithms on microscopic image data

    Czech Academy of Sciences Publication Activity Database

    Beneš, Miroslav; Zitová, Barbara

    2015-01-01

    Roč. 275, č. 1 (2015), s. 65-85 ISSN 0022-2720 R&D Projects: GA ČR GAP103/12/2211 Institutional support: RVO:67985556 Keywords : image segmentation * performance evaluation * microscopic images Subject RIV: JC - Computer Hardware ; Software Impact factor: 2.136, year: 2015 http://library.utia.cas.cz/separaty/2014/ZOI/zitova-0434809-DOI.pdf

  2. Coherence revivals in two-photon frequency combs

    International Nuclear Information System (INIS)

    Torres-Company, Victor; Lancis, Jesus; Lajunen, Hanna; Friberg, Ari T.

    2011-01-01

    We describe and theoretically analyze the self-imaging Talbot effect of entangled photon pairs in the time domain. Rich phenomena are observed in coherence propagation along dispersive media of mode-locked two-photon states with frequency entanglement exhibiting a comblike correlation function. Our results can be used to remotely transfer frequency standards through optical fiber networks with two-photon light, avoiding the requirement of dispersion compensation.

  3. Quantitative methods for the analysis of electron microscope images

    DEFF Research Database (Denmark)

    Skands, Peter Ulrik Vallø

    1996-01-01

    The topic of this thesis is an general introduction to quantitative methods for the analysis of digital microscope images. The images presented are primarily been acquired from Scanning Electron Microscopes (SEM) and interfermeter microscopes (IFM). The topic is approached though several examples...... foundation of the thesis fall in the areas of: 1) Mathematical Morphology; 2) Distance transforms and applications; and 3) Fractal geometry. Image analysis opens in general the possibility of a quantitative and statistical well founded measurement of digital microscope images. Herein lies also the conditions...

  4. Uniform silica nanoparticles encapsulating two-photon absorbing fluorescent dye

    International Nuclear Information System (INIS)

    Wu Weibing; Liu Chang; Wang Mingliang; Huang Wei; Zhou Shengrui; Jiang Wei; Sun Yueming; Cui Yiping; Xu Chunxinag

    2009-01-01

    We have prepared uniform silica nanoparticles (NPs) doped with a two-photon absorbing zwitterionic hemicyanine dye by reverse microemulsion method. Obvious solvatochromism on the absorption spectra of dye-doped NPs indicates that solvents can partly penetrate into the silica matrix and then affect the ground and excited state of dye molecules. For dye-doped NP suspensions, both one-photon and two-photon excited fluorescence are much stronger and recorded at shorter wavelength compared to those of free dye solutions with comparative overall dye concentration. This behavior is possibly attributed to the restricted twisted intramolecular charge transfer (TICT), which reduces fluorescence quenching when dye molecules are trapped in the silica matrix. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells with low cytotoxicity. - Graphical abstract: Water-soluble silica NPs doped with a two-photon absorbing zwitterionic hemicyanine dye were prepared. They were found of enhanced one-photon and two-photon excited fluorescence compared to free dye solutions. Images from two-photon laser scanning fluorescence microscopy demonstrate that the dye-doped silica NPs can be actively uptaken by Hela cells.

  5. Adaptive optical microscope for brain imaging in vivo

    Science.gov (United States)

    Wang, Kai

    2017-04-01

    The optical heterogeneity of biological tissue imposes a major limitation to acquire detailed structural and functional information deep in the biological specimens using conventional microscopes. To restore optimal imaging performance, we developed an adaptive optical microscope based on direct wavefront sensing technique. This microscope can reliably measure and correct biological samples induced aberration. We demonstrated its performance and application in structural and functional brain imaging in various animal models, including fruit fly, zebrafish and mouse.

  6. Cluster secondary ion mass spectrometry microscope mode mass spectrometry imaging.

    Science.gov (United States)

    Kiss, András; Smith, Donald F; Jungmann, Julia H; Heeren, Ron M A

    2013-12-30

    Microscope mode imaging for secondary ion mass spectrometry is a technique with the promise of simultaneous high spatial resolution and high-speed imaging of biomolecules from complex surfaces. Technological developments such as new position-sensitive detectors, in combination with polyatomic primary ion sources, are required to exploit the full potential of microscope mode mass spectrometry imaging, i.e. to efficiently push the limits of ultra-high spatial resolution, sample throughput and sensitivity. In this work, a C60 primary source was combined with a commercial mass microscope for microscope mode secondary ion mass spectrometry imaging. The detector setup is a pixelated detector from the Medipix/Timepix family with high-voltage post-acceleration capabilities. The system's mass spectral and imaging performance is tested with various benchmark samples and thin tissue sections. The high secondary ion yield (with respect to 'traditional' monatomic primary ion sources) of the C60 primary ion source and the increased sensitivity of the high voltage detector setup improve microscope mode secondary ion mass spectrometry imaging. The analysis time and the signal-to-noise ratio are improved compared with other microscope mode imaging systems, all at high spatial resolution. We have demonstrated the unique capabilities of a C60 ion microscope with a Timepix detector for high spatial resolution microscope mode secondary ion mass spectrometry imaging. Copyright © 2013 John Wiley & Sons, Ltd.

  7. Remote Histology Learning from Static versus Dynamic Microscopic Images

    Science.gov (United States)

    Mione, Sylvia; Valcke, Martin; Cornelissen, Maria

    2016-01-01

    Histology is the study of microscopic structures in normal tissue sections. Curriculum redesign in medicine has led to a decrease in the use of optical microscopes during practical classes. Other imaging solutions have been implemented to facilitate remote learning. With advancements in imaging technologies, learning material can now be digitized.…

  8. A method for fast automated microscope image stitching.

    Science.gov (United States)

    Yang, Fan; Deng, Zhen-Sheng; Fan, Qiu-Hong

    2013-05-01

    Image stitching is an important technology to produce a panorama or larger image by combining several images with overlapped areas. In many biomedical researches, image stitching is highly desirable to acquire a panoramic image which represents large areas of certain structures or whole sections, while retaining microscopic resolution. In this study, we develop a fast normal light microscope image stitching algorithm based on feature extraction. At first, an algorithm of scale-space reconstruction of speeded-up robust features (SURF) was proposed to extract features from the images to be stitched with a short time and higher repeatability. Then, the histogram equalization (HE) method was employed to preprocess the images to enhance their contrast for extracting more features. Thirdly, the rough overlapping zones of the images preprocessed were calculated by phase correlation, and the improved SURF was used to extract the image features in the rough overlapping areas. Fourthly, the features were corresponded by matching algorithm and the transformation parameters were estimated, then the images were blended seamlessly. Finally, this procedure was applied to stitch normal light microscope images to verify its validity. Our experimental results demonstrate that the improved SURF algorithm is very robust to viewpoint, illumination, blur, rotation and zoom of the images and our method is able to stitch microscope images automatically with high precision and high speed. Also, the method proposed in this paper is applicable to registration and stitching of common images as well as stitching the microscope images in the field of virtual microscope for the purpose of observing, exchanging, saving, and establishing a database of microscope images. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Holographic Two-Photon Induced Photopolymerization

    Data.gov (United States)

    Federal Laboratory Consortium — Holographic two-photon-induced photopolymerization (HTPIP) offers distinct advantages over conventional one-photon-induced photopolymerization and current techniques...

  10. Advances and challenges in label-free nonlinear optical imaging using two-photon excitation fluorescence and second harmonic generation for cancer research

    NARCIS (Netherlands)

    Thomas, Giju; van Voskuilen, Johan; Gerritsen, Hans C.; Sterenborg, H. J. C. M.

    2014-01-01

    Nonlinear optical imaging (NLOI) has emerged to be a promising tool for bio-medical imaging in recent times. Among the various applications of NLOI, its utility is the most significant in the field of pre-clinical and clinical cancer research. This review begins by briefly covering the core

  11. Early skin tumor detection from microscopic images through image processing

    International Nuclear Information System (INIS)

    Siddiqi, A.A.; Narejo, G.B.; Khan, A.M.

    2017-01-01

    The research is done to provide appropriate detection technique for skin tumor detection. The work is done by using the image processing toolbox of MATLAB. Skin tumors are unwanted skin growth with different causes and varying extent of malignant cells. It is a syndrome in which skin cells mislay the ability to divide and grow normally. Early detection of tumor is the most important factor affecting the endurance of a patient. Studying the pattern of the skin cells is the fundamental problem in medical image analysis. The study of skin tumor has been of great interest to the researchers. DIP (Digital Image Processing) allows the use of much more complex algorithms for image processing, and hence, can offer both more sophisticated performance at simple task, and the implementation of methods which would be impossibly by analog means. It allows much wider range of algorithms to be applied to the input data and can avoid problems such as build up of noise and signal distortion during processing. The study shows that few works has been done on cellular scale for the images of skin. This research allows few checks for the early detection of skin tumor using microscopic images after testing and observing various algorithms. After analytical evaluation the result has been observed that the proposed checks are time efficient techniques and appropriate for the tumor detection. The algorithm applied provides promising results in lesser time with accuracy. The GUI (Graphical User Interface) that is generated for the algorithm makes the system user friendly. (author)

  12. The optics of microscope image formation.

    Science.gov (United States)

    Wolf, David E

    2013-01-01

    Although geometric optics gives a good understanding of how the microscope works, it fails in one critical area, which is explaining the origin of microscope resolution. To accomplish this, one must consider the microscope from the viewpoint of physical optics. This chapter describes the theory of the microscope-relating resolution to the highest spatial frequency that a microscope can collect. The chapter illustrates how Huygens' principle or construction can be used to explain the propagation of a plane wave. It is shown that this limit increases with increasing numerical aperture (NA). As a corollary to this, resolution increases with decreasing wavelength because of how NA depends on wavelength. The resolution is higher for blue light than red light. Resolution is dependent on contrast, and the higher the contrast, the higher the resolution. This last point relates to issues of signal-to-noise and dynamic range. The use of video and new digital cameras has necessitated redefining classical limits such as those of Rayleigh's criterion. Copyright © 2007 Elsevier Inc. All rights reserved.

  13. Quarkonium two-photon decays in QCD

    International Nuclear Information System (INIS)

    Dulyan, L.S.; Khodjamirian, A.Yu.; Magakian, A.D.

    1989-01-01

    The two-photon decay of tensor charmonium χ c2 → 2 γ is calculated with account of gluon condensate effects. The result is in good agreement with experiment. The two-photon width of pseudoscalar b-quarkonium η b → 2 γ is estimated. 19 refs.; 1 fig.; 1 tab

  14. Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and "Big Data" Analysis.

    Science.gov (United States)

    Savtchouk, Iaroslav; Carriero, Giovanni; Volterra, Andrea

    2018-01-01

    Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca 2+ imaging experiments in neurons, they are less useful for analyzing Ca 2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca 2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.

  15. Scanless two-photon excitation of channelrhodopsin-2

    DEFF Research Database (Denmark)

    Papagiakoumou, E.; Anselmi, F.; Bègue, A.

    2010-01-01

    developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups...... of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2–expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and...

  16. Stitching Grid-wise Atomic Force Microscope Images

    DEFF Research Database (Denmark)

    Vestergaard, Mathias Zacho; Bengtson, Stefan Hein; Pedersen, Malte

    2016-01-01

    Atomic Force Microscopes (AFM) are able to capture images with a resolution in the nano metre scale. Due to this high resolution, the covered area per image is relatively small, which can be problematic when surveying a sample. A system able to stitch AFM images has been developed to solve this p...

  17. Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.

    Science.gov (United States)

    Hayashi, Shinichi; Okada, Yasushi

    2015-05-01

    Most current superresolution (SR) microscope techniques surpass the diffraction limit at the expense of temporal resolution, compromising their applications to live-cell imaging. Here we describe a new SR fluorescence microscope based on confocal microscope optics, which we name the spinning disk superresolution microscope (SDSRM). Theoretically, the SDSRM is equivalent to a structured illumination microscope (SIM) and achieves a spatial resolution of 120 nm, double that of the diffraction limit of wide-field fluorescence microscopy. However, the SDSRM is 10 times faster than a conventional SIM because SR signals are recovered by optical demodulation through the stripe pattern of the disk. Therefore a single SR image requires only a single averaged image through the rotating disk. On the basis of this theory, we modified a commercial spinning disk confocal microscope. The improved resolution around 120 nm was confirmed with biological samples. The rapid dynamics of micro-tubules, mitochondria, lysosomes, and endosomes were observed with temporal resolutions of 30-100 frames/s. Because our method requires only small optical modifications, it will enable an easy upgrade from an existing spinning disk confocal to a SR microscope for live-cell imaging. © 2015 Hayashi and Okada. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Characteristics of different frequency ranges in scanning electron microscope images

    International Nuclear Information System (INIS)

    Sim, K. S.; Nia, M. E.; Tan, T. L.; Tso, C. P.; Ee, C. S.

    2015-01-01

    We demonstrate a new approach to characterize the frequency range in general scanning electron microscope (SEM) images. First, pure frequency images are generated from low frequency to high frequency, and then, the magnification of each type of frequency image is implemented. By comparing the edge percentage of the SEM image to the self-generated frequency images, we can define the frequency ranges of the SEM images. Characterization of frequency ranges of SEM images benefits further processing and analysis of those SEM images, such as in noise filtering and contrast enhancement

  19. Characteristics of different frequency ranges in scanning electron microscope images

    Energy Technology Data Exchange (ETDEWEB)

    Sim, K. S., E-mail: kssim@mmu.edu.my; Nia, M. E.; Tan, T. L.; Tso, C. P.; Ee, C. S. [Faculty of Engineering and Technology, Multimedia University, 75450 Melaka (Malaysia)

    2015-07-22

    We demonstrate a new approach to characterize the frequency range in general scanning electron microscope (SEM) images. First, pure frequency images are generated from low frequency to high frequency, and then, the magnification of each type of frequency image is implemented. By comparing the edge percentage of the SEM image to the self-generated frequency images, we can define the frequency ranges of the SEM images. Characterization of frequency ranges of SEM images benefits further processing and analysis of those SEM images, such as in noise filtering and contrast enhancement.

  20. Microscopic imaging through turbid media Monte Carlo modeling and applications

    CERN Document Server

    Gu, Min; Deng, Xiaoyuan

    2015-01-01

    This book provides a systematic introduction to the principles of microscopic imaging through tissue-like turbid media in terms of Monte-Carlo simulation. It describes various gating mechanisms based on the physical differences between the unscattered and scattered photons and method for microscopic image reconstruction, using the concept of the effective point spread function. Imaging an object embedded in a turbid medium is a challenging problem in physics as well as in biophotonics. A turbid medium surrounding an object under inspection causes multiple scattering, which degrades the contrast, resolution and signal-to-noise ratio. Biological tissues are typically turbid media. Microscopic imaging through a tissue-like turbid medium can provide higher resolution than transillumination imaging in which no objective is used. This book serves as a valuable reference for engineers and scientists working on microscopy of tissue turbid media.

  1. Charm production in two-photon collisions

    International Nuclear Information System (INIS)

    Linde, F.L.

    1988-01-01

    The analysis focuses on the production of charmonium resonances η c , χ 0 and χ 2 in two-photon interactions. The measurement of the inclusive production of charged D* mesons is described. 97 refs.; 54 figs.; 15 tabs

  2. Exclusive hadron production in two photon reactions

    International Nuclear Information System (INIS)

    Poppe, M.

    1986-02-01

    This paper summarises experimental results on exclusive hadron production in two photon collisions at electron positron storage rings and attempts some interpretation. Experimental know how is described and new suggestions are made for future analyses. New model calculations on resonance form factors and pair production amplitudes are presented. The two photon vertex is decomposed such that experiments can be parameterised with the minimal number of free parameters. Selection rules for off shell photon collisions are given in addition to Yang's theorems. (orig.)

  3. Stimulated emission depletion following two photon excitation

    OpenAIRE

    Marsh, R. J.; Armoogum, D. A.; Bain, A. J.

    2002-01-01

    The technique of stimulated emission depletion of fluorescence (STED) from a two photon excited molecular population is demonstrated in the S, excited state of fluorescein in ethylene glycol and methanol. Two photon excitation (pump) is achieved using the partial output of a regeneratively amplified Ti:Sapphire laser in conjunction with an optical parametric amplifier whose tuneable output provides a synchronous depletion (dump) pulse. Time resolved fluorescence intensity and anisotropy measu...

  4. Biomedical image retrieval using microscopic configuration with ...

    Indian Academy of Sciences (India)

    G DEEP

    2018-03-10

    Mar 10, 2018 ... The selection of feature descriptors affects the image .... Example of obtaining LBP for 3 9 3 neighbourhoods (adopted from Ojala et al [9]). 20 Page 2 of 13 ...... Directional binary wavelet patterns for biomedical image indexing ...

  5. A New Multichannel Spectral Imaging Laser Scanning Confocal Microscope

    Directory of Open Access Journals (Sweden)

    Yunhai Zhang

    2013-01-01

    Full Text Available We have developed a new multichannel spectral imaging laser scanning confocal microscope for effective detection of multiple fluorescent labeling in the research of biological tissues. In this paper, the design and key technologies of the system are introduced. Representative results on confocal imaging, 3-dimensional sectioning imaging, and spectral imaging are demonstrated. The results indicated that the system is applicable to multiple fluorescent labeling in biological experiments.

  6. Electrically Induced Two-Photon Transparency in Semiconductor Quantum Wells

    International Nuclear Information System (INIS)

    Hayat, Alex; Nevet, Amir; Orenstein, Meir

    2009-01-01

    We demonstrate experimentally two-photon transparency, achieved by current injection into a semiconductor quantum-well structure which exhibits two-photon emission. The two-photon induced luminescence is progressively reduced by the injected current, reaching the point of two-photon transparency - a necessary condition for semiconductor two-photon gain and lasing. These results agree with our calculations.

  7. In vivo cellular imaging with microscopes enabled by MEMS scanners

    Science.gov (United States)

    Ra, Hyejun

    High-resolution optical imaging plays an important role in medical diagnosis and biomedical research. Confocal microscopy is a widely used imaging method for obtaining cellular and sub-cellular images of biological tissue in reflectance and fluorescence modes. Its characteristic optical sectioning capability also enables three-dimensional (3-D) image reconstruction. However, its use has mostly been limited to excised tissues due to the requirement of high numerical aperture (NA) lenses for cellular resolution. Microscope miniaturization can enable in vivo imaging to make possible early cancer diagnosis and biological studies in the innate environment. In this dissertation, microscope miniaturization for in vivo cellular imaging is presented. The dual-axes confocal (DAC) architecture overcomes limitations of the conventional single-axis confocal (SAC) architecture to allow for miniaturization with high resolution. A microelectromechanical systems (MEMS) scanner is the central imaging component that is key in miniaturization of the DAC architecture. The design, fabrication, and characterization of the two-dimensional (2-D) MEMS scanner are presented. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer and is actuated by self-aligned vertical electrostatic combdrives. The imaging performance of the MEMS scanner in a DAC configuration is shown in a breadboard microscope setup, where reflectance and fluorescence imaging is demonstrated. Then, the MEMS scanner is integrated into a miniature DAC microscope. The whole imaging system is integrated into a portable unit for research in small animal models of human biology and disease. In vivo 3-D imaging is demonstrated on mouse skin models showing gene transfer and siRNA silencing. The siRNA silencing process is sequentially imaged in one mouse over time.

  8. Single-cell magnetic imaging using a quantum diamond microscope.

    Science.gov (United States)

    Glenn, D R; Lee, K; Park, H; Weissleder, R; Yacoby, A; Lukin, M D; Lee, H; Walsworth, R L; Connolly, C B

    2015-08-01

    We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

  9. Laser speckle contrast imaging using light field microscope approach

    Science.gov (United States)

    Ma, Xiaohui; Wang, Anting; Ma, Fenghua; Wang, Zi; Ming, Hai

    2018-01-01

    In this paper, a laser speckle contrast imaging (LSCI) system using light field (LF) microscope approach is proposed. As far as we known, it is first time to combine LSCI with LF. To verify this idea, a prototype consists of a modified LF microscope imaging system and an experimental device was built. A commercially used Lytro camera was modified for microscope imaging. Hollow glass tubes with different depth fixed in glass dish were used to simulate the vessels in brain and test the performance of the system. Compared with conventional LSCI, three new functions can be realized by using our system, which include refocusing, extending the depth of field (DOF) and gathering 3D information. Experiments show that the principle is feasible and the proposed system works well.

  10. 3D widefield light microscope image reconstruction without dyes

    Science.gov (United States)

    Larkin, S.; Larson, J.; Holmes, C.; Vaicik, M.; Turturro, M.; Jurkevich, A.; Sinha, S.; Ezashi, T.; Papavasiliou, G.; Brey, E.; Holmes, T.

    2015-03-01

    3D image reconstruction using light microscope modalities without exogenous contrast agents is proposed and investigated as an approach to produce 3D images of biological samples for live imaging applications. Multimodality and multispectral imaging, used in concert with this 3D optical sectioning approach is also proposed as a way to further produce contrast that could be specific to components in the sample. The methods avoid usage of contrast agents. Contrast agents, such as fluorescent or absorbing dyes, can be toxic to cells or alter cell behavior. Current modes of producing 3D image sets from a light microscope, such as 3D deconvolution algorithms and confocal microscopy generally require contrast agents. Zernike phase contrast (ZPC), transmitted light brightfield (TLB), darkfield microscopy and others can produce contrast without dyes. Some of these modalities have not previously benefitted from 3D image reconstruction algorithms, however. The 3D image reconstruction algorithm is based on an underlying physical model of scattering potential, expressed as the sample's 3D absorption and phase quantities. The algorithm is based upon optimizing an objective function - the I-divergence - while solving for the 3D absorption and phase quantities. Unlike typical deconvolution algorithms, each microscope modality, such as ZPC or TLB, produces two output image sets instead of one. Contrast in the displayed image and 3D renderings is further enabled by treating the multispectral/multimodal data as a feature set in a mathematical formulation that uses the principal component method of statistics.

  11. Mass spectrometry imaging: Towards a lipid microscope?

    Science.gov (United States)

    Touboul, David; Brunelle, Alain; Laprévote, Olivier

    2011-01-01

    Biological imaging techniques are the most efficient way to locally measure the variation of different parameters on tissue sections. These analyses are gaining increasing interest since 20 years and allow observing extremely complex biological phenomena at lower and lower time and resolution scale. Nevertheless, most of them only target very few compounds of interest, which are chosen a priori, due to their low resolution power and sensitivity. New chemical imaging technique has to be introduced in order to overcome these limitations, leading to more informative and sensitive analyses for biologists and physicians. Two major mass spectrometry methods can be efficiently used to generate the distribution of biological compounds over a tissue section. Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) needs the co-crystallization of the sample with a matrix before to be irradiated by a laser, whereas the analyte is directly desorbed by a primary ion bombardment for Secondary Ion Mass Spectrometry (SIMS) experiments. In both cases, energy used for desorption/ionization is locally deposited -some tens of microns for the laser and some hundreds of nanometers for the ion beam- meaning that small areas over the surface sample can be separately analyzed. Step by step analysis allows spectrum acquisitions over the tissue sections and the data are treated by modern informatics software in order to create ion density maps, i.e., the intensity plot of one specific ion versus the (x,y) position. Main advantages of SIMS and MALDI compared to other chemical imaging techniques lie in the simultaneous acquisition of a large number of biological compounds in mixture with an excellent sensitivity obtained by Time-of-Flight (ToF) mass analyzer. Moreover, data treatment is done a posteriori, due to the fact that no compound is selectively marked, and let us access to the localization of different lipid classes in only one complete acquisition. Copyright © 2010

  12. Two-photon superradiance in extended medium

    International Nuclear Information System (INIS)

    Branzan, V.; Enache, N.

    1993-01-01

    The possibility of collectivization of an ensemble of atoms of an extended system (the distance between atoms is larger or equal to the wave-length of a spontaneous emitted radiation) during two-photon spontaneous decay is theoretically investigated. It is demonstrated that such systems of inverted atoms should emit phase-correlated pairs of photons. The time-space correlation among atoms is realized due to the two-photon exchanging through the electromagnetic field's vacuum. An increase of the spontaneous decay rate of the two-atom inverted ensemble is demonstrated. The dependence of two-photon superradiance on the sample geometry is investigated. A non-equilibrium method of the elimination of the atoms level Fermi-operators is proposed. (Author)

  13. Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

    Science.gov (United States)

    Wang, Xuhua; Yao, Sheng; Ahn, Hyo-Yang; Zhang, Yuanwei; Bondar, Mykhailo V.; Torres, Joseph A.; Belfield, Kevin D.

    2010-01-01

    Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated. PMID:21258480

  14. Sensitive and rapid detection of endogenous hydrogen sulfide distributing in different mouse viscera via a two-photon fluorescent probe

    International Nuclear Information System (INIS)

    Chen, Qian; Yang, Jinfeng; Li, Yinhui; Zheng, Jing; Yang, Ronghua

    2015-01-01

    Development of efficient methods for detection of endogenous H 2 S in living cells and tissues is of considerable significance for better understanding the biological and pathological functions of H 2 S. Two-photon (TP) fluorescent probes are favorable as powerful molecular tools for studying physiological process due to its non-invasiveness, high spatiotemporal resolution and deep-tissues imaging. Up to date, several TP probes for intracellular H 2 S imaging have been designed, but real-time imaging of endogenous H 2 S-related biological processes in tissues is hampered due to low sensitivity, long response time and interference from other biothiols. To address this issue, we herein report a novel two-photon fluorescent probe (TPP-H 2 S) for highly sensitive and fast monitoring and imaging H 2 S levels in living cells and tissues. In the presence of H 2 S, it exhibits obviously improved sensitivity (LOD: 0.12 μM) and fast response time (about 2 min) compared with the reported two-photon H 2 S probes. With two-photon excitation, TPP-H 2 S displays high signal-to-noise ratio and sensitivity even no interference in cell growth media. As further application, TPP-H 2 S is applied for fast imaging of H 2 S in living cells and different fresh tissues by two-photon confocal microscope. Most importantly we first measured the endogenous H 2 S level in different viscera by vivisection and found that the distribution of endogenous H 2 S mostly in brain, liver and lung. The excellent sensing properties of TPP-H 2 S make it a practically useful tool for further studying biological roles of H 2 S. - Highlights: • This two-photon probe exhibits an improved sensitivity and response time to H 2 S. • This probe shows excellent membrane permeability and fast visualization of H 2 S in living cells and tissues. • This probe is successfully applied to measure the endogenously produced H 2 S levels in different viscera of mouse.

  15. Squeezing via two-photon transitions

    Science.gov (United States)

    Savage, C. M.; Walls, D. F.

    1986-05-01

    The squeezing spectrum for a cavity field mode interacting with an ensemble of three-level 'Lambda-configuration' atoms by an effective two-photon transition is calculated. The advantage of the three-level Lambda system as a squeezing medium, that is, optical nonlinearity without atomic saturation, has recently been pointed out by Reid, Walls, and Dalton. Perfect squeezing is predicted at the turning points for dispersive optical bistability and good squeezing for a range of other cases. Three-level ladder atoms interacting by an effective two-photon transition are also shown to give perfect squeezing in the dispersive limit.

  16. Two-photon excitation of argon

    International Nuclear Information System (INIS)

    Pindzola, P.S.; Payne, M.C.

    1982-01-01

    The authors calculate two photon excitation parameters for various excited states of argon assuming the absorption of near resonance broad-bandwidth laser radiation. Results are given for the case of two photons absorbed for the same laser beam as well as the case of absorbing photons of different frequency from each of two laser beams. The authors use multiconfiguration Hartree-Fock wave functions to evaluate the second-order sums over matrix elements. Various experimental laser schemes are suggested for the efficient excitation and subsequent ionization of argon

  17. A review of two photon physics

    International Nuclear Information System (INIS)

    Cooper, S.

    1982-08-01

    This talk is intended as an introduction for those not yet expert in two-photon physics, especially those e + e - one-photon physicists who still think of two-photon events as background. I concentrate on the physics questions involved, especially emphasizing the areas where I feel progress can be made in the near future, and of necessity leaving most experimental details to be found in the references. After a quick survey of the field and a few words about kinematics, I discuss in detail two major fields: the photon structure function and resonance production. (orig.)

  18. Large scale serial two-photon microscopy to investigate local vascular changes in whole rodent brain models of Alzheimer's disease

    Science.gov (United States)

    Delafontaine-Martel, P.; Lefebvre, J.; Damseh, R.; Castonguay, A.; Tardif, P.; Lesage, F.

    2018-02-01

    In this study, an automated serial two-photon microscope was used to image a fluorescent gelatin filled rodent's brain in 3D. A method to compute vascular density using automatic segmentation was combined with coregistration techniques to build group-level vasculature metrics. By studying the medial prefrontal cortex and the hippocampal formation of 3 age groups (2, 4.5 and 8 months old), we compared vascular density for both WT and an Alzheimer model transgenic brain (APP/PS1). We observe a loss of vascular density caused by the ageing process and we propose further analysis to confirm our results.

  19. Chemical imaging of structured SAMs with a novel SFG microscope

    Science.gov (United States)

    Hoffmann, Dominik M. P.; Kuhnke, Klaus; Kern, Klaus

    2002-11-01

    We present a newly developed microscope for sum frequency generation (SFG) imaging of opaque and reflecting interfaces. The sample is viewed at an angle of 60° with respect to the surface normal in order to increase the collected SFG intensity. Our setup is designed to keep the whole field of view (FOV) in focus and to compensate for the distortion usually related to oblique imaging by means of a blazed grating. The separation of the SFG intensity and the reflected visible beam is accomplished by a suitable combination of spectral filters. The sum frequency microscope (SFM) is capable of in-situ chemically selective imaging by tuning the IR-beam to vibrational transitions of the respective molecules. The SFM is applied to imaging of structured self-assembled monolayers (SAM) of thiol molecules on a gold surface.

  20. Structure Identification in High-Resolution Transmission Electron Microscopic Images

    DEFF Research Database (Denmark)

    Vestergaard, Jacob Schack; Kling, Jens; Dahl, Anders Bjorholm

    2014-01-01

    A connection between microscopic structure and macroscopic properties is expected for almost all material systems. High-resolution transmission electron microscopy is a technique offering insight into the atomic structure, but the analysis of large image series can be time consuming. The present ...

  1. Scanning tunneling microscope for magneto-optical imaging

    NARCIS (Netherlands)

    Prins, M.W.J.; Groeneveld, R.H.M.; Abraham, D.L.; Schad, R.; Kempen, van H.; Kesteren, van H.W.

    1996-01-01

    Images of magnetic bits written in a Pt/Co multilayer are presented. Using photosensitive semiconducting tips in a scanning tunneling microscope the surface topography as well as the polarization-dependent optical transmission are measured. Magnetic contrast is achieved by detection of the Faraday

  2. The Reaction Microscope: Imaging and Pulse Shaping Control in Photodynamics

    NARCIS (Netherlands)

    Vredenborg, A.; Lehmann, C.S.; Irimia, D.; Roeterdink, W.; Janssen, M.H.M.

    2011-01-01

    Herein, we review the current capabilities and potential of advanced single-particle imaging techniques to study photodynamics in isolated molecules. These reaction microscopes are able to measure the full three-dimensional energy and angular distribution of (correlated) particles such as electrons

  3. Computer processing of microscopic images of bacteria : morphometry and fluorimetry

    NARCIS (Netherlands)

    Wilkinson, Michael H.F.; Jansen, Gijsbert J.; Waaij, Dirk van der

    1994-01-01

    Several techniques that use computer analysis of microscopic images have been developed to study the complicated microbial flora in the human intestine, including measuring the shape and fluorescence intensity of bacteria. These techniques allow rapid assessment of changes in the intestinal flora

  4. The Digital Image Processing And Quantitative Analysis In Microscopic Image Characterization

    International Nuclear Information System (INIS)

    Ardisasmita, M. Syamsa

    2000-01-01

    Many electron microscopes although have produced digital images, but not all of them are equipped with a supporting unit to process and analyse image data quantitatively. Generally the analysis of image has to be made visually and the measurement is realized manually. The development of mathematical method for geometric analysis and pattern recognition, allows automatic microscopic image analysis with computer. Image processing program can be used for image texture and structure periodic analysis by the application of Fourier transform. Because the development of composite materials. Fourier analysis in frequency domain become important for measure the crystallography orientation. The periodic structure analysis and crystal orientation are the key to understand many material properties like mechanical strength. stress, heat conductivity, resistance, capacitance and other material electric and magnetic properties. In this paper will be shown the application of digital image processing in microscopic image characterization and analysis in microscopic image

  5. Review of Two-Photon Interactions

    International Nuclear Information System (INIS)

    Urner, David

    2004-01-01

    Presented are recent results of two-photon interactions. Topics include photon structure functions, inclusive hadron production, differential cross sections derived from tagged γγ fusion events and results in exclusive hadron production, particularly the observations of the η'c

  6. Two-photon transitions to exciton polaritons

    International Nuclear Information System (INIS)

    Hassan, A.R.

    1979-08-01

    A semiclassical theory for the creation of excitonic polariton states by two-photon absorption, via an intermediate exciton state, is given. A band model has been introduced which gives the dominant contribution to this process. A numerical calculation is found to be in good agreement with a recent observation in CuCl. (author)

  7. Pion-pair production by two photons

    International Nuclear Information System (INIS)

    Terazawa, Hidezumi.

    1994-07-01

    The cross section for pion-pair production by two photons is calculated approximately by using the low energy theorem previously derived from partially-conserved-axial-vector-current hypothesis and current algebra, and found to agree very well with the experimental data recently obtained by the Mark II, TPC/Two-Gamma and CLEO Collaborations. (author)

  8. Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

    Science.gov (United States)

    Benninger, Richard K.P.; Piston, David W.

    2013-01-01

    Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. PMID:23728746

  9. High-resolution three-dimensional imaging of a depleted CMOS sensor using an edge Transient Current Technique based on the Two Photon Absorption process (TPA-eTCT)

    CERN Document Server

    García, Marcos Fernández; Echeverría, Richard Jaramillo; Moll, Michael; Santos, Raúl Montero; Moya, David; Pinto, Rogelio Palomo; Vila, Iván

    2016-01-01

    For the first time, the deep n-well (DNW) depletion space of a High Voltage CMOS sensor has been characterized using a Transient Current Technique based on the simultaneous absorption of two photons. This novel approach has allowed to resolve the DNW implant boundaries and therefore to accurately determine the real depleted volume and the effective doping concentration of the substrate. The unprecedented spatial resolution of this new method comes from the fact that measurable free carrier generation in two photon mode only occurs in a micrometric scale voxel around the focus of the beam. Real three-dimensional spatial resolution is achieved by scanning the beam focus within the sample.

  10. High-resolution three-dimensional imaging of a depleted CMOS sensor using an edge Transient Current Technique based on the Two Photon Absorption process (TPA-eTCT)

    Energy Technology Data Exchange (ETDEWEB)

    García, Marcos Fernández; Sánchez, Javier González; Echeverría, Richard Jaramillo [Instituto de Física de Cantabria (CSIC-UC), Avda. los Castros s/n, E-39005 Santander (Spain); Moll, Michael [CERN, Organisation europénne pour la recherche nucléaire, CH-1211 Genéve 23 (Switzerland); Santos, Raúl Montero [SGIker Laser Facility, UPV/EHU, Sarriena, s/n - 48940 Leioa-Bizkaia (Spain); Moya, David [Instituto de Física de Cantabria (CSIC-UC), Avda. los Castros s/n, E-39005 Santander (Spain); Pinto, Rogelio Palomo [Departamento de Ingeniería Electrónica, Escuela Superior de Ingenieros Universidad de Sevilla (Spain); Vila, Iván [Instituto de Física de Cantabria (CSIC-UC), Avda. los Castros s/n, E-39005 Santander (Spain)

    2017-02-11

    For the first time, the deep n-well (DNW) depletion space of a High Voltage CMOS sensor has been characterized using a Transient Current Technique based on the simultaneous absorption of two photons. This novel approach has allowed to resolve the DNW implant boundaries and therefore to accurately determine the real depleted volume and the effective doping concentration of the substrate. The unprecedented spatial resolution of this new method comes from the fact that measurable free carrier generation in two photon mode only occurs in a micrometric scale voxel around the focus of the beam. Real three-dimensional spatial resolution is achieved by scanning the beam focus within the sample.

  11. Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements

    DEFF Research Database (Denmark)

    Beutler, Martin; Heisterkamp, Ines M.; Piltz, Bastian

    2014-01-01

    by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen......Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular...... states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence...

  12. Can Two-Photon Interference be Considered the Interference of Two Photons?

    International Nuclear Information System (INIS)

    Pittman, T.B.; Strekalov, D.V.; Migdall, A.; Rubin, M.H.; Sergienko, A.V.; Shih, Y.H.

    1996-01-01

    We report on a open-quote open-quote postponed compensation close-quote close-quote experiment in which the observed two-photon entangled state interference cannot be pictured in terms of the overlap of the two individual photon wave packets of a parametric down-conversion pair on a beam splitter. In the sense of a quantum eraser, the distinguishability of the different two-photon Feynman amplitudes leading to a coincidence detection is removed by delaying the compensation until after the output of an unbalanced two-photon interferometer. copyright 1996 The American Physical Society

  13. Foucault imaging by using non-dedicated transmission electron microscope

    International Nuclear Information System (INIS)

    Taniguchi, Yoshifumi; Matsumoto, Hiroaki; Harada, Ken

    2012-01-01

    An electron optical system for observing Foucault images was constructed using a conventional transmission electron microscope without any special equipment for Lorentz microscopy. The objective lens was switched off and an electron beam was converged by a condenser optical system to the crossover on the selected area aperture plane. The selected area aperture was used as an objective aperture to select the deflected beam for Foucault mode, and the successive image-forming lenses were controlled for observation of the specimen images. The irradiation area on the specimen was controlled by selecting the appropriate diameter of the condenser aperture.

  14. Foucault imaging by using non-dedicated transmission electron microscope

    Science.gov (United States)

    Taniguchi, Yoshifumi; Matsumoto, Hiroaki; Harada, Ken

    2012-08-01

    An electron optical system for observing Foucault images was constructed using a conventional transmission electron microscope without any special equipment for Lorentz microscopy. The objective lens was switched off and an electron beam was converged by a condenser optical system to the crossover on the selected area aperture plane. The selected area aperture was used as an objective aperture to select the deflected beam for Foucault mode, and the successive image-forming lenses were controlled for observation of the specimen images. The irradiation area on the specimen was controlled by selecting the appropriate diameter of the condenser aperture.

  15. Foucault imaging by using non-dedicated transmission electron microscope

    Energy Technology Data Exchange (ETDEWEB)

    Taniguchi, Yoshifumi [Science and Medical Systems Business Group, Hitachi High-Technologies Corp., Ichige, Hitachinaka, Ibaraki 312-8504 (Japan); Matsumoto, Hiroaki [Corporate Manufacturing Strategy Group, Hitachi High-Technologies Corp., Ishikawa-cho, Hitachinaka, Ibaraki 312-1991 (Japan); Harada, Ken [Central Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395 (Japan)

    2012-08-27

    An electron optical system for observing Foucault images was constructed using a conventional transmission electron microscope without any special equipment for Lorentz microscopy. The objective lens was switched off and an electron beam was converged by a condenser optical system to the crossover on the selected area aperture plane. The selected area aperture was used as an objective aperture to select the deflected beam for Foucault mode, and the successive image-forming lenses were controlled for observation of the specimen images. The irradiation area on the specimen was controlled by selecting the appropriate diameter of the condenser aperture.

  16. Electric field effects in scanning tunneling microscope imaging

    DEFF Research Database (Denmark)

    Stokbro, Kurt; Quaade, Ulrich; Grey, Francois

    1998-01-01

    We present a high-voltage extension of the Tersoff-Hamann theory of scanning tunneling microscope (STM) images, which includes the effect of the electric field between the tip and the sample. The theoretical model is based on first-principles electronic structure calculations and has no adjustable...... parameters. We use the method to calculate theoretical STM images of the monohydrate Si(100)-H(2x1) surface with missing hydrogen defects at -2V and find an enhanced corrugation due to the electric field, in good agreement with experimental images....

  17. Assessing microscope image focus quality with deep learning.

    Science.gov (United States)

    Yang, Samuel J; Berndl, Marc; Michael Ando, D; Barch, Mariya; Narayanaswamy, Arunachalam; Christiansen, Eric; Hoyer, Stephan; Roat, Chris; Hung, Jane; Rueden, Curtis T; Shankar, Asim; Finkbeiner, Steven; Nelson, Philip

    2018-03-15

    Large image datasets acquired on automated microscopes typically have some fraction of low quality, out-of-focus images, despite the use of hardware autofocus systems. Identification of these images using automated image analysis with high accuracy is important for obtaining a clean, unbiased image dataset. Complicating this task is the fact that image focus quality is only well-defined in foreground regions of images, and as a result, most previous approaches only enable a computation of the relative difference in quality between two or more images, rather than an absolute measure of quality. We present a deep neural network model capable of predicting an absolute measure of image focus on a single image in isolation, without any user-specified parameters. The model operates at the image-patch level, and also outputs a measure of prediction certainty, enabling interpretable predictions. The model was trained on only 384 in-focus Hoechst (nuclei) stain images of U2OS cells, which were synthetically defocused to one of 11 absolute defocus levels during training. The trained model can generalize on previously unseen real Hoechst stain images, identifying the absolute image focus to within one defocus level (approximately 3 pixel blur diameter difference) with 95% accuracy. On a simpler binary in/out-of-focus classification task, the trained model outperforms previous approaches on both Hoechst and Phalloidin (actin) stain images (F-scores of 0.89 and 0.86, respectively over 0.84 and 0.83), despite only having been presented Hoechst stain images during training. Lastly, we observe qualitatively that the model generalizes to two additional stains, Hoechst and Tubulin, of an unseen cell type (Human MCF-7) acquired on a different instrument. Our deep neural network enables classification of out-of-focus microscope images with both higher accuracy and greater precision than previous approaches via interpretable patch-level focus and certainty predictions. The use of

  18. Sensing of Streptococcus mutans by microscopic imaging ellipsometry

    Science.gov (United States)

    Khaleel, Mai Ibrahim; Chen, Yu-Da; Chien, Ching-Hang; Chang, Yia-Chung

    2017-05-01

    Microscopic imaging ellipsometry is an optical technique that uses an objective and sensing procedure to measure the ellipsometric parameters Ψ and Δ in the form of microscopic maps. This technique is well known for being noninvasive and label-free. Therefore, it can be used to detect and characterize biological species without any impact. Microscopic imaging ellipsometry was used to measure the optical response of dried Streptococcus mutans cells on a glass substrate. The ellipsometric Ψ and Δ maps were obtained with the Optrel Multiskop system for specular reflection in the visible range (λ=450 to 750 nm). The Ψ and Δ images at 500, 600, and 700 nm were analyzed using three different theoretical models with single-bounce, two-bounce, and multibounce light paths to obtain the optical constants and height distribution. The obtained images of the optical constants show different aspects when comparing the single-bounce analysis with the two-bounce or multibounce analysis in detecting S. mutans samples. Furthermore, the height distributions estimated by two-bounce and multibounce analyses of S. mutans samples were in agreement with the thickness values measured by AFM, which implies that the two-bounce and multibounce analyses can provide information complementary to that obtained by a single-bounce light path.

  19. A new clustering algorithm for scanning electron microscope images

    Science.gov (United States)

    Yousef, Amr; Duraisamy, Prakash; Karim, Mohammad

    2016-04-01

    A scanning electron microscope (SEM) is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with the sample atoms, producing various signals that are collected by detectors. The gathered signals contain information about the sample's surface topography and composition. The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image. The most common configuration for an SEM produces a single value per pixel, with the results usually rendered as grayscale images. The captured images may be produced with insufficient brightness, anomalous contrast, jagged edges, and poor quality due to low signal-to-noise ratio, grained topography and poor surface details. The segmentation of the SEM images is a tackling problems in the presence of the previously mentioned distortions. In this paper, we are stressing on the clustering of these type of images. In that sense, we evaluate the performance of the well-known unsupervised clustering and classification techniques such as connectivity based clustering (hierarchical clustering), centroid-based clustering, distribution-based clustering and density-based clustering. Furthermore, we propose a new spatial fuzzy clustering technique that works efficiently on this type of images and compare its results against these regular techniques in terms of clustering validation metrics.

  20. Probing two-photon exchange with OLYMPUS

    International Nuclear Information System (INIS)

    Kohl, M.

    2014-01-01

    Two-photon exchange is believed to be responsible for the discrepancies in the proton electric to magnetic form factor ratio found with the Rosenbluth and polarization transfer methods. If this explanation is correct, one expects significant differences in the lepton-proton cross sections between positrons and electrons. The OLYMPUS experiment at DESY in Hamburg, Germany was designed to measure the ratio of unpolarized positron-proton and electron-proton elastic scattering cross sections over a wide kinematic range with high precision, in order to quantify the effect of two-photon exchange. The experiment used intense beams of electrons and positrons stored in the DORIS ring at 2.0 GeV interacting with an internal windowless hydrogen gas target. The current status of OLYMPUS will be discussed. (authors)

  1. Symmetry adaptation in two-photon spectroscopy

    International Nuclear Information System (INIS)

    Kibler, M.

    1991-11-01

    Symmetry adaptation techniques are applied to the determination of the intensity of two-photon transitions for transition ions in finite symmetry environments. The case of intra-configurational transitions are discussed with some details and some results on inter-configurational transitions are briefly reported. In particular, for intra-configurational transitions, a model is described which takes into account the following ingredients: (symmetry, second- plus third-order mechanisms, S-, L- and J-mixings). (author) 20 refs

  2. Two-photon spin generation and detection

    Energy Technology Data Exchange (ETDEWEB)

    Miah, M Idrish, E-mail: m.miah@griffith.edu.a [Nanoscale Science and Technology Centre, Griffith University, Nathan, Brisbane, QLD 4111 (Australia)

    2009-02-21

    A time- and polarization-resolved two-photon pump-probe investigation is performed in lightly doped GaAs. We generate spin-polarized electrons in bulk GaAs at various temperatures using right-circularly polarized two-photon excitation and detect them by probing the spin-dependent transmission of the sample. The spin polarization (P) of conduction band electrons, as measured using probe pulses with the same (right) and opposite (left) circular polarization, is measured in dependences of pump-probe delay ({Delta}t), lattice temperature (T{sub L}), doping density (n) as well as of the excess photon energy {Delta}E{sub 2{omega}}= {h_bar}2{omega} - E{sub g}, where E{sub g} is the band gap energy. P is found to be decayed with {Delta}t and enhanced with the decrease in T{sub L} or the increase in n. It is also found that P decreases with the increase in {Delta}E{sub 2{omega}}and depolarizes rapidly for {Delta}E{sub 2{omega}}> {Delta}E{sub SO}, where {Delta}E{sub SO} is the spin-orbit splitting energy. The results demonstrate that due to a much longer absorption depth highly polarized spins can be generated optically by two-photon pumping of bulk semiconductors.

  3. Two-photon spin generation and detection

    International Nuclear Information System (INIS)

    Miah, M Idrish

    2009-01-01

    A time- and polarization-resolved two-photon pump-probe investigation is performed in lightly doped GaAs. We generate spin-polarized electrons in bulk GaAs at various temperatures using right-circularly polarized two-photon excitation and detect them by probing the spin-dependent transmission of the sample. The spin polarization (P) of conduction band electrons, as measured using probe pulses with the same (right) and opposite (left) circular polarization, is measured in dependences of pump-probe delay (Δt), lattice temperature (T L ), doping density (n) as well as of the excess photon energy ΔE 2ω = ℎ2ω - E g , where E g is the band gap energy. P is found to be decayed with Δt and enhanced with the decrease in T L or the increase in n. It is also found that P decreases with the increase in ΔE 2ω and depolarizes rapidly for ΔE 2ω > ΔE SO , where ΔE SO is the spin-orbit splitting energy. The results demonstrate that due to a much longer absorption depth highly polarized spins can be generated optically by two-photon pumping of bulk semiconductors.

  4. Two-photon excited fluorescence emission from hemoglobin

    Science.gov (United States)

    Sun, Qiqi; Zeng, Yan; Zhang, Wei; Zheng, Wei; Luo, Yi; Qu, Jianan Y.

    2015-03-01

    Hemoglobin, one of the most important proteins in blood, is responsible for oxygen transportation in almost all vertebrates. Recently, we discovered two-photon excited hemoglobin fluorescence and achieved label-free microvascular imaging based on the hemoglobin fluorescence. However, the mechanism of its fluorescence emission still remains unknown. In this work, we studied the two-photon excited fluorescence properties of the hemoglobin subunits, heme/hemin (iron (II)/(III) protoporphyrin IX) and globin. We first studied the properties of heme and the similar spectral and temporal characteristics of heme and hemoglobin fluorescence provide strong evidence that heme is the fluorophore in hemoglobin. Then we studied the fluorescence properties of hemin, globin and methemoglobin, and found that the hemin may have the main effect on the methemoglobin fluorescence and that globin has tryptophan fluorescence like other proteins. Finally, since heme is a centrosymmetric molecule, that the Soret band fluorescence of heme and hemoglobin was not observed in the single photon process in the previous study may be due to the parity selection rule. The discovery of heme two-photon excited fluorescence may open a new window for heme biology research, since heme as a cofactor of hemoprotein has many functions, including chemical catalysis, electron transfer and diatomic gases transportation.

  5. Hard X-ray Microscopic Imaging Of Human Breast Tissues

    Science.gov (United States)

    Park, Sung H.; Kim, Hong T.; Kim, Jong K.; Jheon, Sang H.; Youn, Hwa S.

    2007-01-01

    X-ray microscopy with synchrotron radiation will be a useful tool for innovation of x-ray imaging in clinical and laboratory settings. It helps us observe detailed internal structure of material samples non-invasively in air. And, it also has the potential to solve some tough problems of conventional breast imaging if it could evaluate various conditions of breast tissue effectively. A new hard x-ray microscope with a spatial resolution better than 100 nm was installed at Pohang Light Source, a third generation synchrotron radiation facility in Pohang, Korea. The x-ray energy was set at 6.95 keV, and the x-ray beam was monochromatized by W/B4C monochromator. Condenser and objective zone plates were used as x-ray lenses. Zernike phase plate next to condenser zone plate was introduced for improved contrast imaging. The image of a sample was magnified 30 times by objective zone plate and 20 times by microscope objective, respectively. After additional 10 times digital magnification, the total magnifying power was up to 6000 times in the end. Phase contrast synchrotron images of 10-μm-thick female breast tissue of the normal, fibroadenoma, fibrocystic change and carcinoma cases were obtained. By phase contrast imaging, hard x-rays enable us to observe many structures of breast tissue without sample preparations such as staining or fixation.

  6. Hard X-ray Microscopic Imaging Of Human Breast Tissues

    International Nuclear Information System (INIS)

    Park, Sung H.; Kim, Hong T.; Kim, Jong K.; Jheon, Sang H.; Youn, Hwa S.

    2007-01-01

    X-ray microscopy with synchrotron radiation will be a useful tool for innovation of x-ray imaging in clinical and laboratory settings. It helps us observe detailed internal structure of material samples non-invasively in air. And, it also has the potential to solve some tough problems of conventional breast imaging if it could evaluate various conditions of breast tissue effectively. A new hard x-ray microscope with a spatial resolution better than 100 nm was installed at Pohang Light Source, a third generation synchrotron radiation facility in Pohang, Korea. The x-ray energy was set at 6.95 keV, and the x-ray beam was monochromatized by W/B4C monochromator. Condenser and objective zone plates were used as x-ray lenses. Zernike phase plate next to condenser zone plate was introduced for improved contrast imaging. The image of a sample was magnified 30 times by objective zone plate and 20 times by microscope objective, respectively. After additional 10 times digital magnification, the total magnifying power was up to 6000 times in the end. Phase contrast synchrotron images of 10-μm-thick female breast tissue of the normal, fibroadenoma, fibrocystic change and carcinoma cases were obtained. By phase contrast imaging, hard x-rays enable us to observe many structures of breast tissue without sample preparations such as staining or fixation

  7. Microscopic validation of whole mouse micro-metastatic tumor imaging agents using cryo-imaging and sliding organ image registration

    OpenAIRE

    Liu, Yiqiao; Zhou, Bo; Qutaish, Mohammed; Wilson, David L.

    2016-01-01

    We created a metastasis imaging, analysis platform consisting of software and multi-spectral cryo-imaging system suitable for evaluating emerging imaging agents targeting micro-metastatic tumor. We analyzed CREKA-Gd in MRI, followed by cryo-imaging which repeatedly sectioned and tiled microscope images of the tissue block face, providing anatomical bright field and molecular fluorescence, enabling 3D microscopic imaging of the entire mouse with single metastatic cell sensitivity. To register ...

  8. Morphometric Evaluation of Preeclamptic Placenta Using Light Microscopic Images

    Directory of Open Access Journals (Sweden)

    Rashmi Mukherjee

    2014-01-01

    Full Text Available Deficient trophoblast invasion and anomalies in placental development generally lead to preeclampsia (PE but the inter-relationship between placental function and morphology in PE still remains unknown. The aim of this study was to evaluate the morphometric features of placental villi and capillaries in preeclamptic and normal placentae. The study included light microscopic images of placental tissue sections of 40 preeclamptic and 35 normotensive pregnant women. Preprocessing and segmentation of these images were performed to characterize the villi and capillaries. Fisher’s linear discriminant analysis (FLDA, hierarchical cluster analysis (HCA, and principal component analysis (PCA were applied to identify the most significant placental (morphometric features from microscopic images. A total of 10 morphometric features were extracted, of which the villous parameters were significantly altered in PE. FLDA identified 5 highly significant morphometric features (>90% overall discrimination accuracy. Two large subclusters were clearly visible in HCA based dendrogram. PCA returned three most significant principal components cumulatively explaining 98.4% of the total variance based on these 5 significant features. Hence, quantitative microscopic evaluation revealed that placental morphometry plays an important role in characterizing PE, where the villous is the major component that is affected.

  9. A portable fluorescence microscopic imaging system for cholecystectomy

    Science.gov (United States)

    Ye, Jian; Yang, Chaoyu; Gan, Qi; Ma, Rong; Zhang, Zeshu; Chang, Shufang; Shao, Pengfei; Zhang, Shiwu; Liu, Chenhai; Xu, Ronald

    2016-03-01

    In this paper we proposed a portable fluorescence microscopic imaging system to prevent iatrogenic biliary injuries from occurring during cholecystectomy due to misidentification of the cystic structures. The system consisted of a light source module, a CMOS camera, a Raspberry Pi computer and a 5 inch HDMI LCD. Specifically, the light source module was composed of 690 nm and 850 nm LEDs, allowing the CMOS camera to simultaneously acquire both fluorescence and background images. The system was controlled by Raspberry Pi using Python programming with the OpenCV library under Linux. We chose Indocyanine green(ICG) as a fluorescent contrast agent and then tested fluorescence intensities of the ICG aqueous solution at different concentration levels by our fluorescence microscopic system compared with the commercial Xenogen IVIS system. The spatial resolution of the proposed fluorescence microscopic imaging system was measured by a 1951 USAF resolution target and the dynamic response was evaluated quantitatively with an automatic displacement platform. Finally, we verified the technical feasibility of the proposed system in mouse models of bile duct, performing both correct and incorrect gallbladder resection. Our experiments showed that the proposed system can provide clear visualization of the confluence between the cystic duct and common bile duct or common hepatic duct, suggesting that this is a potential method for guiding cholecystectomy. The proposed portable system only cost a total of $300, potentially promoting its use in resource-limited settings.

  10. Nanoscale X-Ray Microscopic Imaging of Mammalian Mineralized Tissue

    OpenAIRE

    Andrews, Joy C.; Almeida, Eduardo; van der Meulen, Marjolein C.H.; Alwood, Joshua S.; Lee, Chialing; Liu, Yijin; Chen, Jie; Meirer, Florian; Feser, Michael; Gelb, Jeff; Rudati, Juana; Tkachuk, Andrei; Yun, Wenbing; Pianetta, Piero

    2010-01-01

    A novel hard transmission X-ray microscope (TXM) at the Stanford Synchrotron Radiation Light-source operating from 5 to 15 keV X-ray energy with 14 to 30 µm2 field of view has been used for high-resolution (30–40 nm) imaging and density quantification of mineralized tissue. TXM is uniquely suited for imaging of internal cellular structures and networks in mammalian mineralized tissues using relatively thick (50 µm), untreated samples that preserve tissue micro- and nanostructure. To test this...

  11. Evaluating EUV mask pattern imaging with two EUV microscopes

    International Nuclear Information System (INIS)

    Goldberg, Kenneth A.; Takase, Kei; Naulleau, Patrick P.; Han, Hakseung; Barty, Anton; Kinoshita, Hiroo; Hamamoto, Kazuhiro

    2008-01-01

    Aerial image measurement plays a key role in the development of patterned reticles for each generation of lithography. Studying the field transmitted (reflected) from EUV masks provides detailed information about potential disruptions caused by mask defects, and the performance of defect repair strategies, without the complications of photoresist imaging. Furthermore, by measuring the continuously varying intensity distribution instead of a thresholded, binary resist image, aerial image measurement can be used as feedback to improve mask and lithography system modeling methods. Interest in EUV, at-wavelength, aerial image measurement lead to the creation of several research tools worldwide. These tools are used in advanced mask development work, and in the evaluation of the need for commercial at-wavelength inspection tools. They describe performance measurements of two such tools, inspecting the same EUV mask in a series of benchmarking tests that includes brightfield and darkfield patterns. One tool is the SEMATECH Berkeley Actinic Inspection Tool (AIT) operating on a bending magnet beamline at Lawrence Berkeley National Laboratory's Advanced Light Source. The AIT features an EUV Fresnel zoneplate microscope that emulates the numerical aperture of a 0.25-NA stepper, and projects the aerial image directly onto a CCD camera, with 700x magnification. The second tool is an EUV microscope (EUVM) operating at the NewSUBARU synchrotron in Hyogo, Japan. The NewSUBARU tool projects the aerial image using a reflective, 30x Schwarzschild objective lens, followed by a 10-200x x-ray zooming tube. The illumination conditions and the imaging etendue are different for the two tools. The benchmarking measurements were used to determine many imaging and performance properties of the tools, including resolution, modulation transfer function (MTF), aberration magnitude, aberration field-dependence (including focal-plane tilt), illumination uniformity, line-edge roughness, and flare

  12. Thermal diffusivity imaging with the thermal lens microscope.

    Science.gov (United States)

    Dada, Oluwatosin O; Feist, Peter E; Dovichi, Norman J

    2011-12-01

    A coaxial thermal lens microscope was used to generate images based on both the absorbance and thermal diffusivity of histological samples. A pump beam was modulated at frequencies ranging from 50 kHz to 5 MHz using an acousto-optic modulator. The pump and a CW probe beam were combined with a dichroic mirror, directed into an inverted microscope, and focused onto the specimen. The change in the transmitted probe beam's center intensity was detected with a photodiode. The photodiode's signal and a reference signal from the modulator were sent to a high-speed lock-in amplifier. The in-phase and quadrature signals were recorded as a sample was translated through the focused beams and used to generate images based on the amplitude and phase of the lock-in amplifier's signal. The amplitude is related to the absorbance and the phase is related to the thermal diffusivity of the sample. Thin sections of stained liver and bone tissues were imaged; the contrast and signal-to-noise ratio of the phase image was highest at frequencies from 0.1-1 MHz and dropped at higher frequencies. The spatial resolution was 2.5 μm for both amplitude and phase images, limited by the pump beam spot size. © 2011 Optical Society of America

  13. Development of the water window imaging x-ray microscope

    International Nuclear Information System (INIS)

    Hoover, R.B.; Shealy, D.L.; Baker, P.C.; Barbee, T.W. Jr.; Walker, A.B.C. Jr.

    1991-01-01

    This paper reports on the Water Window Imaging X-ray Microscopy which is currently being developed by a consortium from the Marshall Space Flight Center, the University of Alabama at Birmingham, Baker Consulting, the Lawrence Livermore National Laboratory, and Stanford University. The high quality solar images achieved during the Stanford/MSFC/LLNL Rocket X-ray Spectroheliograph flight conclusively established that excellent imaging could be obtained with doubly reflecting multilayer optical systems. Theoretical studies carried out as part of the MSFC X-ray Microscopy Program, demonstrated that high quality, high resolution multilayer x-ray imaging microscopes could be achieved with spherical optics in the Schwarzschild configuration and with Aspherical optical systems. Advanced Flow Polishing methods have been used to fabricate substrates for multilayer optics. On hemlite grade Sapphire, the authors have achieved microscopy mirror substrates on concave and convex spherical surfaces with 0.5 Angstrom rms surface smoothness, as measured by the Zygo profilometer. In this paper the authors report on the current status of fabrication and testing of the optical and mechanical subsystems for the Water Window Imaging X-ray Microscope

  14. High-speed atomic force microscope imaging: Adaptive multiloop mode

    Science.gov (United States)

    Ren, Juan; Zou, Qingze; Li, Bo; Lin, Zhiqun

    2014-07-01

    In this paper, an imaging mode (called the adaptive multiloop mode) of atomic force microscope (AFM) is proposed to substantially increase the speed of tapping mode (TM) imaging while preserving the advantages of TM imaging over contact mode (CM) imaging. Due to its superior image quality and less sample disturbances over CM imaging, particularly for soft materials such as polymers, TM imaging is currently the most widely used imaging technique. The speed of TM imaging, however, is substantially (over an order of magnitude) lower than that of CM imaging, becoming the major bottleneck of this technique. Increasing the speed of TM imaging is challenging as a stable probe tapping on the sample surface must be maintained to preserve the image quality, whereas the probe tapping is rather sensitive to the sample topography variation. As a result, the increase of imaging speed can quickly lead to loss of the probe-sample contact and/or annihilation of the probe tapping, resulting in image distortion and/or sample deformation. The proposed adaptive multiloop mode (AMLM) imaging overcomes these limitations of TM imaging through the following three efforts integrated together: First, it is proposed to account for the variation of the TM deflection when quantifying the sample topography; second, an inner-outer feedback control loop to regulate the TM deflection is added on top of the tapping-feedback control loop to improve the sample topography tracking; and, third, an online iterative feedforward controller is augmented to the whole control system to further enhance the topography tracking, where the next-line sample topography is predicted and utilized to reduce the tracking error. The added feedback regulation of the TM deflection ensures the probe-sample interaction force remains near the minimum for maintaining a stable probe-sample interaction. The proposed AMLM imaging is tested and demonstrated by imaging a poly(tert-butyl acrylate) sample in experiments. The

  15. Modulus design multiwavelength polarization microscope for transmission Mueller matrix imaging.

    Science.gov (United States)

    Zhou, Jialing; He, Honghui; Chen, Zhenhua; Wang, Ye; Ma, Hui

    2018-01-01

    We have developed a polarization microscope based on a commercial transmission microscope. We replace the halogen light source by a collimated LED light source module of six different colors. We use achromatic polarized optical elements that can cover the six different wavelength ranges in the polarization state generator (PSG) and polarization state analyzer (PSA) modules. The dual-rotating wave plate method is used to measure the Mueller matrix of samples, which requires the simultaneous rotation of the two quarter-wave plates in both PSG and PSA at certain angular steps. A scientific CCD detector is used as the image receiving module. A LabView-based software is developed to control the rotation angels of the wave plates and the exposure time of the detector to allow the system to run fully automatically in preprogrammed schedules. Standard samples, such as air, polarizers, and quarter-wave plates, are used to calibrate the intrinsic Mueller matrix of optical components, such as the objectives, using the eigenvalue calibration method. Errors due to the images walk-off in the PSA are studied. Errors in the Mueller matrices are below 0.01 using air and polarizer as standard samples. Data analysis based on Mueller matrix transformation and Mueller matrix polarization decomposition is used to demonstrate the potential application of this microscope in pathological diagnosis. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

  16. Magnetoacoustic microscopic imaging of conductive objects and nanoparticles distribution

    Science.gov (United States)

    Liu, Siyu; Zhang, Ruochong; Luo, Yunqi; Zheng, Yuanjin

    2017-09-01

    Magnetoacoustic tomography has been demonstrated as a powerful and low-cost multi-wave imaging modality. However, due to limited spatial resolution and detection efficiency of magnetoacoustic signal, full potential of the magnetoacoustic imaging remains to be tapped. Here we report a high-resolution magnetoacoustic microscopy method, where magnetic stimulation is provided by a compact solenoid resonance coil connected with a matching network, and acoustic reception is realized by using a high-frequency focused ultrasound transducer. Scanning the magnetoacoustic microscopy system perpendicularly to the acoustic axis of the focused transducer would generate a two-dimensional microscopic image with acoustically determined lateral resolution. It is analyzed theoretically and demonstrated experimentally that magnetoacoustic generation in this microscopic system depends on the conductivity profile of conductive objects and localized distribution of superparamagnetic iron magnetic nanoparticles, based on two different but related implementations. The lateral resolution is characterized. Directional nature of magnetoacoustic vibration and imaging sensitivity for mapping magnetic nanoparticles are also discussed. The proposed microscopy system offers a high-resolution method that could potentially map intrinsic conductivity distribution in biological tissue and extraneous magnetic nanoparticles.

  17. A super-oscillatory lens optical microscope for subwavelength imaging.

    Science.gov (United States)

    Rogers, Edward T F; Lindberg, Jari; Roy, Tapashree; Savo, Salvatore; Chad, John E; Dennis, Mark R; Zheludev, Nikolay I

    2012-03-25

    The past decade has seen an intensive effort to achieve optical imaging resolution beyond the diffraction limit. Apart from the Pendry-Veselago negative index superlens, implementation of which in optics faces challenges of losses and as yet unattainable fabrication finesse, other super-resolution approaches necessitate the lens either to be in the near proximity of the object or manufactured on it, or work only for a narrow class of samples, such as intensely luminescent or sparse objects. Here we report a new super-resolution microscope for optical imaging that beats the diffraction limit of conventional instruments and the recently demonstrated near-field optical superlens and hyperlens. This non-invasive subwavelength imaging paradigm uses a binary amplitude mask for direct focusing of laser light into a subwavelength spot in the post-evanescent field by precisely tailoring the interference of a large number of beams diffracted from a nanostructured mask. The new technology, which--in principle--has no physical limits on resolution, could be universally used for imaging at any wavelength and does not depend on the luminescence of the object, which can be tens of micrometres away from the mask. It has been implemented as a straightforward modification of a conventional microscope showing resolution better than λ/6.

  18. Radiative corrections to two photon physics

    International Nuclear Information System (INIS)

    Neervan, W.L. van; Vermaseren, J.A.M.

    1983-06-01

    The authors develop a method to calculate radiative corrections to two photon reactions of the type e + e - →e + e - X where X is an arbitrary final state. To illustrate this they take the example where X stands for a point-like pseudoscalar. It will be shown that the method is an improvement on the standard way in adding real and virtual photon contributions to the (differential) cross-sections. This makes it possible to compute differential and total cross-sections to a very high precision and with a minimum of computer time which is not possible by using more conventional methods. (Auth.)

  19. Inclusive two-photon reactions at TRISTAN

    International Nuclear Information System (INIS)

    Drees, M.

    1995-01-01

    After briefly reviewing past accomplishments of TRISTAN experiments in the field of inclusive two-photon reactions, I discuss open problems in the Monte Carlo simulation of such reactions. The main emphasis is on multiple scattering, i.e. events where at least two pairs of partons scatter within the same γγ collision to form at least four (mini)jets. The cross section for such events might just be observable at TRISTAN. While theoretical arguments for the existence of such events are strong, they have not yet been directly observed experimentally, thereby potentially opening a new opportunity for TRISTAN experiments. (author)

  20. Two-photon physics at LEP

    International Nuclear Information System (INIS)

    Ginzburg, I.F.

    1988-01-01

    The two-photon production of hadrons in e + e - collisions e + e - →e + e - h from which the γγ→h cross sections are extracted is discussed. The common features of these processes are: hadrons move, as a rule, along e + e - beam axis, their total transverse momentum K perpendicular or perpendicular to is small; the total hadron energy is usually less than √S/2. Physical problems of soft processes, exotics, hard processes, semihard processes are considered. New possibilities of LEP, the most interesting and real are presented

  1. Two-photon cooling of magnesium atoms

    DEFF Research Database (Denmark)

    Malossi, N.; Damkjær, S.; Hansen, P. L.

    2005-01-01

    A two-photon mechanism for cooling atoms below the Doppler temperature is analyzed. We consider the magnesium ladder system (3s2)S01¿(3s3p)P11 at 285.2nm followed by the (3s3p)P11¿(3s3d)D21 transition at 880.7nm . For the ladder system quantum coherence effects may become important. Combined with...... and experiment is excellent. In addition, by properly choosing the Rabi frequencies of the two optical transitions a velocity independent atomic dark state is observed....

  2. A series of fluorene-based two-photon absorbing molecules: synthesis, linear and nonlinear characterization, and bioimaging

    Science.gov (United States)

    Andrade, Carolina D.; Yanez, Ciceron O.; Rodriguez, Luis; Belfield, Kevin D.

    2010-01-01

    The synthesis, structural, and photophysical characterization of a series of new fluorescent donor–acceptor and acceptor-acceptor molecules, based on the fluorenyl ring system, with two-photon absorbing properties is described. These new compounds exhibited large Stokes shifts, high fluorescent quantum yields, and, significantly, high two-photon absorption cross sections, making them well suited for two-photon fluorescence microscopy (2PFM) imaging. Confocal and two-photon fluorescence microscopy imaging of COS-7 and HCT 116 cells incubated with probe I showed endosomal selectivity, demonstrating the potential of this class of fluorescent probes in multiphoton fluorescence microscopy. PMID:20481596

  3. Resonant production in two photon collisions

    International Nuclear Information System (INIS)

    Butler, F.

    1988-12-01

    Using 220 picobarn/sup /minus/1/ of data collected by the Mark II detector at PEP, we have measured the width of the /eta/' for quasi real photons. The width is separately measured in the three reactions e + e/sup /minus// → e + e/sup /minus///eta/ 0 π + π/sup /minus//; /eta/ 0 → γγ, e + e/sup /minus// → e + e/sup /minus///eta/ 0 π + π/sup /minus//; /eta/ 0 → π + π/sup /minus//(π 0 ,γ) and e + e/sup /minus// → e + e/sup /minus///rho/ 0 γ, giving a statistically weighted average two-photon width of Γ/sub /eta/'→γγ/ = 5. 21+-0.28 keV. As a by-product of the measurement of Γ/sub /eta/'→γγ/ using the decay mode /eta/' → /rho/γ, we also measured a two-photon decay width for the a 2 (1320) of Γ/sub a 2(1320)→γγ/ = 1.17+-0. 15+-0.25 keV. 47 refs., 50 figs., 8 tabs

  4. Imaging properties and its improvements of scanning/imaging x-ray microscope

    International Nuclear Information System (INIS)

    Takeuchi, Akihisa; Uesugi, Kentaro; Suzuki, Yoshio

    2016-01-01

    A scanning / imaging X-ray microscope (SIXM) system has been developed at SPring-8. The SIXM consists of a scanning X-ray microscope with a one-dimensional (1D) X-ray focusing device and an imaging (full-field) X-ray microscope with a 1D X-ray objective. The motivation of the SIXM system is to realize a quantitative and highly-sensitive multimodal 3D X-ray tomography by taking advantages of both the scanning X-ray microscope using multi-pixel detector and the imaging X-ray microscope. Data acquisition process of a 2D image is completely different between in the horizontal direction and in the vertical direction; a 1D signal is obtained with the linear-scanning while the other dimensional signal is obtained with the imaging optics. Such condition have caused a serious problem on the imaging properties that the imaging quality in the vertical direction has been much worse than that in the horizontal direction. In this paper, two approaches to solve this problem will be presented. One is introducing a Fourier transform method for phase retrieval from one phase derivative image, and the other to develop and employ a 1D diffuser to produce an asymmetrical coherent illumination

  5. Red Blood Cell Count Automation Using Microscopic Hyperspectral Imaging Technology.

    Science.gov (United States)

    Li, Qingli; Zhou, Mei; Liu, Hongying; Wang, Yiting; Guo, Fangmin

    2015-12-01

    Red blood cell counts have been proven to be one of the most frequently performed blood tests and are valuable for early diagnosis of some diseases. This paper describes an automated red blood cell counting method based on microscopic hyperspectral imaging technology. Unlike the light microscopy-based red blood count methods, a combined spatial and spectral algorithm is proposed to identify red blood cells by integrating active contour models and automated two-dimensional k-means with spectral angle mapper algorithm. Experimental results show that the proposed algorithm has better performance than spatial based algorithm because the new algorithm can jointly use the spatial and spectral information of blood cells.

  6. Software for imaging phase-shift interference microscope

    Science.gov (United States)

    Malinovski, I.; França, R. S.; Couceiro, I. B.

    2018-03-01

    In recent years absolute interference microscope was created at National Metrology Institute of Brazil (INMETRO). The instrument by principle of operation is imaging phase-shifting interferometer (PSI) equipped with two stabilized lasers of different colour as traceable reference wavelength sources. We report here some progress in development of the software for this instrument. The status of undergoing internal validation and verification of the software is also reported. In contrast with standard PSI method, different methodology of phase evaluation is applied. Therefore, instrument specific procedures for software validation and verification are adapted and discussed.

  7. On the kinematics of the two-photon Cherenkov effect

    International Nuclear Information System (INIS)

    Afanas'ev, G.N.; Stepanovskij, Yu.P.

    2003-01-01

    We study the kinematics of the two-photon Cherenkov effect. In the general case, the emission angles of two photons satisfy certain inequalities and the corresponding radiation intensities are rather diffused. In special cases, when the above inequalities reduce to equalities, the emission angles of two photons are fixed and the corresponding radiation intensities should have sharp maxima at these angles. This makes easier the experimental study of the two-photon Cherenkov effect

  8. Details of the Collagen and Elastin Architecture in the Human Limbal Conjunctiva, Tenon's Capsule and Sclera Revealed by Two-Photon Excited Fluorescence Microscopy.

    Science.gov (United States)

    Park, Choul Yong; Marando, Catherine M; Liao, Jason A; Lee, Jimmy K; Kwon, Jiwon; Chuck, Roy S

    2016-10-01

    To investigate the architecture and distribution of collagen and elastin in human limbal conjunctiva, Tenon's capsule, and sclera. The limbal conjunctiva, Tenon's capsule, and sclera of human donor corneal buttons were imaged with an inverted two-photon excited fluorescence microscope. No fixation process was necessary. The laser (Ti:sapphire) was tuned at 850 nm for two-photon excitation. Backscatter signals of second harmonic generation (SHG) and autofluorescence (AF) were collected through a 425/30-nm and a 525/45-nm emission filter, respectively. Multiple, consecutive, and overlapping (z-stack) images were acquired. Collagen signals were collected with SHG, whereas elastin signals were collected with AF. The size and density of collagen bundles varied widely depending on depth: increasing from conjunctiva to sclera. In superficial image planes, collagen bundles were image planes (episclera and superficial sclera), collagen bundles were thicker (near 100 μm in width) and densely packed. Comparatively, elastin fibers were thinner and sparse. The orientation of elastin fibers was independent of collagen fibers in superficial layers; but in deep sclera, elastin fibers wove through collagen interbundle gaps. At the limbus, both collagen and elastin fibers were relatively compact and were distributed perpendicular to the limbal annulus. Two-photon excited fluorescence microscopy has enabled us to understand in greater detail the collagen and elastin architecture of the human limbal conjunctiva, Tenon's capsule, and sclera.

  9. Optimizing pulse compressibility in completely all-fibered Ytterbium chirped pulse amplifiers for in vivo two photon laser scanning microscopy.

    Science.gov (United States)

    Fernández, A; Grüner-Nielsen, L; Andreana, M; Stadler, M; Kirchberger, S; Sturtzel, C; Distel, M; Zhu, L; Kautek, W; Leitgeb, R; Baltuska, A; Jespersen, K; Verhoef, A

    2017-08-01

    A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.

  10. On the detection of early osteoarthritis by quantitative microscopic imaging

    Science.gov (United States)

    Mittelstaedt, Daniel John

    measurements. These studies demonstrate the ability to use two quantitative microscopic imaging techniques, microCT and microMRI, to detect microscopic changes in collagen and GAG from healthy, biochemically degraded, and early OA cartilage. The capability for microscopic imaging to detect alterations at the earliest stages of OA will ultimately improve the understanding of degradation and may help aid in the detection for the prevention of disease and repair of damaged cartilage.

  11. Two-photon excited UV fluorescence for protein crystal detection

    International Nuclear Information System (INIS)

    Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

    2011-01-01

    Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC

  12. Adaptation of commercial microscopes for advanced imaging applications

    Science.gov (United States)

    Brideau, Craig; Poon, Kelvin; Stys, Peter

    2015-03-01

    Today's commercially available microscopes offer a wide array of options to accommodate common imaging experiments. Occasionally, an experimental goal will require an unusual light source, filter, or even irregular sample that is not compatible with existing equipment. In these situations the ability to modify an existing microscopy platform with custom accessories can greatly extend its utility and allow for experiments not possible with stock equipment. Light source conditioning/manipulation such as polarization, beam diameter or even custom source filtering can easily be added with bulk components. Custom and after-market detectors can be added to external ports using optical construction hardware and adapters. This paper will present various examples of modifications carried out on commercial microscopes to address both atypical imaging modalities and research needs. Violet and near-ultraviolet source adaptation, custom detection filtering, and laser beam conditioning and control modifications will be demonstrated. The availability of basic `building block' parts will be discussed with respect to user safety, construction strategies, and ease of use.

  13. Two-photon interference : spatial aspects of two-photon entanglement, diffraction, and scattering

    NARCIS (Netherlands)

    Peeters, Wouter Herman

    2010-01-01

    This dissertation contains scientific research within the realm of quantum optics, which is a branch of physics. An experimental and theoretical study is made of two-photon interference phenomena in various optical systems. Spatially entangled photon pairs are produced via the nonlinear optical

  14. Neural Network for Nanoscience Scanning Electron Microscope Image Recognition.

    Science.gov (United States)

    Modarres, Mohammad Hadi; Aversa, Rossella; Cozzini, Stefano; Ciancio, Regina; Leto, Angelo; Brandino, Giuseppe Piero

    2017-10-16

    In this paper we applied transfer learning techniques for image recognition, automatic categorization, and labeling of nanoscience images obtained by scanning electron microscope (SEM). Roughly 20,000 SEM images were manually classified into 10 categories to form a labeled training set, which can be used as a reference set for future applications of deep learning enhanced algorithms in the nanoscience domain. The categories chosen spanned the range of 0-Dimensional (0D) objects such as particles, 1D nanowires and fibres, 2D films and coated surfaces, and 3D patterned surfaces such as pillars. The training set was used to retrain on the SEM dataset and to compare many convolutional neural network models (Inception-v3, Inception-v4, ResNet). We obtained compatible results by performing a feature extraction of the different models on the same dataset. We performed additional analysis of the classifier on a second test set to further investigate the results both on particular cases and from a statistical point of view. Our algorithm was able to successfully classify around 90% of a test dataset consisting of SEM images, while reduced accuracy was found in the case of images at the boundary between two categories or containing elements of multiple categories. In these cases, the image classification did not identify a predominant category with a high score. We used the statistical outcomes from testing to deploy a semi-automatic workflow able to classify and label images generated by the SEM. Finally, a separate training was performed to determine the volume fraction of coherently aligned nanowires in SEM images. The results were compared with what was obtained using the Local Gradient Orientation method. This example demonstrates the versatility and the potential of transfer learning to address specific tasks of interest in nanoscience applications.

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

    DEFF Research Database (Denmark)

    Piksarv, Peeter; Marti, Dominik; Le, Tuan

    2017-01-01

    We demonstrate the first light sheet microscope using propagation invariant, accelerating Airy beams that operates both in single- and two-photon modes. The use of the Airy beam permits us to develop an ultra compact, high resolution light sheet system without beam scanning. In two-photon mode......, an increase in the field of view over the use of a standard Gaussian beam by a factor of six is demonstrated. This implementation for light sheet microscopy opens up new possibilities across a wide range of biomedical applications, especially for the study of neuronal processes....

  16. A spirobifluorene-based two-photon fluorescence probe for mercury ions and its applications in living cells

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Haibo, E-mail: xiaohb@shnu.edu.cn; Zhang, Yanzhen; Zhang, Wu; Li, Shaozhi; Tan, Jingjing; Han, Zhongying

    2017-05-01

    A novel spirobifluorene derivative SPF-TMS, which containing dithioacetal groups and triphenylamine units, was synthesized. The probing behaviors toward various metal ions were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. The results indicated that SPF-TMS exhibits high sensitivity and selectivity for mercury ions. The detection limit was at least 8.6 × 10{sup −8}M, which is excellent comparing with other optical sensors for Hg{sup 2+}. When measured by two-photon excited fluorescence technique in THF at 800 nm, the two-photon cross-section of SPF-TMS is 272 GM. Especially, upon reaction with mercury species, SPF-TMS yielded another two-photon dye SPF-DA. Both SPF-TMS and SPF-DA emit strong two-photon induced fluorescence and can be applied in cell imaging by two-photon microscopy. - Highlights: • We report a spirobifluorene-based molecule as two-photon fluorescent probe with large two-photon cross-section. • The molecule has exclusive selectivity and sensitivity for mercury species. • The molecule has large two-photon emission changes before and after addition of Hg{sup 2+}. • Both the probe and the mercury ion-promoted reaction product can be applied in cell imaging by two-photon microscopy.

  17. Imaging optical scattering of butterfly wing scales with a microscope.

    Science.gov (United States)

    Fu, Jinxin; Yoon, Beom-Jin; Park, Jung Ok; Srinivasarao, Mohan

    2017-08-06

    A new optical method is proposed to investigate the reflectance of structurally coloured objects, such as Morpho butterfly wing scales and cholesteric liquid crystals. Using a reflected-light microscope and a digital single-lens reflex (DSLR) camera, we have successfully measured the two-dimensional reflection pattern of individual wing scales of Morpho butterflies. We demonstrate that this method enables us to measure the bidirectional reflectance distribution function (BRDF). The scattering image observed in the back focal plane of the objective is projected onto the camera sensor by inserting a Bertrand lens in the optical path of the microscope. With monochromatic light illumination, we quantify the angle-dependent reflectance spectra from the wing scales of Morpho rhetenor by retrieving the raw signal from the digital camera sensor. We also demonstrate that the polarization-dependent reflection of individual wing scales is readily observed using this method, using the individual wing scales of Morpho cypris . In an effort to show the generality of the method, we used a chiral nematic fluid to illustrate the angle-dependent reflectance as seen by this method.

  18. Two photon processes in surface photovoltage spectroscopy

    International Nuclear Information System (INIS)

    Craig, R.P.; Thurgate, S.M.

    1996-01-01

    Full text: A significant mid-gap effect has been found in Surface Photovoltage Spectroscopy measurements of cleaved GaAs, InP and Si wafer which is normally interpreted as arising from transitions between surface states and band edges. This large mid-gap effect common to various materials is puzzling as such a high proportion of mid-gap states seems unlikely. Most theories of surface states predict states that tail from the band edges into the gap or states that have a well defined energy in the gap. None propose a large state exactly at E G /2. We recently investigated the variation in SPS spectra with flux density. We find a non-linear correlation in the magnitude of Band-Bending arising from mid-gap and band-gap photon energies. We suggest that the mid-gap feature is due to a two photon absorption process leading to carrier pair generation mediated by mid-gap states in the continuum of band-gap surface states

  19. Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment

    Science.gov (United States)

    Nimchuk, Zachary L.; Perdue, Tony D.

    2017-01-01

    Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory. PMID:28579995

  20. Live Imaging of Shoot Meristems on an Inverted Confocal Microscope Using an Objective Lens Inverter Attachment.

    Science.gov (United States)

    Nimchuk, Zachary L; Perdue, Tony D

    2017-01-01

    Live imaging of above ground meristems can lead to new insights in plant development not possible from static imaging of fixed tissue. The use of an upright confocal microscope offers several technical and biological advantages for live imaging floral or shoot meristems. However, many departments and core facilities possess only inverted confocal microscopes and lack the funding for an additional upright confocal microscope. Here we show that imaging of living apical meristems can be performed on existing inverted confocal microscopes with the use of an affordable and detachable InverterScope accessory.

  1. Enhanced two-photon emission from a dressed biexciton

    International Nuclear Information System (INIS)

    Sánchez Muñoz, Carlos; Laussy, Fabrice P; Tejedor, Carlos; Valle, Elena del

    2015-01-01

    Radiative two-photon cascades from biexcitons in semiconductor quantum dots under resonant two-photon excitation are promising candidates for the generation of photon pairs. In this work, we propose a scheme to obtain two-photon emission that allows us to operate under very intense driving fields. This approach relies on the Purcell enhancement of two-photon virtual transitions between states of the biexciton dressed by the laser. The richness provided by the biexcitonic level structure allows to reach a variety of regimes, from antibunched and bunched photon pairs with polarization orthogonal to the driving field, to polarization entangled two-photon emission. This provides evidence that the general paradigm of two-photon emission from a ladder of dressed states can find interesting, particular implementations in a variety of systems. (paper)

  2. Clinical multiphoton tomography and clinical two-photon microendoscopy

    Science.gov (United States)

    König, Karsten; Bückle, Rainer; Weinigel, Martin; Elsner, Peter; Kaatz, Martin

    2009-02-01

    We report on applications of high-resolution clinical multiphoton tomography based on the femtosecond laser system DermaInspectTM with its flexible mirror arm in Australia, Asia, and Europe. Applications include early detection of melanoma, in situ tracing of pharmacological and cosmetical compounds including ZnO nanoparticles in the epidermis and upper dermis, the determination of the skin aging index SAAID as well as the study of the effects of anti-aging products. In addition, first clinical studies with novel rigid high-NA two-photon 1.6 mm GRIN microendoscopes have been conducted to study the effect of wound healing in chronic wounds (ulcus ulcera) as well as to perform intrabody imaging with subcellular resolution in small animals.

  3. Molecular engineering of two-photon fluorescent probes for bioimaging applications

    Science.gov (United States)

    Liu, Hong-Wen; Liu, Yongchao; Wang, Peng; Zhang, Xiao-Bing

    2017-03-01

    During the past two decades, two-photon microscopy (TPM), which utilizes two near-infrared photons as the excitation source, has emerged as a novel, attractive imaging tool for biological research. Compared with one-photon microscopy, TPM offers several advantages, such as lowering background fluorescence in living cells and tissues, reducing photodamage to biosamples, and a photobleaching phenomenon, offering better 3D spatial localization, and increasing penetration depth. Small-molecule-based two-photon fluorescent probes have been well developed for the detection and imaging of various analytes in biological systems. In this review, we will give a general introduction of molecular engineering of two-photon fluorescent probes based on different fluorescence response mechanisms for bioimaging applications during the past decade. Inspired by the desired advantages of small-molecule two-photon fluorescent probes in biological imaging applications, we expect that more attention will be devoted to the development of new two-photon fluorophores and applications of TPM in areas of bioanalysis and disease diagnosis.

  4. Two-photon decay in heavy atoms and ions

    International Nuclear Information System (INIS)

    Mokler, P.H.; Dunford, R.W

    2003-08-01

    We review the status of and comment on current developments in the field of two-photon decay in atomic physics research. Recent work has focused on two-photon decays in highly-charged ions and two-photon decay of inner-shell vacancies in heavy neutral atoms. We emphasize the importance of measuring the shape of the continuum emission in two-photon decay as a probe of relativistic effects in the strong central fields found in heavy atomic systems. New experimental approaches and their consequences will be discussed. (orig.)

  5. A fully-automated multiscale kernel graph cuts based particle localization scheme for temporal focusing two-photon microscopy

    Science.gov (United States)

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

    2017-03-01

    The temporal focusing two-photon microscope (TFM) is developed to perform depth resolved wide field fluorescence imaging by capturing frames sequentially. However, due to strong nonignorable noises and diffraction rings surrounding particles, further researches are extremely formidable without a precise particle localization technique. In this paper, we developed a fully-automated scheme to locate particles positions with high noise tolerance. Our scheme includes the following procedures: noise reduction using a hybrid Kalman filter method, particle segmentation based on a multiscale kernel graph cuts global and local segmentation algorithm, and a kinematic estimation based particle tracking method. Both isolated and partial-overlapped particles can be accurately identified with removal of unrelated pixels. Based on our quantitative analysis, 96.22% isolated particles and 84.19% partial-overlapped particles were successfully detected.

  6. Resonance production in two-photon interactions

    International Nuclear Information System (INIS)

    Roe, N.A.

    1989-02-01

    Resonance production in two-photon interactions is studied using data collected with the ASP detector at the PEP e + e/sup /minus// storage ring located at the Stanford Linear Accelerator Center. The ASP detector is a non-magnetic lead-glass calorimeter constructed from 632 lead-glass bars. It covers 94% of 4π in solid angle, extending to within 20/degree/ of the beamline. Lead-scintillator calorimeters extend the coverage to within 21 mr of the beamline on both sides. Energy resolution of √E/10%, where E is the energy is GeV, is achieved for electrons and photons in the lead-glass calorimeter, and particle trajectories are reconstructed with high efficiency. A total luminosity of 108 pb/sup /minus/1/ was collected with the ASP detector at a center-of-mass energy of 29 GeV. The observed process is e + e/sup /minus// → e + e/sup /minus//γ*γ* → e + e/sup /minus//X, is a pseudoscalar resonance (J/sup PC/ = 0/sup /minus/+/) and γ* is a virtual (mass /ne/ 0) photon. The outgoing electrons scatter down the beampipe and are not detected. The observed resonances are the /eta/ and /eta/' mesons, with masses of 549 and 958 MeV, respectively. They are detected in the γγ decay mode; a total of 2380 +- 49 /eta/ → γγ and 568 +- 26 /eta/' → γγ events are observed. From the number of events, the detection efficiency, and the calculated production cross sections the radiative widths, Γ/sub γγ/, of the /eta/ and /eta/' were measured and found to be: Γ/sub γγ/(/eta/) = .481 +- .010 +- .047keV and Γ/sub γγ/(/eta/') = 4.71 +- .22 +- .70keV. These results are in good agreement with the world average values. 67 refs., 42 figs., 20 tabs

  7. Resonance production in two-photon interactions

    Energy Technology Data Exchange (ETDEWEB)

    Roe, N.A.

    1989-02-01

    Resonance production in two-photon interactions is studied using data collected with the ASP detector at the PEP e/sup +/e/sup /minus// storage ring located at the Stanford Linear Accelerator Center. The ASP detector is a non-magnetic lead-glass calorimeter constructed from 632 lead-glass bars. It covers 94% of 4..pi.. in solid angle, extending to within 20/degree/ of the beamline. Lead-scintillator calorimeters extend the coverage to within 21 mr of the beamline on both sides. Energy resolution of ..sqrt..E/10%, where E is the energy is GeV, is achieved for electrons and photons in the lead-glass calorimeter, and particle trajectories are reconstructed with high efficiency. A total luminosity of 108 pb/sup /minus/1/ was collected with the ASP detector at a center-of-mass energy of 29 GeV. The observed process is e/sup +/e/sup /minus// ..-->.. e/sup +/e/sup /minus//..gamma..*..gamma..* ..-->.. e/sup +/e/sup /minus//X, is a pseudoscalar resonance (J/sup PC/ = 0/sup /minus/+/) and ..gamma..* is a virtual (mass /ne/ 0) photon. The outgoing electrons scatter down the beampipe and are not detected. The observed resonances are the /eta/ and /eta/' mesons, with masses of 549 and 958 MeV, respectively. They are detected in the ..gamma gamma.. decay mode; a total of 2380 +- 49 /eta/ ..-->.. ..gamma gamma.. and 568 +- 26 /eta/' ..-->.. ..gamma gamma.. events are observed. From the number of events, the detection efficiency, and the calculated production cross sections the radiative widths, GAMMA/sub ..gamma gamma../, of the /eta/ and /eta/' were measured and found to be: GAMMA/sub ..gamma gamma../(/eta/) = .481 +- .010 +- .047keV and GAMMA/sub ..gamma gamma../(/eta/') = 4.71 +- .22 +- .70keV. These results are in good agreement with the world average values. 67 refs., 42 figs., 20 tabs.

  8. Optical design and system characterization of an imaging microscope at 121.6 nm

    Science.gov (United States)

    Gao, Weichuan; Finan, Emily; Kim, Geon-Hee; Kim, Youngsik; Milster, Thomas D.

    2018-03-01

    We present the optical design and system characterization of an imaging microscope prototype at 121.6 nm. System engineering processes are demonstrated through the construction of a Schwarzschild microscope objective, including tolerance analysis, fabrication, alignment, and testing. Further improvements on the as-built system with a correction phase plate are proposed and analyzed. Finally, the microscope assembly and the imaging properties of the prototype are demonstrated.

  9. Hyperspectral microscope imaging methods to classify gram-positive and gram-negative foodborne pathogenic bacteria

    Science.gov (United States)

    An acousto-optic tunable filter-based hyperspectral microscope imaging method has potential for identification of foodborne pathogenic bacteria from microcolony rapidly with a single cell level. We have successfully developed the method to acquire quality hyperspectral microscopic images from variou...

  10. Molecular photosensitisers for two-photon photodynamic therapy.

    Science.gov (United States)

    Bolze, F; Jenni, S; Sour, A; Heitz, V

    2017-11-30

    Two-photon excitation has attracted the attention of biologists, especially after the development of two-photon excited microscopy in the nineties. Since then, new applications have rapidly emerged such as the release of biologically active molecules and photodynamic therapy (PDT) using two-photon excitation. PDT, which requires a light-activated drug (photosensitiser), is a clinically approved and minimally invasive treatment for cancer and for non-malignant diseases. This feature article focuses on the engineering of molecular two-photon photosensitisers for PDT, which should bring important benefits to the treatment, increase the treatment penetration depth with near-infrared light excitation, improve the spatial selectivity and reduce the photodamage to healthy tissues. After an overview of the two-photon absorption phenomenon and the methods to evaluate two-photon induced phototoxicity on cell cultures, the different classes of photosensitisers described in the literature are discussed. The two-photon PDT performed with historical one-photon sensitisers are briefly presented, followed by specifically engineered cyclic tetrapyrrole photosensitisers, purely organic photosensitisers and transition metal complexes. Finally, targeted two-photon photosensitisers and theranostic agents that should enhance the selectivity and efficiency of the treatment are discussed.

  11. Two-photon stimulated emission and pulse amplification

    International Nuclear Information System (INIS)

    Yuen, H.P.

    1975-01-01

    Threshold conditions are given for the sustained operation of standing-wave and long-pulse traveling-wave two-photon lasers. Pulse shortening in long-pulse two-photon amplification, a behavior absent in the one-photon case, is also demonstrated analytically. (U.S.)

  12. Two-photon Photoactivation to Measure Histone Exchange Dynamics in Plant Root Cells.

    Science.gov (United States)

    Rosa, Stefanie; Shaw, Peter

    2015-10-20

    Chromatin-binding proteins play a crucial role in chromatin structure and gene expression. Direct binding of chromatin proteins both maintains and regulates transcriptional states. It is therefore important to study the binding properties of these proteins in vivo within the natural environment of the nucleus. Photobleaching, photoactivation and photoconversion (photoswitching) can provide a non-invasive experimental approach to study dynamic properties of living cells and organisms. We used photoactivation to determine exchange dynamics of histone H2B in plant stem cells of the root (Rosa et al. , 2014). The stem cells of the root are located in the middle of the tissue, which made it impossible to carry out photoactivation of sufficiently small and well-defined sub-cellular regions with conventional laser illumination in the confocal microscope, mainly because scattering and refraction effects within the root tissue dispersed the focal spot and caused photoactivation of too large a region. We therefore used 2-photon activation, which has much better inherent resolution of the illuminated region. This is because the activation depends on simultaneous absorption of two or more photons, which in turns depends on the square (or higher power) of the intensity-a much sharper peak. In this protocol we will describe the experimental procedure to perform two-photon photoactivation experiments and the corresponding image analysis. This protocol can be used for nuclear proteins tagged with photoactivable GFP (PA-GFP) expressed in root tissues.

  13. Novel triphenylamine-cored two-photon absorbing dyes for labeling of biomolecules

    International Nuclear Information System (INIS)

    Xiao Haibo; Mei Chong; Wang Yaochuan; Li, Hui; Qian Shixiong; Yin Hongyao; Xu Zhisong

    2011-01-01

    Highlights: → Two novel triphenylamine-cored chromophores were synthesized. → These two dyes have sizable two-photon absorption cross-section at 800 nm. → They possess reasonable water solubility and are suitable as labels in aqueous biological environments. → These dyes have strong chelating ability. → They display a large set of reactivity for coupling to biomolecules. - Abstract: Two novel, V-shaped and Y-shaped dipicolinate derivatives branched from triphenylamine, {4-[(E)-2-(2,6-dimethoxycarbonylpyridin-4-yl)vinyl]}-N-phenyl-N-{4- [(E)-2-(2,6-dimethoxycarbonylpyridin-4-yl)vinylphenyl]}aniline (1) and {4-[(E)-2-(2,6-dimethoxycarbonylpyridin-4-yl) vinyl]}-N,N-bis {4-[(E)-2-(2,6-dimethoxycarbonyl pyridin-4-yl)vinylphenyl]}aniline (2) were synthesized. These compounds were designed for large two-photon absorption and in particular for labeling of biomolecules. Their linear absorption, fluorescence properties and their two-photon absorption properties as well as two-photon fluorescence cell imaging were examined. When excited at 800 nm, the two-photon absorption cross-section values of chromophores 1 and 2 in THF were 208 GM, 376 GM, respectively. These two-photon absorbing dyes possess reasonable water solubility, strong chelating ability and display a large set of reactivity for coupling to biomolecules, which are apparently due to the two methoxycarbonyl groups in pyridine ring. This work suggests that chromophores 1 and 2 are promising labels potentially applicable for the tracking of biomolecules using two-photon scanning microscopy.

  14. The development of efficient two-photon singlet oxygen sensitizers

    DEFF Research Database (Denmark)

    Nielsen, Christian Benedikt

    The development of efficient two-photon singlet oxygen sensitizers is addressed focusing on organic synthesis. Photophysical measurements were carried out on new lipophilic molecules, where two-photon absorption cross sections and singlet oxygen quantumyields were measured. Design principles...... for making efficient two-photon singlet oxygen sensitizers were then constructed from these results. Charge-transfer in the excited state of the prepared molecules was shown to play a pivotal role in the generationof singlet oxygen. This was established through studies of substituent effects on both...... the singlet oxygen yield and the two-photon absorption cross section, where it was revealed that a careful balancing of the amount of charge transfer present in theexcited state of the sensitizer is necessary to obtain both a high singlet oxygen quantum yield and a high two-photon cross section. An increasing...

  15. Microscopic hyperspectral imaging studies of normal and diabetic retina of rats

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A microscopic hyperspectral imager was developed based on the microscopic technology and the spectral imaging technology. Some microscopic hyperspectral images of retina sections of the normal, the diabetic, and the treated rats were collected by the new imager. Single-band images and pseudo-color images of each group were obtained and the typical transmittance spectrums were ex-tracted. The results showed that the transmittance of outer nuclear layer cells of the diabetic group was generally higher than that of the normal. A small absorption peak appeared near the 180th band in the spectrum of the diabetic group and this peak weakened or disappeared in the spectrum of the treated group. Our findings indicate that the microscopic hyperspectral images include wealthy information of retina sections which is helpful for the ophthalmologist to reveal the pathogenesis of diabetic reti-nopathy and explore the therapeutic effect of drugs.

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

    Science.gov (United States)

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

    2013-08-01

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

  17. Polarization resolved imaging with a reflection near-field optical microscope

    DEFF Research Database (Denmark)

    Bozhevolnyi, Sergey I.; Xiao, Mufei; Hvam, Jørn Märcher

    1999-01-01

    Using a rigorous microscopic point-dipole description of probe-sample interactions, we study imaging with a reflection scanning near-field optical microscope. Optical content, topographical artifacts, sensitivity window-i.e., the scale on which near-field optical images represent mainly optical...... configuration is preferable to the cross-linear one, since it ensures more isotropic (in the surface plane) near-field imaging of surface features. The numerical results are supported with experimental near-field images obtained by using a reflection microscope with an uncoated fiber tip....

  18. A simple water-immersion condenser for imaging living brain slices on an inverted microscope.

    Science.gov (United States)

    Prusky, G T

    1997-09-05

    Due to some physical limitations of conventional condensers, inverted compound microscopes are not optimally suited for imaging living brain slices with transmitted light. Herein is described a simple device that converts an inverted microscope into an effective tool for this application by utilizing an objective as a condenser. The device is mounted on a microscope in place of the condenser, is threaded to accept a water immersion objective, and has a slot for a differential interference contrast (DIC) slider. When combined with infrared video techniques, this device allows an inverted microscope to effectively image living cells within thick brain slices in an open perfusion chamber.

  19. Circular dichroism probed by two-photon fluorescence microscopy in enantiopure chiral polyfluorene thin films

    NARCIS (Netherlands)

    Savoini, M.; Wu, Xiaofei; Celebrano, M.; Ziegler, J.; Biagioni, P.; Meskers, S.C.J.; Duo, L.; Hecht, B.; Finazzi, M.

    2012-01-01

    Two-photon fluorescence scanning confocal microscopy sensitive to circular dichroism with a diffraction-limited resolution well below 500 nm is demonstrated. With this method, the spatial variation of the circular dichroism of thermally annealed chiral polyfluorene thin films has been imaged. We

  20. Dependence of the two-photon photoluminescence yield of gold nanostructures on the laser pulse duration

    DEFF Research Database (Denmark)

    Biagioni, P.; Celebrano, M.; Savoini, M.

    2009-01-01

    Two-photon photoluminescence (TPPL) from gold nanostructures is becoming one of the most relevant tools for plasmon-assisted biological imaging and photothermal therapy as well as for the investigation of plasmonic devices. Here we study the yield of TPPL as a function of the temporal width δ of ...

  1. An image processing approach to analyze morphological features of microscopic images of muscle fibers.

    Science.gov (United States)

    Comin, Cesar Henrique; Xu, Xiaoyin; Wang, Yaming; Costa, Luciano da Fontoura; Yang, Zhong

    2014-12-01

    We present an image processing approach to automatically analyze duo-channel microscopic images of muscular fiber nuclei and cytoplasm. Nuclei and cytoplasm play a critical role in determining the health and functioning of muscular fibers as changes of nuclei and cytoplasm manifest in many diseases such as muscular dystrophy and hypertrophy. Quantitative evaluation of muscle fiber nuclei and cytoplasm thus is of great importance to researchers in musculoskeletal studies. The proposed computational approach consists of steps of image processing to segment and delineate cytoplasm and identify nuclei in two-channel images. Morphological operations like skeletonization is applied to extract the length of cytoplasm for quantification. We tested the approach on real images and found that it can achieve high accuracy, objectivity, and robustness. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Two-photon processes in highly charged ions

    International Nuclear Information System (INIS)

    Jahrsetz, Thorsten

    2015-01-01

    Two-photon processes are atomic processes in which an atom interacts simultaneously with two photons. Such processes describe a wide range of phenomena, such as two-photon decay and elastic or inelastic scattering of photons. In recent years two-photon processes involving highly charged heavy ions have become an active area of research. Such studies do not only consider the total transition or scattering rates but also their angular and polarization dependence. To support such examinations in this thesis I present a theoretical framework to describe these properties in all two-photon processes with bound initial and final states and involving heavy H-like or He-like ions. I demonstrate how this framework can be used in some detailed studies of different two-photon processes. Specifically a detailed analysis of two-photon decay of H-like and He-like ions in strong external electromagnetic fields shows the importance of considering the effect of such fields for the physics of such systems. Furthermore I studied the elastic Rayleigh as well as inelastic Raman scattering by heavy H-like ions. I found a number of previously unobserved phenomena in the angular and polarization dependence of the scattering cross-sections that do not only allow to study interesting details of the electronic structure of the ion but might also be useful for the measurement of weak physical effects in such systems.

  3. Two-photon processes in highly charged ions

    Energy Technology Data Exchange (ETDEWEB)

    Jahrsetz, Thorsten

    2015-03-05

    Two-photon processes are atomic processes in which an atom interacts simultaneously with two photons. Such processes describe a wide range of phenomena, such as two-photon decay and elastic or inelastic scattering of photons. In recent years two-photon processes involving highly charged heavy ions have become an active area of research. Such studies do not only consider the total transition or scattering rates but also their angular and polarization dependence. To support such examinations in this thesis I present a theoretical framework to describe these properties in all two-photon processes with bound initial and final states and involving heavy H-like or He-like ions. I demonstrate how this framework can be used in some detailed studies of different two-photon processes. Specifically a detailed analysis of two-photon decay of H-like and He-like ions in strong external electromagnetic fields shows the importance of considering the effect of such fields for the physics of such systems. Furthermore I studied the elastic Rayleigh as well as inelastic Raman scattering by heavy H-like ions. I found a number of previously unobserved phenomena in the angular and polarization dependence of the scattering cross-sections that do not only allow to study interesting details of the electronic structure of the ion but might also be useful for the measurement of weak physical effects in such systems.

  4. A Simple Metric for Determining Resolution in Optical, Ion, and Electron Microscope Images.

    Science.gov (United States)

    Curtin, Alexandra E; Skinner, Ryan; Sanders, Aric W

    2015-06-01

    A resolution metric intended for resolution analysis of arbitrary spatially calibrated images is presented. By fitting a simple sigmoidal function to pixel intensities across slices of an image taken perpendicular to light-dark edges, the mean distance over which the light-dark transition occurs can be determined. A fixed multiple of this characteristic distance is then reported as the image resolution. The prefactor is determined by analysis of scanning transmission electron microscope high-angle annular dark field images of Si. This metric has been applied to optical, scanning electron microscope, and helium ion microscope images. This method provides quantitative feedback about image resolution, independent of the tool on which the data were collected. In addition, our analysis provides a nonarbitrary and self-consistent framework that any end user can utilize to evaluate the resolution of multiple microscopes from any vendor using the same metric.

  5. High-resolution, high-throughput imaging with a multibeam scanning electron microscope.

    Science.gov (United States)

    Eberle, A L; Mikula, S; Schalek, R; Lichtman, J; Knothe Tate, M L; Zeidler, D

    2015-08-01

    Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from biological brain tissue to semiconductor wafers. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  6. Image enhancement of x-ray microscope using frequency spectrum analysis

    International Nuclear Information System (INIS)

    Li Wenjie; Chen Jie; Tian Jinping; Zhang Xiaobo; Liu Gang; Tian Yangchao; Liu Yijin; Wu Ziyu

    2009-01-01

    We demonstrate a new method for x-ray microscope image enhancement using frequency spectrum analysis. Fine sample characteristics are well enhanced with homogeneous visibility and better contrast from single image. This method is easy to implement and really helps to improve the quality of image taken by our imaging system.

  7. Image enhancement of x-ray microscope using frequency spectrum analysis

    Energy Technology Data Exchange (ETDEWEB)

    Li Wenjie; Chen Jie; Tian Jinping; Zhang Xiaobo; Liu Gang; Tian Yangchao [National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei, Anhui 230029 (China); Liu Yijin; Wu Ziyu, E-mail: wuzy@ihep.ac.c, E-mail: ychtian@ustc.edu.c [Institute of High Energy Physics, Chinese Academy of Science, Beijing 100049 (China)

    2009-09-01

    We demonstrate a new method for x-ray microscope image enhancement using frequency spectrum analysis. Fine sample characteristics are well enhanced with homogeneous visibility and better contrast from single image. This method is easy to implement and really helps to improve the quality of image taken by our imaging system.

  8. Exciton molecule in semiconductors by two-photon absorption

    International Nuclear Information System (INIS)

    Arya, K.; Hassan, A.R.

    1976-07-01

    Direct creation of bi-exciton states by two-photon absorption in direct gap semiconductors is investigated theoretically. A numerical application to the case of CuCl shows that the two-photon absorption coefficient for bi-excitonic transitions is larger than that for two-photon interband transitions by three orders of magnitude. It becomes comparable to that for one-photon excitonic transitions for available laser intensities. The main contribution to this enhancement of the absorption coefficient for the transitions to the bi-exciton states is found to be from the resonance effect

  9. Microscopic image processing system for measuring nonuniform film thickness profiles: Image scanning ellipsometry

    International Nuclear Information System (INIS)

    Liu, A.H.; Plawsky, J.L.; Wayner, P.C. Jr.

    1993-01-01

    The long-term objective of this research program is to determine the stability and heat transfer characteristics of evaporating thin films. The current objective is to develop and use a microscopic image-processing system (IPS) which has two parts: an image analyzing interferometer (IAI) and an image scanning ellipsometer (ISE). The primary purpose of this paper is to present the basic concept of ISE, which is a novel technique to measure the two dimensional thickness profile of a non-uniform, thin film, from several nm up to several μm, in a steady state as well as in a transient state. It is a full-field imaging technique which can study every point on the surface simultaneously with high spatial resolution and thickness sensitivity, i.e., it can measure and map the 2-D film thickness profile. The ISE was tested by measuring the thickness profile and the refractive index of a nonuniform solid film

  10. An image processing pipeline to detect and segment nuclei in muscle fiber microscopic images.

    Science.gov (United States)

    Guo, Yanen; Xu, Xiaoyin; Wang, Yuanyuan; Wang, Yaming; Xia, Shunren; Yang, Zhong

    2014-08-01

    Muscle fiber images play an important role in the medical diagnosis and treatment of many muscular diseases. The number of nuclei in skeletal muscle fiber images is a key bio-marker of the diagnosis of muscular dystrophy. In nuclei segmentation one primary challenge is to correctly separate the clustered nuclei. In this article, we developed an image processing pipeline to automatically detect, segment, and analyze nuclei in microscopic image of muscle fibers. The pipeline consists of image pre-processing, identification of isolated nuclei, identification and segmentation of clustered nuclei, and quantitative analysis. Nuclei are initially extracted from background by using local Otsu's threshold. Based on analysis of morphological features of the isolated nuclei, including their areas, compactness, and major axis lengths, a Bayesian network is trained and applied to identify isolated nuclei from clustered nuclei and artifacts in all the images. Then a two-step refined watershed algorithm is applied to segment clustered nuclei. After segmentation, the nuclei can be quantified for statistical analysis. Comparing the segmented results with those of manual analysis and an existing technique, we find that our proposed image processing pipeline achieves good performance with high accuracy and precision. The presented image processing pipeline can therefore help biologists increase their throughput and objectivity in analyzing large numbers of nuclei in muscle fiber images. © 2014 Wiley Periodicals, Inc.

  11. Design of a normal incidence multilayer imaging X-ray microscope

    Science.gov (United States)

    Shealy, David L.; Gabardi, David R.; Hoover, Richard B.; Walker, Arthur B. C., Jr.; Lindblom, Joakim F.

    Normal incidence multilayer Cassegrain X-ray telescopes were flown on the Stanford/MSFC Rocket X-ray Spectroheliograph. These instruments produced high spatial resolution images of the sun and conclusively demonstrated that doubly reflecting multilayer X-ray optical systems are feasible. The images indicated that aplanatic imaging soft X-ray/EUV microscopes should be achievable using multilayer optics technology. A doubly reflecting normal incidence multilayer imaging X-ray microscope based on the Schwarzschild configuration has been designed. The design of the microscope and the results of the optical system ray trace analysis are discussed. High resolution aplanatic imaging X-ray microscopes using normal incidence multilayer X-ray mirrors should have many important applications in advanced X-ray astronomical instrumentation, X-ray lithography, biological, biomedical, metallurgical, and laser fusion research.

  12. Electromagnetically induced two-photon transparency in rubidium atoms

    International Nuclear Information System (INIS)

    Wang, D.; Gao, J.Y.; Xu, J.H.; Bassani, F.; La Rocca, G.C.; Salerno Univ.

    2001-01-01

    We present an experimental demonstration of electromagnetically induced two-photon transparency (EITT) in room temperature rubidium vapor. The 8S 1/2 to 5P 1/2 fluorescence is used to monitor the 5S 1/2 (F = 3) to 8S 1/2 (F = 3) two-photon absorption near resonance with the intermediate state 5P 3/2 . A controlling pump laser beam is employed to coherently couple the 5P 3/2 and 5D 5/2 states, thus producing two dressed intermediate states which give rise to destructive interference in the two-photon transition. An induced two-photon transparency of about 80% has been obtained at resonance; our experimental findings are in good agreement with the general theory of Agarwal et al. (1996), when the appropriate spectroscopic parameters are used. (orig.)

  13. Standard Model Higgs decay for two Photons in CMS

    CERN Multimedia

    Daniel Denegri

    2000-01-01

    Simulated two-photon mass distribution for SM Higgs and expected background in the CMS PbW04 crystal calorimeter for an integrated luminosity of 10 . 5 pb-1, with detailed simulation of calorimeter response.

  14. All-optical femtosecond switch using two-photon absorption

    International Nuclear Information System (INIS)

    Yavuz, D. D.

    2006-01-01

    Utilizing a two-photon absorption scheme in an alkali-metal vapor cell, we suggest a technique where a strong laser beam switches off another laser beam of different wavelength in femtosecond time scales

  15. Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  16. Three Dimensional Imaging of Cold Atoms in a Magneto Optical Trap with a Light Field Microscope

    Science.gov (United States)

    2017-09-14

    with a Light Field Microscope Gordon E. Lott Follow this and additional works at: https://scholar.afit.edu/etd Part of the Atomic, Molecular and......https://scholar.afit.edu/etd/774 THREE-DIMENSIONAL IMAGING OF COLD ATOMS IN A MAGNETO-OPTICAL TRAP WITH A LIGHT FIELD MICROSCOPE DISSERTATION Gordon E

  17. Interferometric and optical tests of water window imaging x ray microscopes

    Science.gov (United States)

    Johnson, R. Barry

    1993-01-01

    Interferometric tests of Schwarzchild X-ray Microscope are performed to evaluate the optical properties and alignment of the components. Photographic measurements of the spatial resolution, focal properties, and vignetting characteristics of the prototype Water Window Imaging X-ray Microscope are made and analyzed.

  18. Imaging properties of the mesooptical Fourier transform microscope for nuclear research emulsion

    International Nuclear Information System (INIS)

    Bencze, Gy.L.; Soroko, L.M.

    1987-01-01

    The optical signal transformation in the Mesooptical Fourier Transform Microscope (MFTM) for nuclear emulsion is treated in terms of Fourier Optics. A continuous conversion of the traditional optical microscope into the MFTM is followed. The images of dot-like and straight line objects given by the MFTM are discussed

  19. Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules

    OpenAIRE

    Elliott, Jonathan T.; Dsouza, Alisha V.; Marra, Kayla; Pogue, Brian W.; Roberts, David W.; Paulsen, Keith D.

    2016-01-01

    Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system spe...

  20. High contrast imaging and flexible photomanipulation for quantitative in vivo multiphoton imaging with polygon scanning microscope.

    Science.gov (United States)

    Li, Yongxiao; Montague, Samantha J; Brüstle, Anne; He, Xuefei; Gillespie, Cathy; Gaus, Katharina; Gardiner, Elizabeth E; Lee, Woei Ming

    2018-02-28

    In this study, we introduce two key improvements that overcome limitations of existing polygon scanning microscopes while maintaining high spatial and temporal imaging resolution over large field of view (FOV). First, we proposed a simple and straightforward means to control the scanning angle of the polygon mirror to carry out photomanipulation without resorting to high speed optical modulators. Second, we devised a flexible data sampling method directly leading to higher image contrast by over 2-fold and digital images with 100 megapixels (10 240 × 10 240) per frame at 0.25 Hz. This generates sub-diffraction limited pixels (60 nm per pixels over the FOV of 512 μm) which increases the degrees of freedom to extract signals computationally. The unique combined optical and digital control recorded fine fluorescence recovery after localized photobleaching (r ~10 μm) within fluorescent giant unilamellar vesicles and micro-vascular dynamics after laser-induced injury during thrombus formation in vivo. These new improvements expand the quantitative biological-imaging capacity of any polygon scanning microscope system. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Mass distribution for the two-photon channel

    CERN Multimedia

    ATLAS, collaboration

    2012-01-01

    Mass distribution for the two-photon channel. The strongest evidence for this new particle comes from analysis of events containing two photons. The smooth dotted line traces the measured background from known processes. The solid line traces a statistical fit to the signal plus background. The new particle appears as the excess around 126.5 GeV. The full analysis concludes that the probability of such a peak is three chances in a million.

  2. Two-photon quantum interference in a Michelson interferometer

    International Nuclear Information System (INIS)

    Odate, Satoru; Wang Haibo; Kobayashi, Takayoshi

    2005-01-01

    We have observed two-photon quantum interference in a Michelson interferometer. For the first time, we experimentally demonstrated two-photon quantum interference patterns, which show the transition from nonsubwavelength interference fringes to the general subwavelength interference. At the same time, a photon bunching effect was also shown by a postselection. The |1, 1> state with a single photon in a mode corresponding to each arm of the interferometer was exclusively postselected by using path difference between two arms

  3. Two-Photon-Absorption Scheme for Optical Beam Tracking

    Science.gov (United States)

    Ortiz, Gerardo G.; Farr, William H.

    2011-01-01

    A new optical beam tracking approach for free-space optical communication links using two-photon absorption (TPA) in a high-bandgap detector material was demonstrated. This tracking scheme is part of the canonical architecture described in the preceding article. TPA is used to track a long-wavelength transmit laser while direct absorption on the same sensor simultaneously tracks a shorter-wavelength beacon. The TPA responsivity was measured for silicon using a PIN photodiode at a laser beacon wavelength of 1,550 nm. As expected, the responsivity shows a linear dependence with incident power level. The responsivity slope is 4.5 x 10(exp -7) A/W2. Also, optical beam spots from the 1,550-nm laser beacon were characterized on commercial charge coupled device (CCD) and complementary metal-oxide semiconductor (CMOS) imagers with as little as 13.7 microWatts of optical power (see figure). This new tracker technology offers an innovative solution to reduce system complexity, improve transmit/receive isolation, improve optical efficiency, improve signal-to-noise ratio (SNR), and reduce cost for free-space optical communications transceivers.

  4. An orthotopic glioblastoma mouse model maintaining brain parenchymal physical constraints and suitable for intravital two-photon microscopy.

    Science.gov (United States)

    Ricard, Clément; Stanchi, Fabio; Rougon, Geneviève; Debarbieux, Franck

    2014-04-21

    Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date. Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma. Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development. Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease. The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

  5. Spatio-temporal imaging of voltage pulses with an ultrafast scanning tunneling microscope

    DEFF Research Database (Denmark)

    Jensen, Jacob Riis; Keil, Ulrich Dieter Felix; Hvam, Jørn Märcher

    1997-01-01

    Measurements on an ultrafast scanning tunneling microscope with simultaneous spatial and temporal resolution are presented. We show images of picosecond pulses propagating on a coplanar waveguide and resolve their mode structures. The influence of transmission line discontinuities on the mode...

  6. Remote laboratory for phase-aided 3D microscopic imaging and metrology

    Science.gov (United States)

    Wang, Meng; Yin, Yongkai; Liu, Zeyi; He, Wenqi; Li, Boqun; Peng, Xiang

    2014-05-01

    In this paper, the establishment of a remote laboratory for phase-aided 3D microscopic imaging and metrology is presented. Proposed remote laboratory consists of three major components, including the network-based infrastructure for remote control and data management, the identity verification scheme for user authentication and management, and the local experimental system for phase-aided 3D microscopic imaging and metrology. The virtual network computer (VNC) is introduced to remotely control the 3D microscopic imaging system. Data storage and management are handled through the open source project eSciDoc. Considering the security of remote laboratory, the fingerprint is used for authentication with an optical joint transform correlation (JTC) system. The phase-aided fringe projection 3D microscope (FP-3DM), which can be remotely controlled, is employed to achieve the 3D imaging and metrology of micro objects.

  7. Image alignment for tomography reconstruction from synchrotron X-ray microscopic images.

    Directory of Open Access Journals (Sweden)

    Chang-Chieh Cheng

    Full Text Available A synchrotron X-ray microscope is a powerful imaging apparatus for taking high-resolution and high-contrast X-ray images of nanoscale objects. A sufficient number of X-ray projection images from different angles is required for constructing 3D volume images of an object. Because a synchrotron light source is immobile, a rotational object holder is required for tomography. At a resolution of 10 nm per pixel, the vibration of the holder caused by rotating the object cannot be disregarded if tomographic images are to be reconstructed accurately. This paper presents a computer method to compensate for the vibration of the rotational holder by aligning neighboring X-ray images. This alignment process involves two steps. The first step is to match the "projected feature points" in the sequence of images. The matched projected feature points in the x-θ plane should form a set of sine-shaped loci. The second step is to fit the loci to a set of sine waves to compute the parameters required for alignment. The experimental results show that the proposed method outperforms two previously proposed methods, Xradia and SPIDER. The developed software system can be downloaded from the URL, http://www.cs.nctu.edu.tw/~chengchc/SCTA or http://goo.gl/s4AMx.

  8. Image alignment for tomography reconstruction from synchrotron X-ray microscopic images.

    Science.gov (United States)

    Cheng, Chang-Chieh; Chien, Chia-Chi; Chen, Hsiang-Hsin; Hwu, Yeukuang; Ching, Yu-Tai

    2014-01-01

    A synchrotron X-ray microscope is a powerful imaging apparatus for taking high-resolution and high-contrast X-ray images of nanoscale objects. A sufficient number of X-ray projection images from different angles is required for constructing 3D volume images of an object. Because a synchrotron light source is immobile, a rotational object holder is required for tomography. At a resolution of 10 nm per pixel, the vibration of the holder caused by rotating the object cannot be disregarded if tomographic images are to be reconstructed accurately. This paper presents a computer method to compensate for the vibration of the rotational holder by aligning neighboring X-ray images. This alignment process involves two steps. The first step is to match the "projected feature points" in the sequence of images. The matched projected feature points in the x-θ plane should form a set of sine-shaped loci. The second step is to fit the loci to a set of sine waves to compute the parameters required for alignment. The experimental results show that the proposed method outperforms two previously proposed methods, Xradia and SPIDER. The developed software system can be downloaded from the URL, http://www.cs.nctu.edu.tw/~chengchc/SCTA or http://goo.gl/s4AMx.

  9. Lateral resolution testing of a novel developed confocal microscopic imaging system

    Science.gov (United States)

    Zhang, Xin; Zhang, Yunhai; Chang, Jian; Huang, Wei; Xue, Xiaojun; Xiao, Yun

    2015-10-01

    Laser scanning confocal microscope has been widely used in biology, medicine and material science owing to its advantages of high resolution and tomographic imaging. Based on a set of confirmatory experiments and system design, a novel confocal microscopic imaging system is developed. The system is composed of a conventional fluorescence microscope and a confocal scanning unit. In the scanning unit a laser beam coupling module provides four different wavelengths 405nm 488nm 561nm and 638nm which can excite a variety of dyes. The system works in spot-to-spot scanning mode with a two-dimensional galvanometer. A 50 microns pinhole is used to guarantee that stray light is blocked and only the fluorescence signal from the focal point can be received . The three-channel spectral splitter is used to perform fluorescence imaging at three different working wavelengths simultaneously. The rat kidney tissue slice is imaged using the developed confocal microscopic imaging system. Nucleues labeled by DAPI and kidney spherule curved pipe labeled by Alexa Fluor 488 can be imaged clearly and respectively, realizing the distinction between the different components of mouse kidney tissue. The three-dimensional tomographic imaging of mouse kidney tissue is reconstructed by several two-dimensional images obtained in different depths. At last the resolution of the confocal microscopic imaging system is tested quantitatively. The experimental result shows that the system can achieve lateral resolution priority to 230nm.

  10. Confocal Microscope Alignment of Nanocrystals for Coherent Diffraction Imaging

    International Nuclear Information System (INIS)

    Beitra, Loren; Watari, Moyu; Matsuura, Takashi; Shimamoto, Naonobu; Harder, Ross; Robinson, Ian

    2010-01-01

    We have installed and tested an Olympus LEXT confocal microscope at the 34-ID-C beamline of the Advanced Photon Source (APS). The beamline is for Coherent X-ray Diffraction (CXD) experiments in which a nanometre-sized crystal is aligned inside a focussed X-ray beam. The microscope was required for three-dimensional (3D) sample alignment to get around sphere-of-confusion issues when locating Bragg peaks in reciprocal space. In this way, and by use of strategic sample preparations, we have succeeded in measuring six Bragg peaks from a single 200 nm gold crystal and obtained six projections of its internal displacement field. This enables the clear identification of stacking-fault bands within the crystal. The confocal alignment method will allow a full determination of the strain tensor provided three or more Bragg reflections from the same crystal are found.

  11. Ghost microscope imaging system from the perspective of coherent-mode representation

    Science.gov (United States)

    Shen, Qian; Bai, Yanfeng; Shi, Xiaohui; Nan, Suqin; Qu, Lijie; Li, Hengxing; Fu, Xiquan

    2018-03-01

    The coherent-mode representation theory of partially coherent fields is firstly used to analyze a two-arm ghost microscope imaging system. It is shown that imaging quality of the generated images depend crucially on the distribution of the decomposition coefficients of the object imaged when the light source is fixed. This theory is also suitable for demonstrating the effects from the distance the object is moved away from the original plane on imaging quality. Our results are verified theoretically and experimentally.

  12. Angularly-selective transmission imaging in a scanning electron microscope.

    Science.gov (United States)

    Holm, Jason; Keller, Robert R

    2016-08-01

    This work presents recent advances in transmission scanning electron microscopy (t-SEM) imaging control capabilities. A modular aperture system and a cantilever-style sample holder that enable comprehensive angular selectivity of forward-scattered electrons are described. When combined with a commercially available solid-state transmission detector having only basic bright-field and dark-field imaging capabilities, the advances described here enable numerous transmission imaging modes. Several examples are provided that demonstrate how contrast arising from diffraction to mass-thickness can be obtained. Unanticipated image contrast at some imaging conditions is also observed and addressed. Published by Elsevier B.V.

  13. Sensing for intracellular thiols by water-insoluble two-photon fluorescent probe incorporating nanogel

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xudong; Zhang, Xin; Wang, Shuangqing; Li, Shayu [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Hu, Rui, E-mail: hurui@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Li, Yi, E-mail: yili@mail.ipc.ac.cn [Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190 (China); Yang, Guoqiang, E-mail: gqyang@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 (China)

    2015-04-15

    Highlights: • A novel “turn-on” two-photon fluorescent probe based on a π-conjugated triarylboron luminogen was designed and synthesized. • Fast, selective and sensitive detection of biothiols in 100% aqueous solution by simply loaded on a nanogel. • Single-photon and two-photon fluorescent bioimaging of biothiols in NIH/3T3 fibroblasts. - Abstract: A novel “turn-on” two-photon fluorescent probe containing a π-conjugated triarylboron luminogen and a maleimide moiety DMDP-M based on the photo-induced electron transfer (PET) mechanism for biothiol detection was designed and synthesized. By simply loading the hydrophobic DMDP-M on a cross-linked Pluronic{sup ®} F127 nanogel (CL-F127), a probing system DMDP-M/CL-F127 was established, which shows quick response, high selectivity and sensitivity to cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in aqueous phase. The DMDP-M/CL-F127 system presented the fastest response to Cys with a rate constant of 0.56 min{sup −1}, and the detection limit to Cys was calculated to be as low as 0.18 μM. The DMDP-M/CL-F127 system has been successfully applied to the fluorescence imaging of biothiols in NIH/3T3 fibroblasts either with single-photon or two-photon excitation because of its high biocompatibility and cell-membrane permeability. The present work provides a general, simple and efficient strategy for the application of hydrophobic molecules to sensing biothiols in aqueous phase, and a novel sensing system for intracellular biothiols fitted for both single-photon and two-photon fluorescence imaging.

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

    Science.gov (United States)

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

    2017-02-01

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

  15. Polarized two-photon photoselection in EGFP: Theory and experiment.

    Science.gov (United States)

    Masters, T A; Marsh, R J; Blacker, T S; Armoogum, D A; Larijani, B; Bain, A J

    2018-04-07

    In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S 0 → S 1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S 0 → S 1 transition.

  16. Polarized two-photon photoselection in EGFP: Theory and experiment

    Science.gov (United States)

    Masters, T. A.; Marsh, R. J.; Blacker, T. S.; Armoogum, D. A.; Larijani, B.; Bain, A. J.

    2018-04-01

    In this work, we present a complete theoretical description of the excited state order created by two-photon photoselection from an isotropic ground state; this encompasses both the conventionally measured quadrupolar (K = 2) and the "hidden" degree of hexadecapolar (K = 4) transition dipole alignment, their dependence on the two-photon transition tensor and emission transition dipole moment orientation. Linearly and circularly polarized two-photon absorption (TPA) and time-resolved single- and two-photon fluorescence anisotropy measurements are used to determine the structure of the transition tensor in the deprotonated form of enhanced green fluorescent protein. For excitation wavelengths between 800 nm and 900 nm, TPA is best described by a single element, almost completely diagonal, two-dimensional (planar) transition tensor whose principal axis is collinear to that of the single-photon S0 → S1 transition moment. These observations are in accordance with assignments of the near-infrared two-photon absorption band in fluorescent proteins to a vibronically enhanced S0 → S1 transition.

  17. Real-time image processing and control interface for remote operation of a microscope

    Science.gov (United States)

    Leng, Hesong; Wilder, Joseph

    1999-08-01

    A real-time image processing and control interface for remote operation of a microscope is presented in this paper. The system has achieved real-time color image display for 640 X 480 pixel images. Multi-resolution image representation can be provided for efficient transmission through the network. Through the control interface the computer can communicate with the programmable microscope via the RS232 serial ports. By choosing one of three scanning patterns, a sequence of images can be saved as BMP or PGM files to record information on an entire microscope slide. The system will be used by medical and graduate students at the University of Medicine and Dentistry of New Jersey for distance learning. It can be used in many network-based telepathology applications.

  18. Atomic Force Microscope Image Contrast Mechanisms on Supported Lipid Bilayers

    OpenAIRE

    Schneider, James; Dufrêne, Yves F.; Barger Jr., William R.; Lee, Gil U.

    2000-01-01

    This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures o...

  19. Microscope self-calibration based on micro laser line imaging and soft computing algorithms

    Science.gov (United States)

    Apolinar Muñoz Rodríguez, J.

    2018-06-01

    A technique to perform microscope self-calibration via micro laser line and soft computing algorithms is presented. In this technique, the microscope vision parameters are computed by means of soft computing algorithms based on laser line projection. To implement the self-calibration, a microscope vision system is constructed by means of a CCD camera and a 38 μm laser line. From this arrangement, the microscope vision parameters are represented via Bezier approximation networks, which are accomplished through the laser line position. In this procedure, a genetic algorithm determines the microscope vision parameters by means of laser line imaging. Also, the approximation networks compute the three-dimensional vision by means of the laser line position. Additionally, the soft computing algorithms re-calibrate the vision parameters when the microscope vision system is modified during the vision task. The proposed self-calibration improves accuracy of the traditional microscope calibration, which is accomplished via external references to the microscope system. The capability of the self-calibration based on soft computing algorithms is determined by means of the calibration accuracy and the micro-scale measurement error. This contribution is corroborated by an evaluation based on the accuracy of the traditional microscope calibration.

  20. Interference of two photons in parametric down conversion

    International Nuclear Information System (INIS)

    Ghosh, R.; Hong, C.K.; Ou, Z.Y.; Mandel, L.

    1986-01-01

    A theoretical treatment is given of the process in which the two photons produced simultaneously in the parametric frequency splitting of light are allowed to interfere. It is shown that, while there is no interference in the usual sense involving quantities that are of the second order in the field, fourth-order interference effects are present. These may be revealed by measuring the joint probability of detecting two photons at two points x,x' in the interference plane with photoelectric detectors as a function of the separation x-x'. The probability exhibits a cosine modulation with x-x', with visibility that can approach 100%, even though the integration time in the experiment may greatly exceed the reciprocal bandwidth of the photons. The interference effect has a nonclassical origin and implies a violation of local realism in the highly correlated two-photon state

  1. Quantum efficiency and two-photon absorption cross-section of conjugated polyelectrolytes used for protein conformation measurements with applications on amyloid structures

    Energy Technology Data Exchange (ETDEWEB)

    Stabo-Eeg, Frantz [Norwegian University of Science and Technology, N-7491 Trondheim (Norway)], E-mail: Frantz.Stabo-Eeg@phys.ntnu.no; Lindgren, Mikael [Norwegian University of Science and Technology, N-7491 Trondheim (Norway); Nilsson, K. Peter R.; Inganaes, Olle; Hammarstroem, Per [IFM Department of Physics, Chemistry and Biology Linkoeping University, S-581 83 Linkoeping (Sweden)

    2007-07-27

    Amyloid diseases such as Alzheimer's and spongiform encephalopathies evolve from aggregation of proteins due to misfolding of the protein structure. Early disease handling require sophisticated but yet simple techniques to follow the complex properties of the aggregation process. Conjugated polyelectrolytes (CPEs) have shown promising capabilities acting as optical biological sensors, since they can specifically bind to polypeptides both in solution and in solid phase. The structural changes in biomolecules can be monitored by changes of the optical spectra of the CPEs, both in absorption and emission modes. Notably, the studied CPEs possess multi-photon excitation capability, making them potential for in vivo imaging using laser scanning microscopy. Aggregation of proteins depends on concentration, temperature and pH. The optical effect on the molecular probe in various environments must also be investigated if applied in these environments. Here we present the results of quantum efficiency and two-photon absorption cross-section of three CPEs: POMT, POWT and PTAA in three different pH buffer systems. The extinction coefficient and quantum efficiency were measured. POMT was found to have the highest quantum efficiency being approximately 0.10 at pH 2.0. The two-photon absorption cross-section was measured for POMT and POWT and was found to be more than 18-25 times and 7-11 times that of Fluorescein, respectively. We also show how POMT fluorescence can be used to distinguish conformational differences between amyloid fibrils formed from reduced and non-reduced insulin in spectrally resolved images recorded with a laser scanning microscope using both one- and two-photon excitation.

  2. High-resolution imaging in the scanning transmission electron microscope

    International Nuclear Information System (INIS)

    Pennycook, S.J.; Jesson, D.E.

    1992-03-01

    The high-resolution imaging of crystalline materials in the scanning transmission electron microscopy (STEM) is reviewed with particular emphasis on the conditions under which an incoherent image can be obtained. It is shown that a high-angle annular detector can be used to break the coherence of the imaging process, in the transverse plane through the geometry of the detector, or in three dimensions if multiphonon diffuse scattering is detected. In the latter case, each atom can be treated as a highly independent source of high-angle scattering. The most effective fast electron states are therefore tightly bound s-type Bloch states. Furthermore, they add constructively for each incident angle in the coherent STEM probe, so that s states are responsible for practically the entire image contrast. Dynamical effects are largely removed, and almost perfect incoherent imaging is achieved. s states are relatively insensitive to neighboring strings, so that incoherent imaging is maintained for superlattice and interfaces, and supercell calculations are unnecessary. With an optimum probe profile, the incoherent image represents a direct image of the crystal projection, with compositional sensitivity built in through the strong dependence of the scattering cross sections on atomic number Z

  3. Measurement of wavefront aberrations in cortex and peripheral nerve using a two-photon excitation guidestar

    Science.gov (United States)

    Futia, Gregory L.; Fontaine, Arjun; McCullough, Connor; Ozbay, Baris N.; George, Nickolas M.; Caldwell, John; Restrepo, Diego; Weir, Richard; Gibson, Emily A.

    2018-02-01

    Neural-machine interfaces using optogenetics are of interest due to their minimal invasiveness and potential for parallel read in and read out of activity. One possible biological target for such an interface is the peripheral nerve, where axonlevel imaging or stimulation could greatly improve interfacing with artificial limbs or enable neuron/fascicle level neuromodulation in the vagus nerve. Two-photon imaging has been successful in imaging brain activity using genetically encoded calcium or voltage indicators, but in the peripheral nerve, this is severely limited by scattering and aberrations from myelin. We employ a Shack-Hartman wavefront sensor and two-photon excitation guidestar to quantify optical scattering and aberrations in peripheral nerves and cortex. The sciatic and vagus nerves, and cortex from a ChAT-Cre ChR-eYFP transgenic mouse were excised and imaged directly. In peripheral nerves, defocus was the strongest aberration followed by astigmatism and coma. Peripheral nerve had orders of magnitude higher aberration compared with cortex. These results point to the potential of adaptive optics for increasing the depth of two-photon access into peripheral nerves.

  4. Multi-technology Integration Based on Low-contrast Microscopic Image Enhancement

    Directory of Open Access Journals (Sweden)

    Haoge Ma

    2014-01-01

    Full Text Available Microscopic image enhancement is an important issue of image processing technique, which is used to improve the visual quality of image. This paper describes a novel multi resolution image segmentation algorithm for low DOF images. The algorithm is designed to separate a sharply focused object of interest from other foreground or background objects. The algorithm is fully automatic in that all parameters are image in dependent. A multiscale-approach based on high frequency wavelet coefficients and their statistics is used to perform context dependent classification of individual blocks of the image. Compared with the state of the art algorithms, this new algorithm provides better accuracy at higher speed.

  5. The importance of radiographic imaging in the microscopic assessment of bone tumors

    International Nuclear Information System (INIS)

    Larousserie, F.; Kreshak, J.; Gambarotti, M.; Alberghini, M.; Vanel, D.

    2013-01-01

    Introduction: Primary bone tumors are rare and require a multidisciplinary approach. Diagnosis involves primarily the radiologist and the pathologist. Bone lesions are often heterogeneous and the microscopic diagnostic component(s) may be in the minority, especially on core needle biopsies. Reactive processes, benign, and malignant tumors may have similar microscopic aspects. For these challenging cases, the correlation of microscopic and radiologic information is critical, or diagnostic mistakes may be made with severe clinical consequences for the patient. The purpose of this article is to explain how pathologists can best use imaging studies to improve the diagnostic accuracy of bone lesions. Diagnosis: Many bone lesions are microscopically and/or radiographically heterogeneous, especially those with both lytic and matrix components. Final diagnosis may require specific microscopic diagnostic features that may be present in the lesion, but not the biopsy specimen. A review of the imaging helps assess if sampling was adequate. The existence of a pre-existing bone lesion, syndrome (such as Ollier disease or multiple hereditary exostosis), or oncologic history may be of crucial importance. Finally, imaging information is very useful for the pathologist to perform accurate local and regional staging during gross examination. Conclusion: Close teamwork between pathologists, radiologists, and clinicians is of utmost importance in the evaluation and management of bone tumors. These lesions can be very difficult to interpret microscopically; imaging studies therefore play a crucial role in their accurate diagnosis

  6. The importance of radiographic imaging in the microscopic assessment of bone tumors

    Energy Technology Data Exchange (ETDEWEB)

    Larousserie, F., E-mail: frederique.larousserie@cch.aphp.fr [Université Paris Descartes, Sorbonne Paris Cité, Paris (France); Department of pathology, Rizzoli Institute, Bologna (Italy); Kreshak, J.; Gambarotti, M.; Alberghini, M.; Vanel, D. [Department of pathology, Rizzoli Institute, Bologna (Italy)

    2013-12-01

    Introduction: Primary bone tumors are rare and require a multidisciplinary approach. Diagnosis involves primarily the radiologist and the pathologist. Bone lesions are often heterogeneous and the microscopic diagnostic component(s) may be in the minority, especially on core needle biopsies. Reactive processes, benign, and malignant tumors may have similar microscopic aspects. For these challenging cases, the correlation of microscopic and radiologic information is critical, or diagnostic mistakes may be made with severe clinical consequences for the patient. The purpose of this article is to explain how pathologists can best use imaging studies to improve the diagnostic accuracy of bone lesions. Diagnosis: Many bone lesions are microscopically and/or radiographically heterogeneous, especially those with both lytic and matrix components. Final diagnosis may require specific microscopic diagnostic features that may be present in the lesion, but not the biopsy specimen. A review of the imaging helps assess if sampling was adequate. The existence of a pre-existing bone lesion, syndrome (such as Ollier disease or multiple hereditary exostosis), or oncologic history may be of crucial importance. Finally, imaging information is very useful for the pathologist to perform accurate local and regional staging during gross examination. Conclusion: Close teamwork between pathologists, radiologists, and clinicians is of utmost importance in the evaluation and management of bone tumors. These lesions can be very difficult to interpret microscopically; imaging studies therefore play a crucial role in their accurate diagnosis.

  7. Extended morphological processing: a practical method for automatic spot detection of biological markers from microscopic images.

    Science.gov (United States)

    Kimori, Yoshitaka; Baba, Norio; Morone, Nobuhiro

    2010-07-08

    A reliable extraction technique for resolving multiple spots in light or electron microscopic images is essential in investigations of the spatial distribution and dynamics of specific proteins inside cells and tissues. Currently, automatic spot extraction and characterization in complex microscopic images poses many challenges to conventional image processing methods. A new method to extract closely located, small target spots from biological images is proposed. This method starts with a simple but practical operation based on the extended morphological top-hat transformation to subtract an uneven background. The core of our novel approach is the following: first, the original image is rotated in an arbitrary direction and each rotated image is opened with a single straight line-segment structuring element. Second, the opened images are unified and then subtracted from the original image. To evaluate these procedures, model images of simulated spots with closely located targets were created and the efficacy of our method was compared to that of conventional morphological filtering methods. The results showed the better performance of our method. The spots of real microscope images can be quantified to confirm that the method is applicable in a given practice. Our method achieved effective spot extraction under various image conditions, including aggregated target spots, poor signal-to-noise ratio, and large variations in the background intensity. Furthermore, it has no restrictions with respect to the shape of the extracted spots. The features of our method allow its broad application in biological and biomedical image information analysis.

  8. Doppler-free two-photon excitation of 238U

    International Nuclear Information System (INIS)

    Hodgkinson, D.P.; Wort, D.J.H.

    1981-04-01

    A theory of resonantly enhanced two-photon absorption is presented and tested in a number of experiments in which 238 U vapour is excited by two continuous wave dye lasers. Good quantitative agreement between theory and experiment is found. In particular the central prediction of the theory, that antiparallel laser beams of modest intensity can pump an appreciable fraction of the Maxwell velocity distribution, has been checked directly by measuring the spectral width of the fluorescence from the two-photon excited level. (author)

  9. Microscopic validation of whole mouse micro-metastatic tumor imaging agents using cryo-imaging and sliding organ image registration

    Science.gov (United States)

    Liu, Yiqiao; Zhou, Bo; Qutaish, Mohammed; Wilson, David L.

    2016-03-01

    We created a metastasis imaging, analysis platform consisting of software and multi-spectral cryo-imaging system suitable for evaluating emerging imaging agents targeting micro-metastatic tumor. We analyzed CREKA-Gd in MRI, followed by cryo-imaging which repeatedly sectioned and tiled microscope images of the tissue block face, providing anatomical bright field and molecular fluorescence, enabling 3D microscopic imaging of the entire mouse with single metastatic cell sensitivity. To register MRI volumes to the cryo bright field reference, we used our standard mutual information, non-rigid registration which proceeded: preprocess --> affine --> B-spline non-rigid 3D registration. In this report, we created two modified approaches: mask where we registered locally over a smaller rectangular solid, and sliding organ. Briefly, in sliding organ, we segmented the organ, registered the organ and body volumes separately and combined results. Though sliding organ required manual annotation, it provided the best result as a standard to measure other registration methods. Regularization parameters for standard and mask methods were optimized in a grid search. Evaluations consisted of DICE, and visual scoring of a checkerboard display. Standard had accuracy of 2 voxels in all regions except near the kidney, where there were 5 voxels sliding. After mask and sliding organ correction, kidneys sliding were within 2 voxels, and Dice overlap increased 4%-10% in mask compared to standard. Mask generated comparable results with sliding organ and allowed a semi-automatic process.

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

    Science.gov (United States)

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

    2013-02-01

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

  11. Automated Image Analysis of Lung Branching Morphogenesis from Microscopic Images of Fetal Rat Explants

    Science.gov (United States)

    Rodrigues, Pedro L.; Rodrigues, Nuno F.; Duque, Duarte; Granja, Sara; Correia-Pinto, Jorge; Vilaça, João L.

    2014-01-01

    Background. Regulating mechanisms of branching morphogenesis of fetal lung rat explants have been an essential tool for molecular research. This work presents a new methodology to accurately quantify the epithelial, outer contour, and peripheral airway buds of lung explants during cellular development from microscopic images. Methods. The outer contour was defined using an adaptive and multiscale threshold algorithm whose level was automatically calculated based on an entropy maximization criterion. The inner lung epithelium was defined by a clustering procedure that groups small image regions according to the minimum description length principle and local statistical properties. Finally, the number of peripheral buds was counted as the skeleton branched ends from a skeletonized image of the lung inner epithelia. Results. The time for lung branching morphometric analysis was reduced in 98% in contrast to the manual method. Best results were obtained in the first two days of cellular development, with lesser standard deviations. Nonsignificant differences were found between the automatic and manual results in all culture days. Conclusions. The proposed method introduces a series of advantages related to its intuitive use and accuracy, making the technique suitable to images with different lighting characteristics and allowing a reliable comparison between different researchers. PMID:25250057

  12. Automated Image Analysis of Lung Branching Morphogenesis from Microscopic Images of Fetal Rat Explants

    Directory of Open Access Journals (Sweden)

    Pedro L. Rodrigues

    2014-01-01

    Full Text Available Background. Regulating mechanisms of branching morphogenesis of fetal lung rat explants have been an essential tool for molecular research. This work presents a new methodology to accurately quantify the epithelial, outer contour, and peripheral airway buds of lung explants during cellular development from microscopic images. Methods. The outer contour was defined using an adaptive and multiscale threshold algorithm whose level was automatically calculated based on an entropy maximization criterion. The inner lung epithelium was defined by a clustering procedure that groups small image regions according to the minimum description length principle and local statistical properties. Finally, the number of peripheral buds was counted as the skeleton branched ends from a skeletonized image of the lung inner epithelia. Results. The time for lung branching morphometric analysis was reduced in 98% in contrast to the manual method. Best results were obtained in the first two days of cellular development, with lesser standard deviations. Nonsignificant differences were found between the automatic and manual results in all culture days. Conclusions. The proposed method introduces a series of advantages related to its intuitive use and accuracy, making the technique suitable to images with different lighting characteristics and allowing a reliable comparison between different researchers.

  13. Imaging cardiomyocytes in intact tissue with a remote focusing microscope

    Science.gov (United States)

    Corbett, A. D.; Burton, R. A. B.; Bub, G.; Wilson, T.

    2015-03-01

    In cardiac imaging, the spacing between sub-cellular sarcomere structures is of great importance to physiologists in understanding muscle design and performance. Making accurate measurements of the sarcomere length (SL) presents a significant imaging challenge owing to the size of the SL (~2μm) and its naturally low variability (pathological models of chronic hypertension. As well as improving measurement precision, the distribution of α across the field of view provides additional structural information which can be related to disease morphology. To validate this new imaging protocol, the value ofα calculated from the oblique planes provided the input to a rigid model cell which was used to predict the appearance of the cell in the conventional focal plane. The comparison of the model to the image data provided a confidence metric for our measurements. Finally, by considering the optical transfer function, the range of cell orientations for which the method is valid could be calculated.

  14. Automated Analysis of Microscopic Images of Isolated Pancreatic Islets

    Czech Academy of Sciences Publication Activity Database

    Habart, D.; Švihlík, J.; Schier, Jan; Cahová, M.; Girman, P.; Zacharovová, K.; Berková, Z.; Kříž, J.; Fabryová, E.; Kosinová, L.; Papáčková, Z.; Kybic, J.; Saudek, F.

    2016-01-01

    Roč. 25, č. 12 (2016), s. 2145-2156 ISSN 0963-6897 Grant - others:GA ČR(CZ) GA14-10440S Institutional support: RVO:67985556 Keywords : enumeration of islets * image processing * image segmentation * islet transplantation * machine-learning * quality control Subject RIV: IN - Informatics, Computer Science Impact factor: 3.006, year: 2016 http://library.utia.cas.cz/separaty/2016/ZOI/schier-0465945.pdf

  15. Design of an imaging microscope for soft X-ray applications

    Science.gov (United States)

    Hoover, Richard B.; Shealy, David L.; Gabardi, David R.; Walker, Arthur B. C., Jr.; Lindblom, Joakim F.

    1988-01-01

    An imaging soft X-ray microscope with a spatial resolution of 0.1 micron and normal incidence multilayer optics is discussed. The microscope has a Schwarzschild configuration, which consists of two concentric spherical mirrors with radii of curvature which minimize third-order spherical aberration, coma, and astigmatism. The performance of the Stanford/MSFC Cassegrain X-ray telescope and its relevance to the present microscope are addressed. A ray tracing analysis of the optical system indicates that diffraction-limited performance can be expected for an object height of 0.2 mm.

  16. Dual-mode optical microscope based on single-pixel imaging

    OpenAIRE

    Rodríguez Jiménez, Angel David; Clemente Pesudo, Pedro Javier; Tajahuerce, Enrique; Lancis Sáez, Jesús

    2016-01-01

    We demonstrate an inverted microscope that can image specimens in both reflection and transmission modes simultaneously with a single light source. The microscope utilizes a digital micromirror device (DMD) for patterned illumination altogether with two single-pixel photosensors for efficient light detection. The system, a scan-less device with no moving parts, works by sequential projection of a set of binary intensity patterns onto the sample that are codified onto a modified commercial DMD...

  17. Automated and unsupervised detection of malarial parasites in microscopic images

    Directory of Open Access Journals (Sweden)

    Purwar Yashasvi

    2011-12-01

    Full Text Available Abstract Background Malaria is a serious infectious disease. According to the World Health Organization, it is responsible for nearly one million deaths each year. There are various techniques to diagnose malaria of which manual microscopy is considered to be the gold standard. However due to the number of steps required in manual assessment, this diagnostic method is time consuming (leading to late diagnosis and prone to human error (leading to erroneous diagnosis, even in experienced hands. The focus of this study is to develop a robust, unsupervised and sensitive malaria screening technique with low material cost and one that has an advantage over other techniques in that it minimizes human reliance and is, therefore, more consistent in applying diagnostic criteria. Method A method based on digital image processing of Giemsa-stained thin smear image is developed to facilitate the diagnostic process. The diagnosis procedure is divided into two parts; enumeration and identification. The image-based method presented here is designed to automate the process of enumeration and identification; with the main advantage being its ability to carry out the diagnosis in an unsupervised manner and yet have high sensitivity and thus reducing cases of false negatives. Results The image based method is tested over more than 500 images from two independent laboratories. The aim is to distinguish between positive and negative cases of malaria using thin smear blood slide images. Due to the unsupervised nature of method it requires minimal human intervention thus speeding up the whole process of diagnosis. Overall sensitivity to capture cases of malaria is 100% and specificity ranges from 50-88% for all species of malaria parasites. Conclusion Image based screening method will speed up the whole process of diagnosis and is more advantageous over laboratory procedures that are prone to errors and where pathological expertise is minimal. Further this method

  18. Atomic force microscope image contrast mechanisms on supported lipid bilayers.

    Science.gov (United States)

    Schneider, J; Dufrêne, Y F; Barger, W R; Lee, G U

    2000-08-01

    This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.

  19. Fluorescence excitation analysis by two-photon confocal laser scanning microscopy: a new method to identify fluorescent nanoparticles on histological tissue sections

    Directory of Open Access Journals (Sweden)

    Kahn E

    2012-10-01

    Full Text Available Edmond Kahn,1 Nicolas Tissot,3 Perrine Frere,3 Aurélien Dauphin,3 Mohamed Boumhras,2,4 Claude-Marie Bachelet,3 Frédérique Frouin,1 Gérard Lizard21Institut National de la Santé et de la Recherche Médicale (INSERM U678/UMR-S UPMC, CHU Pitié-Salpêtrière, Paris, France; 2Equipe Biochimie du Peroxysome, Inflammation et Métabolisme Lipidique EA7270, Faculté des Sciences Gabriel, Université de Bourgogne-INSERM Dijon, France; 3Plateforme d'Imagerie cellulaire, UPMC, Paris, France; 4Laboratory of Biochemistry and Neuroscience, Applied Toxicology Group, Faculty of Science and Technology, Settat, MoroccoAbstract: In the present study, we make use of the ability of two-photon confocal laser scanning microscopes (CLSMs equipped with tunable lasers to produce spectral excitation image sequences. Furthermore, unmixing, which is usually performed on emission image sequences, is performed on these excitation image sequences. We use factor analysis of medical image sequences (FAMIS, which produces factor images, to unmix spectral image sequences of stained structures in tissue sections to provide images of characterized stained cellular structures. This new approach is applied to histological tissue sections of mouse aorta containing labeled iron nanoparticles stained with Texas Red and counterstained with SYTO13, to obtain visual information about the accumulation of these nanoparticles in the arterial wall. The possible presence of Texas Red is determined using a two-photon CLSM associated with FAMIS via the excitation spectra. Texas Red and SYTO13 are thus differentiated, and corresponding factor images specify their possible presence and cellular localization. In conclusion, the designed protocol shows that sequences of images obtained by excitation in a two-photon CLSM enables characterization of Texas Red-stained nanoparticles and other markers. This methodology offers an alternative and complementary solution to the conventional use of emission

  20. Precision two-photon spectroscopy of alkali elements

    Indian Academy of Sciences (India)

    effect is eliminated if the wave vector of the photons is ka = −kb, i.e., the two beams .... atomic cesium, and the metre is (indirectly) defined from the wavelength of .... plasma absorb radiation, the electrical circuit parameters may vary and this .... two-photon excitation followed by an ionization step in Ca [71], Sr [72] and Gd [73] ...

  1. Two-photon couplings of quarkonia with arbitrary JPC

    International Nuclear Information System (INIS)

    Barnes, T.; Tennessee Univ., Knoxville, TN

    1992-01-01

    We present theoretical results for the two-photon widths of relativistic quarkonium states with arbitrary angular momenta. These relativistic formulas are required to obtain reasonable agreement with the absolute scale of quarkonium decay rates to two photons, and have previously only been derived for spin-singlet q bar q states. We also evaluate these formulas numerically for ell ≤3 q = u, d states in a Coulomb-plus-linear q bar q potential model. Light-quark higher-ell and radially-excited q bar q states should be observable experimentally, as their two-photon widths are typically found to be ∼1 KeV. The radially-excited 1 S 0 higher-mass quarkonium states such as c bar c and b bar b should also be observable in γγ, but orbitally-excited c bar c states with ell>1 and b bar b states with ell>0 are expected to have very small two-photon widths. The helicity structure of the higher-ell q bar q couplings is predicted to be nontrivial, with both λ=0 and λ=2γγ final states contributing significantly; these results may be useful as signatures for q bar q states

  2. Two-photon optics of Bessel-Gaussian modes

    CSIR Research Space (South Africa)

    McLaren, M

    2013-09-01

    Full Text Available In this paper we consider geometrical two-photon optics of Bessel-Gaussian modes generated in spontaneous parametric down-conversion of a Gaussian pump beam. We provide a general theoretical expression for the orbital angular momentum (OAM) spectrum...

  3. Space-time description of the two-photon decay

    International Nuclear Information System (INIS)

    Hrasko, P.

    1981-09-01

    The time correlation of photons in a two-photon decay is shown to depend on the instantaneous nature of the wave-function collapse in an essential way so the latter hypothesis can be verified by the experimental study of these correlations. (author)

  4. Positron two-photon annihilation coincidence technique: difference mode

    Energy Technology Data Exchange (ETDEWEB)

    Karol, P J; Klobuchar, R L [Carnegie-Mellon Univ., Pittsburgh, Pa. (USA). Dept. of Chemistry

    1978-05-01

    A difference (or comparative) mode in the measurement of two-photon coincidences from positron or positronium annihilation has been developed. The method can be used to advantage, particularly in gases, in determining annihilation parameters such as quenching cross sections for low concentration strongly quenching chemical species in a reference medium which is relatively non-quenching.

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

    Science.gov (United States)

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

    2000-04-01

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

  6. A universal fluid cell for the imaging of biological specimens in the atomic force microscope.

    Science.gov (United States)

    Kasas, Sandor; Radotic, Ksenja; Longo, Giovanni; Saha, Bashkar; Alonso-Sarduy, Livan; Dietler, Giovanni; Roduit, Charles

    2013-04-01

    Recently, atomic force microscope (AFM) manufacturers have begun producing instruments specifically designed to image biological specimens. In most instances, they are integrated with an inverted optical microscope, which permits concurrent optical and AFM imaging. An important component of the set-up is the imaging chamber, whose design determines the nature of the experiments that can be conducted. Many different imaging chamber designs are available, usually designed to optimize a single parameter, such as the dimensions of the substrate or the volume of fluid that can be used throughout the experiment. In this report, we present a universal fluid cell, which simultaneously optimizes all of the parameters that are important for the imaging of biological specimens in the AFM. This novel imaging chamber has been successfully tested using mammalian, plant, and microbial cells. Copyright © 2013 Wiley Periodicals, Inc.

  7. Soft X-ray imaging with axisymmetry microscope and electronic readout

    International Nuclear Information System (INIS)

    Sauneuf, A.; Cavailler, C.; Henry, Ph.; Launspach, J.; Mascureau, J. de; Rostaing, M.

    1984-11-01

    An axisymmetric microscope with 10 X magnification has been constructed; its resolution has been measured using severals grids, backlighted by an X-ray source and found to be near 25 μm. So it could be used to make images of laser driven plasmas in the soft X-ray region. In order to see rapidly those images we have associated it with a new detector. It is a small image converter tube with a soft X-ray photocathode and a P20 phosphor deposited on an optic fiber plate. The electronic image appearing on the screen is read by a CCD working in the spectral range. An electronic image readout chain, which is identical to those we use with streak cameras, then processes automatically and immediatly the images given by the microscope

  8. Microscopic imaging ellipsometry of submicron-scale bacterial cells

    African Journals Online (AJOL)

    MIE can detect very thin layers and measure their precise ... for future applications especially in the field of biological imaging since it ... a quarter-wave plate and rotates with the range of angles from 5º ... calibrated first by using a flat substrate like glass,. Si, or Au. .... more sensitive detection of the boundary of the structure ...

  9. Removal of Vesicle Structures from Transmission Electron Microscope Images

    DEFF Research Database (Denmark)

    Jensen, Katrine Hommelhoff; Sigworth, Fred; Brandt, Sami Sebastian

    2015-01-01

    In this paper, we address the problem of imaging membrane proteins for single-particle cryo-electron microscopy reconstruction of the isolated protein structure. More precisely, we propose a method for learning and removing the interfering vesicle signals from the micrograph, prior to reconstruct...

  10. In situ atomic force microscope imaging of supported lipid bilayers

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Leidy, Chad; Ipsen, John Hjorth

    2001-01-01

    In situ AFM images of phospholipase A/sub 2/ (PLA/sub 2/) hydrolysis of mica-supported one- and two-component lipid bilayers are presented. For one-component DPPC bilayers an enhanced enzymatic activity is observed towards preexisting defects in the bilayer. Phase separation is observed in two-co...

  11. 3D microscopic imaging and evaluation of tubular tissue architecture

    Czech Academy of Sciences Publication Activity Database

    Janáček, Jiří; Čapek, Martin; Michálek, Jan; Karen, Petr; Kubínová, Lucie

    2014-01-01

    Roč. 63, Suppl.1 (2014), S49-S55 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH13028; GA ČR(CZ) GA13-12412S Institutional support: RVO:67985823 Keywords : confocal microscopy * capillaries * brain * skeletal muscle * image analysis Subject RIV: EA - Cell Biology Impact factor: 1.293, year: 2014

  12. Microdose fluorescence imaging of ABY-029 on an operating microscope adapted by custom illumination and imaging modules.

    Science.gov (United States)

    Elliott, Jonathan T; Dsouza, Alisha V; Marra, Kayla; Pogue, Brian W; Roberts, David W; Paulsen, Keith D

    2016-09-01

    Fluorescence guided surgery has the potential to positively impact surgical oncology; current operating microscopes and stand-alone imaging systems are too insensitive or too cumbersome to maximally take advantage of new tumor-specific agents developed through the microdose pathway. To this end, a custom-built illumination and imaging module enabling picomolar-sensitive near-infrared fluorescence imaging on a commercial operating microscope is described. The limits of detection and system specifications are characterized, and in vivo efficacy of the system in detecting ABY-029 is evaluated in a rat orthotopic glioma model following microdose injections, showing the suitability of the device for microdose phase 0 clinical trials.

  13. Simulation of high-resolution X-ray microscopic images for improved alignment

    International Nuclear Information System (INIS)

    Song Xiangxia; Zhang Xiaobo; Liu Gang; Cheng Xianchao; Li Wenjie; Guan Yong; Liu Ying; Xiong Ying; Tian Yangchao

    2011-01-01

    The introduction of precision optical elements to X-ray microscopes necessitates fine realignment to achieve optimal high-resolution imaging. In this paper, we demonstrate a numerical method for simulating image formation that facilitates alignment of the source, condenser, objective lens, and CCD camera. This algorithm, based on ray-tracing and Rayleigh-Sommerfeld diffraction theory, is applied to simulate the X-ray microscope beamline U7A of National Synchrotron Radiation Laboratory (NSRL). The simulations and imaging experiments show that the algorithm is useful for guiding experimental adjustments. Our alignment simulation method is an essential tool for the transmission X-ray microscope (TXM) with optical elements and may also be useful for the alignment of optical components in other modes of microscopy.

  14. A multiphoton laser scanning microscope setup for transcranial in vivo brain imaging on mice

    Science.gov (United States)

    Nase, Gabriele; Helm, P. Johannes; Reppen, Trond; Ottersen, Ole Petter

    2005-12-01

    We describe a multiphoton laser scanning microscope setup for transcranial in vivo brain imaging in mice. The modular system is based on a modified industrial standard Confocal Scanning Laser Microscope (CSLM) and is assembled mainly from commercially available components. A special multifunctional stage, which is optimized for both laser scanning microscopic observation and preparative animal surgery, has been developed and built. The detection unit includes a highly efficient photomultiplier tube installed in a Peltier-cooled thermal box shielding the detector from changes in room temperature and from distortions caused by external electromagnetic fields. The images are recorded using a 12-bit analog-to-digital converter. Depending on the characteristics of the staining, individual nerve cells can be imaged down to at least 100μm below the intact cranium and down to at least 200μm below the opened cranium.

  15. NMR imaging: A 'chemical' microscope for coal analysis

    International Nuclear Information System (INIS)

    French, D.C.; Dieckman, S.L.; Gopalsami, N.; Botto, R.E.

    1991-01-01

    This paper presents a new three-dimensional (3-D) nuclear magnetic resonance (NMR) imaging technique for spatially mapping proton distributions in whole coals and solvent-swollen coal samples. The technique is based on a 3-D back-projection protocol for data acquisition, and a reconstruction technique based on 3-D Radon transform inversion. In principle, the 3-D methodology provides higher spatial resolution of solid materials than is possible with conventional slice-selection protocols. The applicability of 3-D NMR imaging has been demonstrated by mapping the maceral phases in Utah Blind Canyon (APCS number-sign 6) coal and the distribution of mobile phases in Utah coal swollen with deuterated and protic pyridine. 7 refs., 5 figs

  16. Automatic analysis of microscopic images of red blood cell aggregates

    Science.gov (United States)

    Menichini, Pablo A.; Larese, Mónica G.; Riquelme, Bibiana D.

    2015-06-01

    Red blood cell aggregation is one of the most important factors in blood viscosity at stasis or at very low rates of flow. The basic structure of aggregates is a linear array of cell commonly termed as rouleaux. Enhanced or abnormal aggregation is seen in clinical conditions, such as diabetes and hypertension, producing alterations in the microcirculation, some of which can be analyzed through the characterization of aggregated cells. Frequently, image processing and analysis for the characterization of RBC aggregation were done manually or semi-automatically using interactive tools. We propose a system that processes images of RBC aggregation and automatically obtains the characterization and quantification of the different types of RBC aggregates. Present technique could be interesting to perform the adaptation as a routine used in hemorheological and Clinical Biochemistry Laboratories because this automatic method is rapid, efficient and economical, and at the same time independent of the user performing the analysis (repeatability of the analysis).

  17. Precision crystal alignment for high-resolution electron microscope imaging

    International Nuclear Information System (INIS)

    Wood, G.J.; Beeching, M.J.

    1990-01-01

    One of the more difficult tasks involved in obtaining quality high-resolution electron micrographs is the precise alignment of a specimen into the required zone. The current accepted procedure, which involves changing to diffraction mode and searching for symmetric point diffraction pattern, is insensitive to small amounts of misalignment and at best qualitative. On-line analysis of the fourier space representation of the image, both for determining and correcting crystal tilt, is investigated. 8 refs., 42 figs

  18. Direct microscopic image and measurement of the atomization process of a port fuel injector

    International Nuclear Information System (INIS)

    Esmail, Mohamed; Kawahara, Nobuyuki; Tomita, Eiji; Sumida, Mamoru

    2010-01-01

    The main objective of this study is to observe and investigate the phenomena of atomization, i.e. the fuel break-up process very close to the nozzle exit of a practical port fuel injector (PFI). In order to achieve this objective, direct microscopic images of the atomization process were obtained using an ultra-high-speed video camera that could record 102 frames at rates of up to 1 Mfps, coupled with a long-distance microscope and Barlow lens. The experiments were carried out using a PFI in a closed chamber at atmospheric pressure. Time-series images of the spray behaviour were obtained with a high temporal resolution using backlighting. The direct microscopic images of a liquid column break-up were compared with experimental results from laser-induced exciplex fluorescence (LIEF), and the wavelength obtained from the experimental results compared with that predicated from the Kelvin–Helmholtz break-up model. The droplet size diameters from a ligament break-up were compared with results predicated from Weber's analysis. Furthermore, experimental results of the mean droplet diameter from a direct microscopic image were compared with the results obtained from phase Doppler anemometry (PDA) experimental results. Three conclusions were obtained from this study. The atomization processes and detailed characterizations of the break-up of a liquid column were identified; the direct microscopic image results were in good agreement with the results obtained from LIEF, experimental results of the wavelength were in good agreement with those from the Kelvin–Helmholtz break-up model. The break-up process of liquid ligaments into droplets was investigated, and Weber's analysis of the predicated droplet diameter from ligament break-up was found to be applicable only at larger wavelengths. Finally, the direct microscopic image method and PDA method give qualitatively similar trends for droplet size distribution and quantitatively similar values of Sauter mean diameter

  19. Direct microscopic image and measurement of the atomization process of a port fuel injector

    Science.gov (United States)

    Esmail, Mohamed; Kawahara, Nobuyuki; Tomita, Eiji; Sumida, Mamoru

    2010-07-01

    The main objective of this study is to observe and investigate the phenomena of atomization, i.e. the fuel break-up process very close to the nozzle exit of a practical port fuel injector (PFI). In order to achieve this objective, direct microscopic images of the atomization process were obtained using an ultra-high-speed video camera that could record 102 frames at rates of up to 1 Mfps, coupled with a long-distance microscope and Barlow lens. The experiments were carried out using a PFI in a closed chamber at atmospheric pressure. Time-series images of the spray behaviour were obtained with a high temporal resolution using backlighting. The direct microscopic images of a liquid column break-up were compared with experimental results from laser-induced exciplex fluorescence (LIEF), and the wavelength obtained from the experimental results compared with that predicated from the Kelvin-Helmholtz break-up model. The droplet size diameters from a ligament break-up were compared with results predicated from Weber's analysis. Furthermore, experimental results of the mean droplet diameter from a direct microscopic image were compared with the results obtained from phase Doppler anemometry (PDA) experimental results. Three conclusions were obtained from this study. The atomization processes and detailed characterizations of the break-up of a liquid column were identified; the direct microscopic image results were in good agreement with the results obtained from LIEF, experimental results of the wavelength were in good agreement with those from the Kelvin-Helmholtz break-up model. The break-up process of liquid ligaments into droplets was investigated, and Weber's analysis of the predicated droplet diameter from ligament break-up was found to be applicable only at larger wavelengths. Finally, the direct microscopic image method and PDA method give qualitatively similar trends for droplet size distribution and quantitatively similar values of Sauter mean diameter.

  20. Internal scanning method as unique imaging method of optical vortex scanning microscope

    Science.gov (United States)

    Popiołek-Masajada, Agnieszka; Masajada, Jan; Szatkowski, Mateusz

    2018-06-01

    The internal scanning method is specific for the optical vortex microscope. It allows to move the vortex point inside the focused vortex beam with nanometer resolution while the whole beam stays in place. Thus the sample illuminated by the focused vortex beam can be scanned just by the vortex point. We show that this method enables high resolution imaging. The paper presents the preliminary experimental results obtained with the first basic image recovery procedure. A prospect of developing more powerful tools for topography recovery with the optical vortex scanning microscope is discussed shortly.

  1. New method for thickness determination and microscopic imaging of graphene-like two-dimensional materials

    International Nuclear Information System (INIS)

    Qin Xudong; Chen Yonghai; Liu Yu; Zhu Laipan; Li Yuan; Wu Qing; Huang Wei

    2016-01-01

    We employed the microscopic reflectance difference spectroscopy (micro-RDS) to determine the layer-number and microscopically image the surface topography of graphene and MoS 2 samples. The contrast image shows the efficiency and reliability of this new clipping technique. As a low-cost, quantifiable, no-contact and non-destructive method, it is not concerned with the characteristic signal of certain materials and can be applied to arbitrary substrates. Therefore it is a perfect candidate for characterizing the thickness of graphene-like two-dimensional materials. (paper)

  2. A widefield fluorescence microscope with a linear image sensor for image cytometry of biospecimens: Considerations for image quality optimization

    Energy Technology Data Exchange (ETDEWEB)

    Hutcheson, Joshua A.; Majid, Aneeka A.; Powless, Amy J.; Muldoon, Timothy J., E-mail: tmuldoon@uark.edu [Department of Biomedical Engineering, University of Arkansas, 120 Engineering Hall, Fayetteville, Arkansas 72701 (United States)

    2015-09-15

    Linear image sensors have been widely used in numerous research and industry applications to provide continuous imaging of moving objects. Here, we present a widefield fluorescence microscope with a linear image sensor used to image translating objects for image cytometry. First, a calibration curve was characterized for a custom microfluidic chamber over a span of volumetric pump rates. Image data were also acquired using 15 μm fluorescent polystyrene spheres on a slide with a motorized translation stage in order to match linear translation speed with line exposure periods to preserve the image aspect ratio. Aspect ratios were then calculated after imaging to ensure quality control of image data. Fluorescent beads were imaged in suspension flowing through the microfluidics chamber being pumped by a mechanical syringe pump at 16 μl min{sup −1} with a line exposure period of 150 μs. The line period was selected to acquire images of fluorescent beads with a 40 dB signal-to-background ratio. A motorized translation stage was then used to transport conventional glass slides of stained cellular biospecimens. Whole blood collected from healthy volunteers was stained with 0.02% (w/v) proflavine hemisulfate was imaged to highlight leukocyte morphology with a 1.56 mm × 1.28 mm field of view (1540 ms total acquisition time). Oral squamous cells were also collected from healthy volunteers and stained with 0.01% (w/v) proflavine hemisulfate to demonstrate quantifiable subcellular features and an average nuclear to cytoplasmic ratio of 0.03 (n = 75), with a resolution of 0.31 μm pixels{sup −1}.

  3. Two-photon absorption and two-photon circular dichroism of hexahelicene derivatives: a study of the effect of the nature of intramolecular charge transfer

    Czech Academy of Sciences Publication Activity Database

    Díaz, C.; Vesga, Y.; Echevarria, L.; Stará, Irena G.; Starý, Ivo; Anger, E.; Shen, C.; Moussa, M. E. S.; Vanthuyne, N.; Crassous, J.; Rizzo, A.; Hernández, F. E.

    2015-01-01

    Roč. 5, č. 23 (2015), s. 17429-17437 ISSN 2046-2069 Institutional support: RVO:61388963 Keywords : [6]helicene * two photon absorption * two photon CD Subject RIV: CC - Organic Chemistry Impact factor: 3.289, year: 2015

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

    Science.gov (United States)

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

    2014-10-01

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

  5. Imaging of a large collection of human embryo using a super-parallel MR microscope

    International Nuclear Information System (INIS)

    Matsuda, Yoshimasa; Ono, Shinya; Otake, Yosuke; Handa, Shinya; Kose, Katsumi; Haishi, Tomoyuki; Yamada, Shigeto; Uwabe, Chikako; Shiota, Kohei

    2007-01-01

    Using 4 and 8-channel super-parallel magnetic resonance (MR) microscopes with a horizontal bore 2.34T superconducting magnet developed for 3-dimensional MR microscopy of the large Kyoto Collection of Human Embryos, we acquired T 1 -weighted 3D images of 1204 embryos at a spatial resolution of (40 μm) 3 to (150 μm) 3 in about 2 years. Similarity of image contrast between the T 1 -weighted images and stained anatomical sections indicated that T 1 -weighted 3D images could be used for an anatomical 3D image database for human embryology. (author)

  6. First images from the Stanford tabletop scanning soft x-ray microscope

    International Nuclear Information System (INIS)

    Trail, J.A.; Byer, R.L.

    1988-01-01

    The authors have constructed a scanning soft x-ray microscope which uses a laser-produced plasma as the soft x-ray source and normal incidence multilayer coated mirrors in a Schwarzschild configuration as the focusing optics. The microscope operates at a wavelength of 140 angstrom, has a spatial resolution of 0.5 μm, and has a soft x-ray photon flux through the focus of 10 4 s -1 when operated with only 170 mW of average laser power. The microscope is compact; the complete system, including the laser, fits on a single optical table. In this paper they describe the microscope and present images of metallic microstructures

  7. The plant virus microscope image registration method based on mismatches removing.

    Science.gov (United States)

    Wei, Lifang; Zhou, Shucheng; Dong, Heng; Mao, Qianzhuo; Lin, Jiaxiang; Chen, Riqing

    2016-01-01

    The electron microscopy is one of the major means to observe the virus. The view of virus microscope images is limited by making specimen and the size of the camera's view field. To solve this problem, the virus sample is produced into multi-slice for information fusion and image registration techniques are applied to obtain large field and whole sections. Image registration techniques have been developed in the past decades for increasing the camera's field of view. Nevertheless, these approaches typically work in batch mode and rely on motorized microscopes. Alternatively, the methods are conceived just to provide visually pleasant registration for high overlap ratio image sequence. This work presents a method for virus microscope image registration acquired with detailed visual information and subpixel accuracy, even when overlap ratio of image sequence is 10% or less. The method proposed focus on the correspondence set and interimage transformation. A mismatch removal strategy is proposed by the spatial consistency and the components of keypoint to enrich the correspondence set. And the translation model parameter as well as tonal inhomogeneities is corrected by the hierarchical estimation and model select. In the experiments performed, we tested different registration approaches and virus images, confirming that the translation model is not always stationary, despite the fact that the images of the sample come from the same sequence. The mismatch removal strategy makes building registration of virus microscope images at subpixel accuracy easier and optional parameters for building registration according to the hierarchical estimation and model select strategies make the proposed method high precision and reliable for low overlap ratio image sequence. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Focal dynamics of multiple filaments: Microscopic imaging and reconstruction

    International Nuclear Information System (INIS)

    Kiran, P. Prem; Bagchi, Suman; Kumar, G. Ravindra; Krishnan, Siva Rama; Arnold, C. L.; Couairon, A.

    2010-01-01

    We observe the complete dynamics of the propagation of very intense, femtosecond laser pulses in air under tight focusing conditions via direct imaging of the entire interaction zone. The whole life history of the focused pulses is then reconstructed by means of numerical simulations. We show that beam breakup leads to a dual-rate increase in filament numbers with laser power. Linearly and circularly polarized pulses give rise to beam breakup and fusion governed by external focusing conditions. For tight focusing conditions, intensity saturation due to plasma generation and nonlinear losses does not limit the intensity growth, thereby giving access to a new propagation regime featured by an efficient laser energy deposition in fully ionized air and intense 10 15 W/cm 2 pulses at the focus.

  10. Improvements in low-cost label-free QPI microscope for live cell imaging

    Science.gov (United States)

    Seniya, C.; Towers, C. E.; Towers, D. P.

    2017-07-01

    This paper reports an improvement in the development of a low-cost QPI microscope offering new capabilities in term of phase measurement accuracy for label-free live samples in the longer term (i.e., hours to days). The spatially separated scattered and non-scattered image light fields are reshaped in the Fourier plane and modulated to form an interference image at a CCD camera. The apertures that enable these two beams to be generated have been optimised by means of laser-cut apertures placed on the mirrors of a Michelson interferometer and has improved the phase measuring and reconstruction capability of the QPI microscope. The microscope was tested with transparent onion cells as an object of interest.

  11. Deformed two-photon squeezed states in noncommutative space

    International Nuclear Information System (INIS)

    Zhang Jianzu

    2004-01-01

    Recent studies on nonperturbation aspects of noncommutative quantum mechanics explored a new type of boson commutation relations at the deformed level, described by deformed annihilation-creation operators in noncommutative space. This correlated boson commutator correlates different degrees of freedom, and shows an essential influence on dynamics. This Letter devotes to the development of formalism of deformed two-photon squeezed states in noncommutative space. General representations of deformed annihilation-creation operators and the consistency condition for the electromagnetic wave with a single mode of frequency in noncommunicative space are obtained. Two-photon squeezed states are studied. One finds that variances of the dimensionless Hermitian quadratures of the annihilation operator in one degree of freedom include variances in the other degree of freedom. Such correlations show the new feature of spatial noncommutativity and allow a deeper understanding of the correlated boson commutator

  12. Meson production in two-photon interactions at LHC energies

    Energy Technology Data Exchange (ETDEWEB)

    Da Silva, D. T.; Goncalves, V. P.; Sauter, W. K. [Instituto de Fisica e Matematica, Universidade Federal de Pelotas, Campus Universitario UFPel, CP 354, 96010-900, Capao do Leao-RS (Brazil)

    2013-03-25

    The LHC opens a new kinematical regime at high energy, where several questions related to the description of the high-energy regime of the Quantum Chromodynamics (QCD) remain without satisfactory answers. Some open questions are the search for non-q-bar q resonances, the determination of the spectrum of q-bar q states and the identification of states with anomalous {gamma}{gamma} couplings. A possible way to study these problems is the study of meson production in two-photon interactions. In this contribution we calculate the meson production in two-photon interactions at LHC energies considering proton - proton collisions and estimate the total cross section for the production of the mesons {pi}, a, f, {eta} and {chi}.

  13. Fast processing of microscopic images using object-based extended depth of field.

    Science.gov (United States)

    Intarapanich, Apichart; Kaewkamnerd, Saowaluck; Pannarut, Montri; Shaw, Philip J; Tongsima, Sissades

    2016-12-22

    Microscopic analysis requires that foreground objects of interest, e.g. cells, are in focus. In a typical microscopic specimen, the foreground objects may lie on different depths of field necessitating capture of multiple images taken at different focal planes. The extended depth of field (EDoF) technique is a computational method for merging images from different depths of field into a composite image with all foreground objects in focus. Composite images generated by EDoF can be applied in automated image processing and pattern recognition systems. However, current algorithms for EDoF are computationally intensive and impractical, especially for applications such as medical diagnosis where rapid sample turnaround is important. Since foreground objects typically constitute a minor part of an image, the EDoF technique could be made to work much faster if only foreground regions are processed to make the composite image. We propose a novel algorithm called object-based extended depths of field (OEDoF) to address this issue. The OEDoF algorithm consists of four major modules: 1) color conversion, 2) object region identification, 3) good contrast pixel identification and 4) detail merging. First, the algorithm employs color conversion to enhance contrast followed by identification of foreground pixels. A composite image is constructed using only these foreground pixels, which dramatically reduces the computational time. We used 250 images obtained from 45 specimens of confirmed malaria infections to test our proposed algorithm. The resulting composite images with all in-focus objects were produced using the proposed OEDoF algorithm. We measured the performance of OEDoF in terms of image clarity (quality) and processing time. The features of interest selected by the OEDoF algorithm are comparable in quality with equivalent regions in images processed by the state-of-the-art complex wavelet EDoF algorithm; however, OEDoF required four times less processing time. This

  14. Two-photon polarization Fourier spectroscopy of metastable atomic hydrogen

    International Nuclear Information System (INIS)

    Duncan, A.J.; Beyer, H.-J.; Kleinpoppen, H.; Sheikh, Z.A,; B-Z Univ., Multan

    1997-01-01

    A novel Fourier-transform spectroscopic method using two-photon polarization to determine the spectral distribution of the two photons emitted in the spontaneous decay of metastable atomic hydrogen is described. The method uses birefringent retardation plates and takes advantage of the subtle interplay between the spectral properties and the entangled polarization properties of the radiation emitted in the decay. Assuming the validity of the theoretical spectral distribution, it is shown that the experimental results agree well with theory. On the other hand, success in solving the inverse problem of determining the spectral distribution from the experimental results is limited by the small number of experimental points. However, making reasonable assumptions it is deduced that the observed spectrum is characterized by a broadband signal of width (0.43 ± 0.06) x 10 16 rad s -1 and centre angular frequency (0.77 ± 0.03) x 10 16 rad s -1 in good agreement with the predictions of 0.489 x 10 16 rad s -1 and 0.775 x 10 16 rad s -1 , respectively, obtained from the theoretical spectral distribution modified to take account of the absorption of the two-photon radiation in air. The values of 1.5 fs for the coherence time and 440 nm for the coherence length for single photons of the two-photon pair which are obtained from the measured bandwidth imply that, in the ideal case, these values are determined by the essentially zero lifetime of the virtual intermediate state of the decay process rather than the long lifetime of the metastable state which, it is suggested, determines the coherence time and coherence length appropriate to certain types of fourth-order interference experiments. (Author)

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Electromagnetic field enhancement (FE) effects occurring in thin gold films 3-12-nm are investigated with two-photon photoluminescence (TPL) and Raman scanning optical microscopies. The samples are characterized using scanning electron microscopy images and linear optical spectroscopy. TPL images...... exhibit a strong increase in the level of TPL signals for films thicknesses 3-8-nm, near the percolation threshold. For some thicknesses, TPL measurements reveal super-cubic dependences on the incident power. We ascribe this feature to the occurrence of very strongly localized and enhanced electromagnetic...

  16. Two-Photon Absorption in Organometallic Bromide Perovskites

    KAUST Repository

    Walters, Grant

    2015-07-21

    Organometallic trihalide perovskites are solution processed semiconductors that have made great strides in third generation thin film light harvesting and light emitting optoelectronic devices. Recently it has been demonstrated that large, high purity single crystals of these perovskites can be synthesized from the solution phase. These crystals’ large dimensions, clean bandgap, and solid-state order, have provided us with a suitable medium to observe and quantify two-photon absorption in perovskites. When CH3NH3PbBr3 single crystals are pumped with intense 800 nm light, we observe band-to-band photoluminescence at 572 nm, indicative of two-photon absorption. We report the nonlinear absorption coefficient of CH3NH3PbBr3 perovskites to be 8.6 cm GW-1 at 800 nm, comparable to epitaxial single crystal semiconductors of similar bandgap. We have leveraged this nonlinear process to electrically autocorrelate a 100 fs pulsed laser using a two-photon perovskite photodetector. This work demonstrates the viability of organometallic trihalide perovskites as a convenient and low-cost nonlinear absorber for applications in ultrafast photonics.

  17. Particle Production in Two-Photon Collisions at Belle

    International Nuclear Information System (INIS)

    Nakazawa, Hideyuki

    2010-01-01

    Experimental study of η η production in two-photon collisions: The differential cross section for the process γ γ → η η has been measured in the kinematic range above the η η threshold, 1.096 GeV -1 data sample collected with the Belle detector at the KEKB e + e - collider. In the W range 1.1-2.0 GeV/c 2 we perform an analysis of resonance amplitudes for various partial waves; at higher energy we extract the contributions of χ cJ charmonia and compare the energy and angular dependence of the cross section with the predictions of theoretical models. Observation of η c (2S) in six-prong final states produced in two-photon collisions: We report the observation of η c (2S), produced in two-photon collisions, and decaying to the six-prong final states 3(π + π - ), K + K - 2(π + π - ), and K 0 S K + ππ + π - (including the charge-conjugate state). This analysis is based on a large data sample accumulated by the Belle experiment at the KEKB asymmetric-energy electron-positron collider. This is the first observation of decay modes of the η c (2S) other than K 0 S K + π - . (author)

  18. Two-photon exchange in elastic electron-nucleon scattering

    International Nuclear Information System (INIS)

    Blunden, P.G.; Melnitchouk, W.; Tjon, J.A.

    2005-01-01

    A detailed study of two-photon exchange in unpolarized and polarized elastic electron-nucleon scattering is presented, taking particular account of nucleon finite size effects. Contributions from nucleon elastic intermediate states are found to have a strong angular dependence, which leads to a partial resolution of the discrepancy between the Rosenbluth and polarization transfer measurements of the proton electric to magnetic form factor ratio, G E /G M . The two-photon exchange contribution to the longitudinal polarization transfer P L is small, whereas the contribution to the transverse polarization transfer P T is enhanced at backward angles by several percent, increasing with Q 2 . This gives rise to a small, E /G M obtained from the polarization transfer ratio P T /P L at large Q 2 . We also compare the two-photon exchange effects with data on the ratio of e + p to e - p cross sections, which is predicted to be enhanced at backward angles. Finally, we evaluate the corrections to the form factors of the neutron and estimate the elastic intermediate state contribution to the 3 He form factors

  19. Two-Photon Absorption in Organometallic Bromide Perovskites

    KAUST Repository

    Walters, Grant; Sutherland, Brandon R; Hoogland, Sjoerd; Shi, Dong; Comin, Riccardo; Sellan, Daniel P.; Bakr, Osman; Sargent, Edward H.

    2015-01-01

    Organometallic trihalide perovskites are solution processed semiconductors that have made great strides in third generation thin film light harvesting and light emitting optoelectronic devices. Recently it has been demonstrated that large, high purity single crystals of these perovskites can be synthesized from the solution phase. These crystals’ large dimensions, clean bandgap, and solid-state order, have provided us with a suitable medium to observe and quantify two-photon absorption in perovskites. When CH3NH3PbBr3 single crystals are pumped with intense 800 nm light, we observe band-to-band photoluminescence at 572 nm, indicative of two-photon absorption. We report the nonlinear absorption coefficient of CH3NH3PbBr3 perovskites to be 8.6 cm GW-1 at 800 nm, comparable to epitaxial single crystal semiconductors of similar bandgap. We have leveraged this nonlinear process to electrically autocorrelate a 100 fs pulsed laser using a two-photon perovskite photodetector. This work demonstrates the viability of organometallic trihalide perovskites as a convenient and low-cost nonlinear absorber for applications in ultrafast photonics.

  20. Imaging of surface plasmon polariton interference using phase-sensitive scanning tunneling microscope

    NARCIS (Netherlands)

    Jose, J.; Segerink, Franciscus B.; Korterik, Jeroen P.; Herek, Jennifer Lynn; Offerhaus, Herman L.

    2011-01-01

    We report the surface plasmon polariton interference, generated via a ‘buried’ gold grating, and imaged using a phase-sensitive Photon Scanning Tunneling Microscope (PSTM). The phase-resolved PSTM measurement unravels the complex surface plasmon polariton interference fields at the gold-air

  1. Chemical imaging of cotton fibers using an infrared microscope and a focal-plane array detector

    Science.gov (United States)

    In this presentation, the chemical imaging of cotton fibers with an infrared microscope and a Focal-Plane Array (FPA) detector will be discussed. Infrared spectroscopy can provide us with information on the structure and quality of cotton fibers. In addition, FPA detectors allow for simultaneous spe...

  2. Classification of gram-positive and gram-negative foodborne pathogenic bacteria with hyperspectral microscope imaging

    Science.gov (United States)

    Optical method with hyperspectral microscope imaging (HMI) has potential for identification of foodborne pathogenic bacteria from microcolonies rapidly with a cell level. A HMI system that provides both spatial and spectral information could be an effective tool for analyzing spectral characteristic...

  3. Rapid identification of salmonella serotypes with stereo and hyperspectral microscope imaging Methods

    Science.gov (United States)

    The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

  4. High resolution imaging of dielectric surfaces with an evanescent field optical microscope

    NARCIS (Netherlands)

    van Hulst, N.F.; Segerink, Franciscus B.; Bölger, B.

    1992-01-01

    An evanescent field optical microscope (EFOM) is presented which employs frustrated total internal reflection o­n a localized scale by scanning a dielectric tip in close proximity to a sample surface. High resolution images of dielectric gratings and spheres containing both topographic and

  5. Acousto-Optic Tunable Filter Hyperspectral Microscope Imaging Method for Characterizing Spectra from Foodborne Pathogens.

    Science.gov (United States)

    Hyperspectral microscope imaging (HMI) method, which provides both spatial and spectral characteristics of samples, can be effective for foodborne pathogen detection. The acousto-optic tunable filter (AOTF)-based HMI method can be used to characterize spectral properties of biofilms formed by Salmon...

  6. MRT letter: Guided filtering of image focus volume for 3D shape recovery of microscopic objects.

    Science.gov (United States)

    Mahmood, Muhammad Tariq

    2014-12-01

    In this letter, a shape from focus (SFF) method is proposed that utilizes the guided image filtering to enhance the image focus volume efficiently. First, image focus volume is computed using a conventional focus measure. Then each layer of image focus volume is filtered using guided filtering. In this work, the all-in-focus image, which can be obtained from the initial focus volume, is used as guidance image. Finally, improved depth map is obtained from the filtered image focus volume by maximizing the focus measure along the optical axis. The proposed SFF method is efficient and provides better depth maps. The improved performance is highlighted by conducting several experiments using image sequences of simulated and real microscopic objects. The comparative analysis demonstrates the effectiveness of the proposed SFF method. © 2014 Wiley Periodicals, Inc.

  7. Atomic imaging of an InSe single-crystal surface with atomic force microscope

    OpenAIRE

    Uosaki, Kohei; Koinuma, Michio

    1993-01-01

    The atomic force microscope was employed to observed in air the surface atomic structure of InSe, one of III-VI compound semiconductors with layered structures. Atomic arrangements were observed in both n-type and p-type materials. The observed structures are in good agreement with those expected from bulk crystal structures. The atomic images became less clear by repeating the imaging process. Wide area imaging after the imaging of small area clearly showed that a mound was created at the sp...

  8. SUPERVISED AUTOMATIC HISTOGRAM CLUSTERING AND WATERSHED SEGMENTATION. APPLICATION TO MICROSCOPIC MEDICAL COLOR IMAGES

    Directory of Open Access Journals (Sweden)

    Olivier Lezoray

    2011-05-01

    Full Text Available In this paper, an approach to the segmentation of microscopic color images is addressed, and applied to medical images. The approach combines a clustering method and a region growing method. Each color plane is segmented independently relying on a watershed based clustering of the plane histogram. The marginal segmentation maps intersect in a label concordance map. The latter map is simplified based on the assumption that the color planes are correlated. This produces a simplified label concordance map containing labeled and unlabeled pixels. The formers are used as an image of seeds for a color watershed. This fast and robust segmentation scheme is applied to several types of medical images.

  9. Inverted light-sheet microscope for imaging mouse pre-implantation development.

    Science.gov (United States)

    Strnad, Petr; Gunther, Stefan; Reichmann, Judith; Krzic, Uros; Balazs, Balint; de Medeiros, Gustavo; Norlin, Nils; Hiiragi, Takashi; Hufnagel, Lars; Ellenberg, Jan

    2016-02-01

    Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.

  10. Switched capacitor charge pump used for low-distortion imaging in atomic force microscope.

    Science.gov (United States)

    Zhang, Jie; Zhang, Lian Sheng; Feng, Zhi Hua

    2015-01-01

    The switched capacitor charge pump (SCCP) is an effective method of linearizing charges on piezoelectric actuators and therefore constitute a significant approach to nano-positioning. In this work, it was for the first time implemented in an atomic force microscope for low-distortion imaging. Experimental results showed that the image quality was improved evidently under the SCCP drive compared with that under traditional linear voltage drive. © Wiley Periodicals, Inc.

  11. Excitation-scanning hyperspectral imaging system for microscopic and endoscopic applications

    Science.gov (United States)

    Mayes, Sam A.; Leavesley, Silas J.; Rich, Thomas C.

    2016-04-01

    Current microscopic and endoscopic technologies for cancer screening utilize white-light illumination sources. Hyper-spectral imaging has been shown to improve sensitivity while retaining specificity when compared to white-light imaging in both microscopy and in vivo imaging. However, hyperspectral imaging methods have historically suffered from slow acquisition times due to the narrow bandwidth of spectral filters. Often minutes are required to gather a full image stack. We have developed a novel approach called excitation-scanning hyperspectral imaging that provides 2-3 orders of magnitude increased signal strength. This reduces acquisition times significantly, allowing for live video acquisition. Here, we describe a preliminary prototype excitation-scanning hyperspectral imaging system that can be coupled with endoscopes or microscopes for hyperspectral imaging of tissues and cells. Our system is comprised of three subsystems: illumination, transmission, and imaging. The illumination subsystem employs light-emitting diode arrays to illuminate at different wavelengths. The transmission subsystem utilizes a unique geometry of optics and a liquid light guide. Software controls allow us to interface with and control the subsystems and components. Digital and analog signals are used to coordinate wavelength intensity, cycling and camera triggering. Testing of the system shows it can cycle 16 wavelengths at as fast as 1 ms per cycle. Additionally, more than 18% of the light transmits through the system. Our setup should allow for hyperspectral imaging of tissue and cells in real time.

  12. Three-dimensional phase-contrast X-ray microtomography with scanning–imaging X-ray microscope optics

    International Nuclear Information System (INIS)

    Takeuchi, Akihisa; Uesugi, Kentaro; Suzuki, Yoshio

    2013-01-01

    A novel three-dimensional X-ray microtomographic micro-imaging system which enables simultaneous measurement of differential phase contrast and absorption contrast has been developed. The optical system consists of a scanning microscope with one-dimensional focusing device and an imaging microscope with one-dimensional objective. A three-dimensional (3D) X-ray tomographic micro-imaging system has been developed. The optical system is based on a scanning–imaging X-ray microscope (SIXM) optics, which is a hybrid system consisting of a scanning microscope optics with a one-dimensional (1D) focusing (line-focusing) device and an imaging microscope optics with a 1D objective. In the SIXM system, each 1D dataset of a two-dimensional (2D) image is recorded independently. An object is illuminated with a line-focused beam. Positional information of the region illuminated by the line-focused beam is recorded with the 1D imaging microscope optics as line-profile data. By scanning the object with the line focus, 2D image data are obtained. In the same manner as for a scanning microscope optics with a multi-pixel detector, imaging modes such as phase contrast and absorption contrast can be arbitrarily configured after the image data acquisition. By combining a tomographic scan method and the SIXM system, quantitative 3D imaging is performed. Results of a feasibility study of the SIXM for 3D imaging are shown

  13. Use of an axisymmetric microscope with electronic readout for collecting soft X-ray images

    International Nuclear Information System (INIS)

    Cavailler, C.; Henry, P.; Launspach, J.; De Mascureau, J.; Millerioux, M.; Rostaing, M.; Sauneuf, R.

    1984-08-01

    The axisymmetric microscope, first discussed by Wolter, provides high resolution and sensitivity for investigating the soft X-ray emission of laser-driven plasmas. Such a device having a 10 X magnification has been constructed. We present a comparison between the images of laser-driven plasmas given by this microscope and by a 10 X pinhole camera. Until now these images were recorded on X-ray film. We have shown that film could be replaced by C.C.D. in a pinhole camera when the photon energy lies within the 1-10 keV range. Below 1 keV the quantum yield is too low so we have used an image converter tube made by RTC. It is a diode-inverter tube with a soft X-ray photocathode and a P20 phosphor deposited on an optic fiber plate. The electronic image appearing on the screen is read by a C.C.D. working in the visible spectral fields. An electronic image readout chain, which is identical to those associated with streak cameras, then processes automatically and immediately the images given by the microscope [fr

  14. Design of a cathodoluminescence image generator using a Raspberry Pi coupled to a scanning electron microscope

    Science.gov (United States)

    Benítez, Alfredo; Santiago, Ulises; Sanchez, John E.; Ponce, Arturo

    2018-01-01

    In this work, an innovative cathodoluminescence (CL) system is coupled to a scanning electron microscope and synchronized with a Raspberry Pi computer integrated with an innovative processing signal. The post-processing signal is based on a Python algorithm that correlates the CL and secondary electron (SE) images with a precise dwell time correction. For CL imaging, the emission signal is collected through an optical fiber and transduced to an electrical signal via a photomultiplier tube (PMT). CL Images are registered in a panchromatic mode and can be filtered using a monochromator connected between the optical fiber and the PMT to produce monochromatic CL images. The designed system has been employed to study ZnO samples prepared by electrical arc discharge and microwave methods. CL images are compared with SE images and chemical elemental mapping images to correlate the emission regions of the sample.

  15. Automatic segmentation of Leishmania parasite in microscopic images using a modified CV level set method

    Science.gov (United States)

    Farahi, Maria; Rabbani, Hossein; Talebi, Ardeshir; Sarrafzadeh, Omid; Ensafi, Shahab

    2015-12-01

    Visceral Leishmaniasis is a parasitic disease that affects liver, spleen and bone marrow. According to World Health Organization report, definitive diagnosis is possible just by direct observation of the Leishman body in the microscopic image taken from bone marrow samples. We utilize morphological and CV level set method to segment Leishman bodies in digital color microscopic images captured from bone marrow samples. Linear contrast stretching method is used for image enhancement and morphological method is applied to determine the parasite regions and wipe up unwanted objects. Modified global and local CV level set methods are proposed for segmentation and a shape based stopping factor is used to hasten the algorithm. Manual segmentation is considered as ground truth to evaluate the proposed method. This method is tested on 28 samples and achieved 10.90% mean of segmentation error for global model and 9.76% for local model.

  16. Imaging of soft and hard materials using a Boersch phase plate in a transmission electron microscope

    Energy Technology Data Exchange (ETDEWEB)

    Alloyeau, D., E-mail: alloyeau.damien@gmail.com [National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, One Cyclotron Road, MS/72, Berkeley, CA 94720 (United States); Hsieh, W.K. [National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, One Cyclotron Road, MS/72, Berkeley, CA 94720 (United States); Anderson, E.H.; Hilken, L. [Center for X-ray Optics, Lawrence Berkeley National Laboratory, Berkeley CA 94720 (United States); Benner, G. [Carl Zeiss NTS GmbH, Oberkochen 73447 (Germany); Meng, X. [Electrical Engineering and Computer Sciences, UC Berkeley, Berkeley, CA 94720-1770 (United States); Chen, F.R. [Department of Engineering and System Science, National Tsing Hua University, Hsinchu, Taiwan (China); Kisielowski, C. [National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, One Cyclotron Road, MS/72, Berkeley, CA 94720 (United States)

    2010-04-15

    Using two levels of electron beam lithography, vapor phase deposition techniques, and FIB etching, we have fabricated an electrostatic Boersch phase plate for contrast enhancement of weak phase objects in a transmission electron microscope. The phase plate has suitable dimensions for the imaging of small biological samples without compromising the high-resolution capabilities of the microscope. A micro-structured electrode allows for phase tuning of the unscattered electron beam, which enables the recording of contrast enhanced in-focus images and in-line holograms. We have demonstrated experimentally that our phase plate improves the contrast of carbon nanotubes while maintaining high-resolution imaging performance, which is demonstrated for the case of an AlGaAs heterostructure. The development opens a new way to study interfaces between soft and hard materials.

  17. Computer Aided Quantification of Pathological Features for Flexor Tendon Pulleys on Microscopic Images

    Directory of Open Access Journals (Sweden)

    Yung-Chun Liu

    2013-01-01

    Full Text Available Quantifying the pathological features of flexor tendon pulleys is essential for grading the trigger finger since it provides clinicians with objective evidence derived from microscopic images. Although manual grading is time consuming and dependent on the observer experience, there is a lack of image processing methods for automatically extracting pulley pathological features. In this paper, we design and develop a color-based image segmentation system to extract the color and shape features from pulley microscopic images. Two parameters which are the size ratio of abnormal tissue regions and the number ratio of abnormal nuclei are estimated as the pathological progression indices. The automatic quantification results show clear discrimination among different levels of diseased pulley specimens which are prone to misjudgments for human visual inspection. The proposed system provides a reliable and automatic way to obtain pathological parameters instead of manual evaluation which is with intra- and interoperator variability. Experiments with 290 microscopic images from 29 pulley specimens show good correspondence with pathologist expectations. Hence, the proposed system has great potential for assisting clinical experts in routine histopathological examinations.

  18. Automatic detection of the hippocampal region associated with Alzheimer's disease from microscopic images of mice brain

    Science.gov (United States)

    Albaidhani, Tahseen; Hawkes, Cheryl; Jassim, Sabah; Al-Assam, Hisham

    2016-05-01

    The hippocampus is the region of the brain that is primarily associated with memory and spatial navigation. It is one of the first brain regions to be damaged when a person suffers from Alzheimer's disease. Recent research in this field has focussed on the assessment of damage to different blood vessels within the hippocampal region from a high throughput brain microscopic images. The ultimate aim of our research is the creation of an automatic system to count and classify different blood vessels such as capillaries, veins, and arteries in the hippocampus region. This work should provide biologists with efficient and accurate tools in their investigation of the causes of Alzheimer's disease. Locating the boundary of the Region of Interest in the hippocampus from microscopic images of mice brain is the first essential stage towards developing such a system. This task benefits from the variation in colour channels and texture between the two sides of the hippocampus and the boundary region. Accordingly, the developed initial step of our research to locating the hippocampus edge uses a colour-based segmentation of the brain image followed by Hough transforms on the colour channel that isolate the hippocampus region. The output is then used to split the brain image into two sides of the detected section of the boundary: the inside region and the outside region. Experimental results on a sufficiently number of microscopic images demonstrate the effectiveness of the developed solution.

  19. Pattern generation using axicon lens beam shaping in two-photon polymerisation

    International Nuclear Information System (INIS)

    Bhuian, B.; Winfield, R.J.; O'Brien, S.; Crean, G.M.

    2007-01-01

    The fabrication of three-dimensional microstructures by two-photon polymerisation has been widely reported as a viable route to the development of photonic crystals, rotors, bridges and other complex artefacts requiring nanoscale resolution. Conventionally, single point serial writing is used to write the structures but recently multipoint beam delivery using beam division optics has been reported as a method of introducing parallel processing. In this paper we present an alternative and novel approach using an axicon lens to give profiled beam delivery. This enables complete three-dimensional annular structure fabrication without the use of scanning stages. In addition, the concept of axicon delivery is developed further to investigate three-dimensional structure as a function of axicon geometry. A Ti:sapphire laser, with wavelength 795 nm, 80 MHz repetition rate, 100 fs pulse duration and an average power of 700 mW, was used to initiate two-photon polymerisation. The axicon was used, in combination with a 100x microscope objective, to form representative three-dimensional structures based on the annular cell with varying diameter. The structures were written in a Zr-loaded resin prepared on a glass substrate using dip coating deposition of a Zr/PMMA hybrid prepared by the sol-gel method. Annuli with diameters up to 50 μm were characterised in terms of topography and surface roughness using SEM and Zygo interferometer. The writing technique was also extended to fabrication of stacked structures

  20. Two photon versus one photon fluorescence excitation in whispering gallery mode microresonators

    International Nuclear Information System (INIS)

    Pastells, Carme; Marco, M.-Pilar; Merino, David; Loza-Alvarez, Pablo; Pasquardini, Laura; Lunelli, Lorenzo; Pederzolli, Cecilia; Daldosso, Nicola; Farnesi, Daniele; Berneschi, Simone; Righini, Giancarlo C.; Quercioli, Franco; Nunzi Conti, Gualtiero; Soria, Silvia

    2016-01-01

    We investigate the feasibility of both one photon and two photon fluorescence excitation using whispering gallery mode microresonators. We report the linear and non linear fluorescence real-time detection of labeled IgG covalently bonded to the surface of a silica whispering gallery mode resonator (WGMR). The immunoreagents have been immobilized onto the surface of the WGMR sensor after being activated with an epoxy silane and an orienting layer. The developed immunosensor presents great potential as a robust sensing device for fast and early detection of immunoreactions. We also investigate the potential of microbubbles as nonlinear enhancement platform. The dyes used in these studies are dylight800, tetramethyl rhodamine isothiocyanate, rhodamine 6G and fluorescein. All measurements were performed in a modified confocal microscope. - Highlights: • One photon fluorescence overlaps with the semiconductor pump laser gain bandwidth. • We report on the feasibility to excite two photon fluorescence in microbubble resonators. • Our functionalization process maintains a good quality factor of the microresonator.

  1. Two photon versus one photon fluorescence excitation in whispering gallery mode microresonators

    Energy Technology Data Exchange (ETDEWEB)

    Pastells, Carme; Marco, M.-Pilar [Nanobiotechnology for Diagnostics Group (Nb4Dg), IQAC-CSIC, 08034 Barcelona (Spain); CIBER de Bioingeniería, Biomateriales y Nanomedicina, 08034 Barcelona (Spain); Merino, David; Loza-Alvarez, Pablo [ICFO-Institut de Ciències Fotòniques, Castelldefels, 08860 Barcelona (Spain); Pasquardini, Laura [Fondazione Bruno Kessler, 38123 Povo, TN (Italy); Lunelli, Lorenzo [Fondazione Bruno Kessler, 38123 Povo, TN (Italy); IBF-CNR, 38123 Povo, TN (Italy); Pederzolli, Cecilia [Fondazione Bruno Kessler, 38123 Povo, TN (Italy); Daldosso, Nicola [Department of Computer Science, University of Verona, Strada le Grazie 15, 37134 Verona (Italy); Farnesi, Daniele [CNR-IFAC “Nello Carrara” Institute of Applied Physics, 50019 Sesto Fiorentino, FI (Italy); Museo Storico della Fisica e Centro Studi e Ricerche “E. Fermi”, 00184 Roma (Italy); Berneschi, Simone [CNR-IFAC “Nello Carrara” Institute of Applied Physics, 50019 Sesto Fiorentino, FI (Italy); Righini, Giancarlo C. [CNR-IFAC “Nello Carrara” Institute of Applied Physics, 50019 Sesto Fiorentino, FI (Italy); Museo Storico della Fisica e Centro Studi e Ricerche “E. Fermi”, 00184 Roma (Italy); Quercioli, Franco [CNR-INO National Institute of Optics, Sesto Fiorentino, FI (Italy); Nunzi Conti, Gualtiero [CNR-IFAC “Nello Carrara” Institute of Applied Physics, 50019 Sesto Fiorentino, FI (Italy); Soria, Silvia, E-mail: s.soria@ifac.cnr.it [CNR-IFAC “Nello Carrara” Institute of Applied Physics, 50019 Sesto Fiorentino, FI (Italy)

    2016-02-15

    We investigate the feasibility of both one photon and two photon fluorescence excitation using whispering gallery mode microresonators. We report the linear and non linear fluorescence real-time detection of labeled IgG covalently bonded to the surface of a silica whispering gallery mode resonator (WGMR). The immunoreagents have been immobilized onto the surface of the WGMR sensor after being activated with an epoxy silane and an orienting layer. The developed immunosensor presents great potential as a robust sensing device for fast and early detection of immunoreactions. We also investigate the potential of microbubbles as nonlinear enhancement platform. The dyes used in these studies are dylight800, tetramethyl rhodamine isothiocyanate, rhodamine 6G and fluorescein. All measurements were performed in a modified confocal microscope. - Highlights: • One photon fluorescence overlaps with the semiconductor pump laser gain bandwidth. • We report on the feasibility to excite two photon fluorescence in microbubble resonators. • Our functionalization process maintains a good quality factor of the microresonator.

  2. NIR-emitting molecular-based nanoparticles as new two-photon absorbing nanotools for single particle tracking

    Science.gov (United States)

    Daniel, J.; Godin, A. G.; Clermont, G.; Lounis, B.; Cognet, L.; Blanchard-Desce, M.

    2015-07-01

    In order to provide a green alternative to QDs for bioimaging purposes and aiming at designing bright nanoparticles combining both large one- and two-photon brightness, a bottom-up route based on the molecular engineering of dedicated red to NIR emitting dyes that spontaneously form fluorescent organic nanoparticles (FONs) has been implemented. These fully organic nanoparticles built from original quadrupolar dyes are prepared using a simple, expeditious and green protocol that yield very small molecular-based nanoparticles (radius ~ 7 nm) suspension in water showing a nice NIR emission (λem=710 nm). These FONs typically have absorption coefficient more than two orders larger than popular NIR-emitting dyes (such as Alexa Fluor 700, Cy5.5 ….) and much larger Stokes shift values (i.e. up to over 5500 cm-1). They also show very large two-photon absorption response in the 800-1050 nm region (up to about 106 GM) of major promise for two-photon excited fluorescence microscopy. Thanks to their brightness and enhanced photostability, these FONs could be imaged as isolated nanoparticles and tracked using wide-field imaging. As such, thanks to their size and composition (absence of heavy metals), they represent highly promising alternatives to NIR-emitting QDs for use in bioimaging and single particle tracking applications. Moreover, efficient FONs coating was achieved by using a polymeric additive built from a long hydrophobic (PPO) and a short hydrophilic (PEO) segment and having a cationic head group able to interact with the highly negative surface of FONs. This electrostatically-driven interaction promotes both photoluminescence and two-photon absorption enhancement leading to an increase of two-photon brightness of about one order of magnitude. This opens the way to wide-field single particle tracking under two-photon excitation

  3. Analytical electron microscope based on scanning transmission electron microscope with wavelength dispersive x-ray spectroscopy to realize highly sensitive elemental imaging especially for light elements

    International Nuclear Information System (INIS)

    Koguchi, Masanari; Tsuneta, Ruriko; Anan, Yoshihiro; Nakamae, Koji

    2017-01-01

    An analytical electron microscope based on the scanning transmission electron microscope with wavelength dispersive x-ray spectroscopy (STEM-WDX) to realize highly sensitive elemental imaging especially for light elements has been developed. In this study, a large-solid-angle multi-capillary x-rays lens with a focal length of 5 mm, long-time data acquisition (e.g. longer than 26 h), and a drift-free system made it possible to visualize boron-dopant images in a Si substrate at a detection limit of 0.2 atomic percent. (paper)

  4. Fermi surface contours obtained from scanning tunneling microscope images around surface point defects

    International Nuclear Information System (INIS)

    Khotkevych-Sanina, N V; Kolesnichenko, Yu A; Van Ruitenbeek, J M

    2013-01-01

    We present a theoretical analysis of the standing wave patterns in scanning tunneling microscope (STM) images, which occur around surface point defects. We consider arbitrary dispersion relations for the surface states and calculate the conductance for a system containing a small-size tunnel contact and a surface impurity. We find rigorous theoretical relations between the interference patterns in the real-space STM images, their Fourier transforms and the Fermi contours of two-dimensional electrons. We propose a new method for reconstructing Fermi contours of surface electron states, directly from the real-space STM images around isolated surface defects. (paper)

  5. New deconvolution method for microscopic images based on the continuous Gaussian radial basis function interpolation model.

    Science.gov (United States)

    Chen, Zhaoxue; Chen, Hao

    2014-01-01

    A deconvolution method based on the Gaussian radial basis function (GRBF) interpolation is proposed. Both the original image and Gaussian point spread function are expressed as the same continuous GRBF model, thus image degradation is simplified as convolution of two continuous Gaussian functions, and image deconvolution is converted to calculate the weighted coefficients of two-dimensional control points. Compared with Wiener filter and Lucy-Richardson algorithm, the GRBF method has an obvious advantage in the quality of restored images. In order to overcome such a defect of long-time computing, the method of graphic processing unit multithreading or increasing space interval of control points is adopted, respectively, to speed up the implementation of GRBF method. The experiments show that based on the continuous GRBF model, the image deconvolution can be efficiently implemented by the method, which also has a considerable reference value for the study of three-dimensional microscopic image deconvolution.

  6. Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

    Science.gov (United States)

    Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

    2013-06-01

    Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

  7. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    Science.gov (United States)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

  8. A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

    Science.gov (United States)

    Hasan, Md Mehedi; Alam, Mohammad Wajih; Wahid, Khan A; Miah, Sayem; Lukong, Kiven Erique

    2016-01-01

    This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

  9. A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability.

    Directory of Open Access Journals (Sweden)

    Md Mehedi Hasan

    Full Text Available This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size.

  10. A Low-Cost Digital Microscope with Real-Time Fluorescent Imaging Capability

    Science.gov (United States)

    Hasan, Md. Mehedi; Wahid, Khan A.; Miah, Sayem; Lukong, Kiven Erique

    2016-01-01

    This paper describes the development of a prototype of a low-cost digital fluorescent microscope built from commercial off-the-shelf (COTS) components. The prototype was tested to detect malignant tumor cells taken from a living organism in a preclinical setting. This experiment was accomplished by using Alexa Fluor 488 conjugate dye attached to the cancer cells. Our prototype utilizes a torch along with an excitation filter as a light source for fluorophore excitation, a dichroic mirror to reflect the excitation and pass the emitted green light from the sample under test and a barrier filter to permit only appropriate wavelength. The system is designed out of a microscope using its optical zooming property and an assembly of exciter filter, dichroic mirror and transmitter filter. The microscope is connected to a computer or laptop through universal serial bus (USB) that allows real-time transmission of captured florescence images; this also offers real-time control of the microscope. The designed system has comparable features of high-end commercial fluorescent microscopes while reducing cost, power, weight and size. PMID:27977709

  11. Inclusive $D*^{+-}$ Production in Two-Photon Collisions at LEP

    CERN Document Server

    Achard, P.; Aguilar-Benitez, M.; Alcaraz, J.; Alemanni, G.; Allaby, J.; Aloisio, A.; Alviggi, M.G.; Anderhub, H.; Andreev, Valery P.; Anselmo, F.; Arefev, A.; Azemoon, T.; Aziz, T.; Bagnaia, P.; Bajo, A.; Baksay, G.; Baksay, L.; Baldew, S.V.; Banerjee, S.; Banerjee, Sw.; Barczyk, A.; Barillere, R.; Bartalini, P.; Basile, M.; Batalova, N.; Battiston, R.; Bay, A.; Becattini, F.; Becker, U.; Behner, F.; Bellucci, L.; Berbeco, R.; Berdugo, J.; Berges, P.; Bertucci, B.; Betev, B.L.; Biasini, M.; Biglietti, M.; Biland, A.; Blaising, J.J.; Blyth, S.C.; Bobbink, G.J.; Bohm, A.; Boldizsar, L.; Borgia, B.; Bottai, S.; Bourilkov, D.; Bourquin, M.; Braccini, S.; Branson, J.G.; Brochu, F.; Burger, J.D.; Burger, W.J.; Cai, X.D.; Capell, M.; Cara Romeo, G.; Carlino, G.; Cartacci, A.; Casaus, J.; Cavallari, F.; Cavallo, N.; Cecchi, C.; Cerrada, M.; Chamizo, M.; Chang, Y.H.; Chemarin, M.; Chen, A.; Chen, G.; Chen, G.M.; Chen, H.F.; Chen, H.S.; Chiefari, G.; Cifarelli, L.; Cindolo, F.; Clare, I.; Clare, R.; Coignet, G.; Colino, N.; Costantini, S.; de la Cruz, B.; Cucciarelli, S.; van Dalen, J.A.; de Asmundis, R.; Deglon, P.; Debreczeni, J.; Degre, A.; Deiters, K.; della Volpe, D.; Delmeire, E.; Denes, P.; DeNotaristefani, F.; De Salvo, A.; Diemoz, M.; Dierckxsens, M.; Dionisi, C.; Dittmar, M.; Doria, A.; Dova, M.T.; Duchesneau, D.; Echenard, B.; Eline, A.; El Mamouni, H.; Engler, A.; Eppling, F.J.; Ewers, A.; Extermann, P.; Falagan, M.A.; Falciano, S.; Favara, A.; Fay, J.; Fedin, O.; Felcini, M.; Ferguson, T.; Fesefeldt, H.; Fiandrini, E.; Field, J.H.; Filthaut, F.; Fisher, P.H.; Fisher, W.; Fisk, I.; Forconi, G.; Freudenreich, K.; Furetta, C.; Galaktionov, Iouri; Ganguli, S.N.; Garcia-Abia, Pablo; Gataullin, M.; Gentile, S.; Giagu, S.; Gong, Z.F.; Grenier, Gerald Jean; Grimm, O.; Gruenewald, M.W.; Guida, M.; van Gulik, R.; Gupta, V.K.; Gurtu, A.; Gutay, L.J.; Haas, D.; Hakobyan, R.S.; Hatzifotiadou, D.; Hebbeker, T.; Herve, Alain; Hirschfelder, J.; Hofer, H.; Hohlmann, M.; Holzner, G.; Hou, S.R.; Hu, Y.; Jin, B.N.; Jones, Lawrence W.; de Jong, P.; Josa-Mutuberria, I.; Kafer, D.; Kaur, M.; Kienzle-Focacci, M.N.; Kim, J.K.; Kirkby, Jasper; Kittel, W.; Klimentov, A.; Konig, A.C.; Kopal, M.; Koutsenko, V.; Kraber, M.; Kraemer, R.W.; Krenz, W.; Kruger, A.; Kunin, A.; Ladron de Guevara, P.; Laktineh, I.; Landi, G.; Lebeau, M.; Lebedev, A.; Lebrun, P.; Lecomte, P.; Lecoq, P.; Le Coultre, P.; Le Goff, J.M.; Leiste, R.; Levtchenko, M.; Levtchenko, P.; Li, C.; Likhoded, S.; Lin, C.H.; Lin, W.T.; Linde, F.L.; Lista, L.; Liu, Z.A.; Lohmann, W.; Longo, E.; Lu, Y.S.; Lubelsmeyer, K.; Luci, C.; Luminari, L.; Lustermann, W.; Ma, W.G.; Malgeri, L.; Malinin, A.; Mana, C.; Mangeol, D.; Mans, J.; Martin, J.P.; Marzano, F.; Mazumdar, K.; McNeil, R.R.; Mele, S.; Merola, L.; Meschini, M.; Metzger, W.J.; Mihul, A.; Milcent, H.; Mirabelli, G.; Mnich, J.; Mohanty, G.B.; Muanza, G.S.; Muijs, A.J.M.; Musicar, B.; Musy, M.; Nagy, S.; Natale, S.; Napolitano, M.; Nessi-Tedaldi, F.; Newman, H.; Niessen, T.; Nisati, A.; Kluge, Hannelies; Ofierzynski, R.; Organtini, G.; Palomares, C.; Pandoulas, D.; Paolucci, P.; Paramatti, R.; Passaleva, G.; Patricelli, S.; Paul, Thomas Cantzon; Pauluzzi, M.; Paus, C.; Pauss, F.; Pedace, M.; Pensotti, S.; Perret-Gallix, D.; Petersen, B.; Piccolo, D.; Pierella, F.; Pioppi, M.; Piroue, P.A.; Pistolesi, E.; Plyaskin, V.; Pohl, M.; Pojidaev, V.; Pothier, J.; Prokofev, D.O.; Prokofiev, D.; Quartieri, J.; Rahal-Callot, G.; Rahaman, M.A.; Raics, P.; Raja, N.; Ramelli, R.; Rancoita, P.G.; Ranieri, R.; Raspereza, A.; Razis, P.; Ren, D.; Rescigno, M.; Reucroft, S.; Riemann, S.; Riles, Keith; Roe, B.P.; Romero, L.; Rosca, A.; Rosier-Lees, S.; Roth, Stefan; Rosenbleck, C.; Roux, B.; Rubio, J.A.; Ruggiero, G.; Rykaczewski, H.; Sakharov, A.; Saremi, S.; Sarkar, S.; Salicio, J.; Sanchez, E.; Sanders, M.P.; Schafer, C.; Schegelsky, V.; Schmidt-Kaerst, S.; Schmitz, D.; Schopper, H.; Schotanus, D.J.; Schwering, G.; Sciacca, C.; Servoli, L.; Shevchenko, S.; Shivarov, N.; Shoutko, V.; Shumilov, E.; Shvorob, A.; Siedenburg, T.; Son, D.; Spillantini, P.; Steuer, M.; Stickland, D.P.; Stoyanov, B.; Straessner, A.; Sudhakar, K.; Sultanov, G.; Sun, L.Z.; Sushkov, S.; Suter, H.; Swain, J.D.; Szillasi, Z.; Tang, X.W.; Tarjan, P.; Tauscher, L.; Taylor, L.; Tellili, B.; Teyssier, D.; Timmermans, Charles; Ting, Samuel C.C.; Ting, S.M.; Tonwar, S.C.; Toth, J.; Tully, C.; Tung, K.L.; Ulbricht, J.; Valente, E.; Van de Walle, R.T.; Veszpremi, V.; Vesztergombi, G.; Vetlitsky, I.; Vicinanza, D.; Viertel, G.; Villa, S.; Vivargent, M.; Vlachos, S.; Vodopianov, I.; Vogel, H.; Vogt, H.; Vorobev, I.; Vorobyov, A.A.; Wadhwa, M.; Wallraff, W.; Wang, X.L.; Wang, Z.M.; Weber, M.; Wienemann, P.; Wilkens, H.; Wynhoff, S.; Xia, L.; Xu, Z.Z.; Yamamoto, J.; Yang, B.Z.; Yang, C.G.; Yang, H.J.; Yang, M.; Yeh, S.C.; Zalite, A.; Zalite, Yu.; Zhang, Z.P.; Zhao, J.; Zhu, G.Y.; Zhu, R.Y.; Zhuang, H.L.; Zichichi, A.; Zilizi, G.; Zimmermann, B.; Zoller, M.

    2002-01-01

    Inclusive D^{*+-} production in two-photon collisions is studied with the L3 detector at LEP, using 683 pb^{-1} of data collected at centre-of-mass energies from 183 to 208 GeV. Differential cross sections are determined as functions of the transverse momentum and pseudorapidity of the D^{*+-} mesons in the kinematic region 1 GeV e^+e^-D^{*+-}X)$ in this kinematical region is measured and the sigma(e^+e^- ---> e^+e^- cc{bar}X) cross section is derived. The measurements are compared with next-to-leading order perturbative QCD calculations.

  12. New two-photon based nanoscopic modalities and optogenetics

    DEFF Research Database (Denmark)

    Glückstad, Jesper

    that can be equipped with optical trapping handles for convenient mechanical control using only optical forces [6]. These microstructures illustrated above can be effectively handled with simultaneous top- and side-view on our BioPhotonics Workstation to undertake six-degree-of-freedom optical actuation...... of two-photon polymerised microstructures equipped with features easily entering the submicron-regime. Aided by European collaborators who fabricated test structures with built-in waveguides for us, we were able to put the idea of optically steerable freestanding waveguides – coined: wave-guided optical...

  13. Two photon production of charm states - a charmonium calculation

    International Nuclear Information System (INIS)

    Suaya, R.

    1980-03-01

    The comparison of the nonrelativistic multichannel calculation with the double Rutherford process shows that duality is satisfied, in the sense that if one averages the actual cross sections for a wide enough range of W one gets the same result as for nonstrongly interacting point like quarks. It has been shown for the case of e + e - annihilation that duality in this sense follows from nonrelativistic potential models; and it has been verified that this proof can be extended to two-photon processes

  14. Two-photon exclusive processes in quantum chromodynamics

    International Nuclear Information System (INIS)

    Brodsky, S.J.

    1986-07-01

    QCD predictions for γγ annihilation into single mesons, meson pairs, and baryon pairs are reviewed. Two-photon exclusive processes provide the most sensitive and practical measure of the distribution amplitudes, and thus a critical confrontation between QCD and experiment. Both the angular distribution and virtual photon mass dependence of these amplitudes are sensitive to the shapes of the phi (chi, Q). Novel effects involving the production of qq anti q anti q states at threshold are also discussed, and a new method is presented for systematically incorporating higher-order QCD corrections in γγ reactions

  15. Two-photon exclusive processes in quantum chromodynamics

    Energy Technology Data Exchange (ETDEWEB)

    Brodsky, S.J.

    1986-07-01

    QCD predictions for ..gamma gamma.. annihilation into single mesons, meson pairs, and baryon pairs are reviewed. Two-photon exclusive processes provide the most sensitive and practical measure of the distribution amplitudes, and thus a critical confrontation between QCD and experiment. Both the angular distribution and virtual photon mass dependence of these amplitudes are sensitive to the shapes of the phi (chi, Q). Novel effects involving the production of qq anti q anti q states at threshold are also discussed, and a new method is presented for systematically incorporating higher-order QCD corrections in ..gamma gamma.. reactions.

  16. Direct and indirect two-photon processes in semiconductors

    International Nuclear Information System (INIS)

    Hassan, A.R.

    1986-07-01

    The expressions describing direct and indirect two-photon absorption in crystals are given. They are valid both near and far from the energy gap. A perturbative approach through two different band models is adopted. The effects of the non-parabolicity and the degeneracy of the energy bands are considered. The numerical results are compared with the other theories and with a recent experimental data in Zn and AgCl. It is shown that the dominant transition mechanisms are of the allowed-allowed type near and far from the gap for both direct and indirect processes. (author)

  17. {Lambda}{bar {Lambda}} production in two-photon interactions

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, S.; Kubota, Y.; Lattery, M.; ONeill, J.J.; Patton, S.; Poling, R.; Riehle, T.; Savinov, V.; Smith, A. [University of Minnesota, Minneapolis, Minnesota 55455 (United States); Alam, M.S.; Athar, S.B.; Ling, Z.; Mahmood, A.H.; Severini, H.; Timm, S.; Wappler, F. [State University of New York at Albany, Albany, New York 12222 (United States); Anastassov, A.; Blinov, S.; Duboscq, J.E.; Fujino, D.; Fulton, R.; Gan, K.K.; Hart, T.; Honscheid, K.; Kagan, H.; Kass, R.; Lee, J.; Spencer, M.B.; Sung, M.; Undrus, A.; Wanke, R.; Wolf, A.; Zoeller, M.M. [Ohio State University, Columbus, Ohio 43210 (United States); Nemati, B.; Richichi, S.J.; Ross, W.R.; Skubic, P.; Wood, M. [University of Oklahoma, Norman, Oklahoma 73019 (United States); Bishai, M.; Fast, J.; Gerndt, E.; Hinson, J.W.; Menon, N.; Miller, D.H.; Shibata, E.I.; Shipsey, I.P.; Yurko, M. [Purdue University, West Lafayette, Indiana 47907 (United States); Gibbons, L.; Johnson, S.D.; Kwon, Y.; Roberts, S.; Thorndike, E.H. [University of Rochester, Rochester, New York 14627 (United States); Jessop, C.P.; Lingel, K.; Marsiske, H.; Perl, M.L.; Schaffner, S.F.; Ugolini, D.; Wang, R.; Zhou, X. [Stanford Linear Accelerator Center, Stanford University, Stanford, California 94309 (United States); Coan, T.E.; Fadeyev, V.; Korolkov, I.; Maravin, Y.; Narsky, I.; Shelkov, V.; Staeck, J.; Stroynowski, R.; Volobouev, I.; Ye, J. [Southern Methodist University, Dallas, Texas 75275 (United States); Artuso, M.; Efimov, A.; Frasconi, F.; Gao, M.; Goldberg, M.; He, D.; Kopp, S.; Moneti, G.C.; Mountain, R.; Mukhin, Y.; Schuh, S.; Skwarnicki, T.; Stone, S.; Viehhauser, G.; Xing, X. [Syracuse University, Syracuse, New York 13244 (United States); Bartelt, J.; Csorna, S.E.; Jain, V.; Marka, S. [Vanderbilt University, Nashville, Tennessee 37235 (United States); Freyberger, A.; Gibaut, D.; Godang, R.; Kinoshita, K.; Lai, I.C.; Pomianowski, P.; Schrenk, S.; and others

    1997-09-01

    Using the CLEO detector at the Cornell e{sup +}e{sup {minus}} storage ring CESR we study the two-photon production of {Lambda}{bar {Lambda}}, making the first observation of {gamma}{gamma}{r_arrow}{Lambda}{bar {Lambda}}. We present the cross section for {gamma}{gamma}{r_arrow}{Lambda}{bar {Lambda}} as a function of the {gamma}{gamma} center of mass energy and compare it to that predicted by the quark-diquark model. {copyright} {ital 1997} {ital The American Physical Society}

  18. Automated detection of analyzable metaphase chromosome cells depicted on scanned digital microscopic images

    Science.gov (United States)

    Qiu, Yuchen; Wang, Xingwei; Chen, Xiaodong; Li, Yuhua; Liu, Hong; Li, Shibo; Zheng, Bin

    2010-02-01

    Visually searching for analyzable metaphase chromosome cells under microscopes is quite time-consuming and difficult. To improve detection efficiency, consistency, and diagnostic accuracy, an automated microscopic image scanning system was developed and tested to directly acquire digital images with sufficient spatial resolution for clinical diagnosis. A computer-aided detection (CAD) scheme was also developed and integrated into the image scanning system to search for and detect the regions of interest (ROI) that contain analyzable metaphase chromosome cells in the large volume of scanned images acquired from one specimen. Thus, the cytogeneticists only need to observe and interpret the limited number of ROIs. In this study, the high-resolution microscopic image scanning and CAD performance was investigated and evaluated using nine sets of images scanned from either bone marrow (three) or blood (six) specimens for diagnosis of leukemia. The automated CAD-selection results were compared with the visual selection. In the experiment, the cytogeneticists first visually searched for the analyzable metaphase chromosome cells from specimens under microscopes. The specimens were also automated scanned and followed by applying the CAD scheme to detect and save ROIs containing analyzable cells while deleting the others. The automated selected ROIs were then examined by a panel of three cytogeneticists. From the scanned images, CAD selected more analyzable cells than initially visual examinations of the cytogeneticists in both blood and bone marrow specimens. In general, CAD had higher performance in analyzing blood specimens. Even in three bone marrow specimens, CAD selected 50, 22, 9 ROIs, respectively. Except matching with the initially visual selection of 9, 7, and 5 analyzable cells in these three specimens, the cytogeneticists also selected 41, 15 and 4 new analyzable cells, which were missed in initially visual searching. This experiment showed the feasibility of

  19. Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope.

    Science.gov (United States)

    Kolomytkin, O V; Golubok, A O; Davydov, D N; Timofeev, V A; Vinogradova, S A; Tipisev SYa

    1991-01-01

    The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 PMID:1712239

  20. Dual-mode optical microscope based on single-pixel imaging

    Science.gov (United States)

    Rodríguez, A. D.; Clemente, P.; Tajahuerce, E.; Lancis, J.

    2016-07-01

    We demonstrate an inverted microscope that can image specimens in both reflection and transmission modes simultaneously with a single light source. The microscope utilizes a digital micromirror device (DMD) for patterned illumination altogether with two single-pixel photosensors for efficient light detection. The system, a scan-less device with no moving parts, works by sequential projection of a set of binary intensity patterns onto the sample that are codified onto a modified commercial DMD. Data to be displayed are geometrically transformed before written into a memory cell to cancel optical artifacts coming from the diamond-like shaped structure of the micromirror array. The 24-bit color depth of the display is fully exploited to increase the frame rate by a factor of 24, which makes the technique practicable for real samples. Our commercial DMD-based LED-illumination is cost effective and can be easily coupled as an add-on module for already existing inverted microscopes. The reflection and transmission information provided by our dual microscope complement each other and can be useful for imaging non-uniform samples and to prevent self-shadowing effects.

  1. Nonlinear behavior of photoluminescence from silicon particles under two-photon excitation

    International Nuclear Information System (INIS)

    Xu Xingsheng; Yokoyama, Shiyoshi

    2011-01-01

    Two-photon excited fluorescence (TPEF) under continuous-wave excitation from silicon particles produced by a pulsed laser is investigated. Spectra and images of TPEF from silicon particles are studied under different excitation intensities and operation modes (continuous wave or pulse). It is found that the photoluminescence depends superlinearly on the excitation intensity and that the spectral shape and peaks vary with different silicon particles. The above phenomena show the nonlinear behavior of TPEF from silicon particles, and stimulated emission is a possible process.

  2. Two-photon mapping of localized field enhancements in thin nanostrip antennas

    DEFF Research Database (Denmark)

    Beermann, I.; Novikov, S.M.; Søndergaard, Thomas

    2008-01-01

    scanning optical microscopy, in which two-photon-excited photoluminescence (TPL) excited with a strongly focused laser beam at the wavelength 745 nm is detected. We use TPL images to map the local field enhancements from individual nanostrips at a resolution of 0.35µm and compare results with theoretical......Resonant scattering and local field enhancements by 11-nm-thin gold nanostrip antennas due to constructive interference of counter propagating slow surface plasmon polaritons is investigated. We characterize nanostrips of widths between 50-530 nm using both reflection spectroscopy and nonlinear...

  3. Intensity-based segmentation and visualization of cells in 3D microscopic images using the GPU

    Science.gov (United States)

    Kang, Mi-Sun; Lee, Jeong-Eom; Jeon, Woong-ki; Choi, Heung-Kook; Kim, Myoung-Hee

    2013-02-01

    3D microscopy images contain abundant astronomical data, rendering 3D microscopy image processing time-consuming and laborious on a central processing unit (CPU). To solve these problems, many people crop a region of interest (ROI) of the input image to a small size. Although this reduces cost and time, there are drawbacks at the image processing level, e.g., the selected ROI strongly depends on the user and there is a loss in original image information. To mitigate these problems, we developed a 3D microscopy image processing tool on a graphics processing unit (GPU). Our tool provides efficient and various automatic thresholding methods to achieve intensity-based segmentation of 3D microscopy images. Users can select the algorithm to be applied. Further, the image processing tool provides visualization of segmented volume data and can set the scale, transportation, etc. using a keyboard and mouse. However, the 3D objects visualized fast still need to be analyzed to obtain information for biologists. To analyze 3D microscopic images, we need quantitative data of the images. Therefore, we label the segmented 3D objects within all 3D microscopic images and obtain quantitative information on each labeled object. This information can use the classification feature. A user can select the object to be analyzed. Our tool allows the selected object to be displayed on a new window, and hence, more details of the object can be observed. Finally, we validate the effectiveness of our tool by comparing the CPU and GPU processing times by matching the specification and configuration.

  4. Super-resolution for everybody: An image processing workflow to obtain high-resolution images with a standard confocal microscope.

    Science.gov (United States)

    Lam, France; Cladière, Damien; Guillaume, Cyndélia; Wassmann, Katja; Bolte, Susanne

    2017-02-15

    In the presented work we aimed at improving confocal imaging to obtain highest possible resolution in thick biological samples, such as the mouse oocyte. We therefore developed an image processing workflow that allows improving the lateral and axial resolution of a standard confocal microscope. Our workflow comprises refractive index matching, the optimization of microscope hardware parameters and image restoration by deconvolution. We compare two different deconvolution algorithms, evaluate the necessity of denoising and establish the optimal image restoration procedure. We validate our workflow by imaging sub resolution fluorescent beads and measuring the maximum lateral and axial resolution of the confocal system. Subsequently, we apply the parameters to the imaging and data restoration of fluorescently labelled meiotic spindles of mouse oocytes. We measure a resolution increase of approximately 2-fold in the lateral and 3-fold in the axial direction throughout a depth of 60μm. This demonstrates that with our optimized workflow we reach a resolution that is comparable to 3D-SIM-imaging, but with better depth penetration for confocal images of beads and the biological sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Scanning tunneling microscopic images and scanning tunneling spectra for coupled rectangular quantum corrals

    International Nuclear Information System (INIS)

    Mitsuoka, Shigenori; Tamura, Akira

    2011-01-01

    Assuming that an electron confined by double δ-function barriers lies in a quasi-stationary state, we derived eigenstates and eigenenergies of the electron. Such an electron has a complex eigenenergy, and the imaginary part naturally leads to the lifetime of the electron associated with tunneling through barriers. We applied this point of view to the electron confined in a rectangular quantum corral (QC) on a noble metal surface, and obtained scanning tunneling microscopic images and a scanning tunneling spectrum consistent with experimental ones. We investigated the electron states confined in coupled QCs and obtained the coupled states constructed with bonding and anti-bonding states. Using those energy levels and wavefunctions we specified scanning tunneling microscope (STM) images and scanning tunneling spectra (STS) for the doubly and triply coupled QCs. In addition we pointed out the feature of resonant electron states associated with the same QCs at both ends of the triply coupled QCs.

  6. Spectro-refractometry of individual microscopic objects using swept-source quantitative phase imaging.

    Science.gov (United States)

    Jung, Jae-Hwang; Jang, Jaeduck; Park, Yongkeun

    2013-11-05

    We present a novel spectroscopic quantitative phase imaging technique with a wavelength swept-source, referred to as swept-source diffraction phase microscopy (ssDPM), for quantifying the optical dispersion of microscopic individual samples. Employing the swept-source and the principle of common-path interferometry, ssDPM measures the multispectral full-field quantitative phase imaging and spectroscopic microrefractometry of transparent microscopic samples in the visible spectrum with a wavelength range of 450-750 nm and a spectral resolution of less than 8 nm. With unprecedented precision and sensitivity, we demonstrate the quantitative spectroscopic microrefractometry of individual polystyrene beads, 30% bovine serum albumin solution, and healthy human red blood cells.

  7. Scanning electron microscope cathodoluminescence imaging of subgrain boundaries, twins and planar deformation features in quartz

    Science.gov (United States)

    Hamers, M. F.; Pennock, G. M.; Drury, M. R.

    2017-04-01

    The study of deformation features has been of great importance to determine deformation mechanisms in quartz. Relevant microstructures in both growth and deformation processes include dislocations, subgrains, subgrain boundaries, Brazil and Dauphiné twins and planar deformation features (PDFs). Dislocations and twin boundaries are most commonly imaged using a transmission electron microscope (TEM), because these cannot directly be observed using light microscopy, in contrast to PDFs. Here, we show that red-filtered cathodoluminescence imaging in a scanning electron microscope (SEM) is a useful method to visualise subgrain boundaries, Brazil and Dauphiné twin boundaries. Because standard petrographic thin sections can be studied in the SEM, the observed structures can be directly and easily correlated to light microscopy studies. In contrast to TEM preparation methods, SEM techniques are non-destructive to the area of interest on a petrographic thin section.

  8. Single-organelle tracking by two-photon conversion

    Science.gov (United States)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Fukui, Kiichi; Shin-Ichi Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Isobe, Keisuke; Itoh, Kazuyoshi

    2007-03-01

    Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

  9. Inclusive D*(+/-) production in two photon collisions at LEP

    CERN Document Server

    Prokofiev, Denis Olegovich

    2001-01-01

    In this thesis I present my results on the measurement of the open charm production in two-photon collision events done with the L3 detector at Large Electron Positron machine (LEP). The data sample was collected from 1997 through 2000 at center-of-mass energies ranging from 183 GeV to 209 GeV, corresponding to a total integrated luminosity of 683.4pb −1. The open charm production in two-photon collision events extrapolated to the full phase space is estimated to be: s&parl0;e+e-&rarrr;e +e-cc&d1;X&parr0;=9 23±69±109±222pb. The differential cross sections d s /dpT(D*±) and d s /d:η(D*±): are also measured as functions of transverse momentum pT(D*±) and the absolute value of pseudorapidity :η(D*±):, respectively. A fit to the data estimating the relative contributions of Direct and Resolved open charm production mechanisms is performed, giving (28.7 ± 5.6)% and (71.3 ± 8.8)%, respectively. Using those relative fractions, the Direct and Resolved process cross sections yield: s&p...

  10. Temperature dependence of the two photon absorption in indium arsenide

    International Nuclear Information System (INIS)

    Berryman, K.W.; Rella, C.W.

    1995-01-01

    Nonlinear optical processes in semiconductors have long been a source of interesting physics. Two photon absorption (TPA) is one such process, in which two photons provide the energy for the creation of an electron-hole pair. Researchers at other FEL centers have studied room temperature TPA in InSb, InAs, and HgCdTe. Working at the Stanford Picosecond FEL Center, we have extended and refined this work by measuring the temperature dependence of the TPA coefficient in InAs over the range from 80 to 350 K at four wavelengths: 4.5, 5.06, 6.01, and 6.3 microns. The measurements validate the functional dependence of recent band structure calculations with enough precision to discriminate parabolic from non-parabolic models, and to begin to observe smaller effects, such as contributions due to the split-off band. These experiments therefore serve as a strong independent test of the Kane band theory, as well as providing a starting point for detailed observations of other nonlinear absorption mechanisms

  11. Two-photon emission and multiphoton absorption by atoms

    International Nuclear Information System (INIS)

    Mu, X.

    1988-01-01

    This thesis consists of investigations of two problems concerning photon-atom interactions. The first topic deals with two-photon transitions in atomic inner shells. An independent-particle model has been used to describe the two-photon transitions between different inner-shell electron states. The first relativistic self-consistent-field calculation of these transition rates in Ag, Mo, and Xe has been carried out. The theoretical results are compared with recent measurements. Good agreement with measured rates is found except in some cases where more reliable experiments still need to be done. The second topic is multiphoton multiionization of atoms. The maximum entropy principle has been employed in this theoretical investigation. A detailed statistical analysis of measured ionic charge distributions produced in strong laser pulses has been carried out. The results of this analysis indicates that the charge-state distribution is a Poissonian, rather than the binomial which prevails under infrared radiation, and hence that ionization occurs stepwise during the pulse. This result is shown to be consistent with experimental data

  12. Monte Carlo simulation of two-photon processes

    International Nuclear Information System (INIS)

    Daverveldt, P.H.W.M.

    1985-01-01

    During the last two decades e + e - collider experiments provided physicists with a wealth of important discoveries concerning elementary particle physics. This thesis explains in detail how the Monte Carlo approach can be applied to establish the comparison between two-photon experiments and theory. The author describes the main motives for and objectives of two-photon research. He defines the kinematics and pays attention to some special kinematical regions. Also a popular approximation for the exact differential cross section is reviewed. Next he discusses the calculation of the complete lowest order cross section for processes with four leptons in the final state and for reactions such as e + e - →e + e - qanti q, e + e - →μ + μ - qanti q. Radiative corrections to the multiperipheral diagrams are considered. The author explains in detail the distinction between soft and hard photon corrections which turns out to be somewhat more tricky than in the case of radiative corrections to one-photon processes. Finally, he presents some results which were obtained by using the event generators. (Auth.)

  13. Preprocessing with Photoshop Software on Microscopic Images of A549 Cells in Epithelial-Mesenchymal Transition.

    Science.gov (United States)

    Ren, Zhou-Xin; Yu, Hai-Bin; Shen, Jun-Ling; Li, Ya; Li, Jian-Sheng

    2015-06-01

    To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

  14. Reducing charging effects in scanning electron microscope images by Rayleigh contrast stretching method (RCS).

    Science.gov (United States)

    Wan Ismail, W Z; Sim, K S; Tso, C P; Ting, H Y

    2011-01-01

    To reduce undesirable charging effects in scanning electron microscope images, Rayleigh contrast stretching is developed and employed. First, re-scaling is performed on the input image histograms with Rayleigh algorithm. Then, contrast stretching or contrast adjustment is implemented to improve the images while reducing the contrast charging artifacts. This technique has been compared to some existing histogram equalization (HE) extension techniques: recursive sub-image HE, contrast stretching dynamic HE, multipeak HE and recursive mean separate HE. Other post processing methods, such as wavelet approach, spatial filtering, and exponential contrast stretching, are compared as well. Overall, the proposed method produces better image compensation in reducing charging artifacts. Copyright © 2011 Wiley Periodicals, Inc.

  15. Noise removal in extended depth of field microscope images through nonlinear signal processing.

    Science.gov (United States)

    Zahreddine, Ramzi N; Cormack, Robert H; Cogswell, Carol J

    2013-04-01

    Extended depth of field (EDF) microscopy, achieved through computational optics, allows for real-time 3D imaging of live cell dynamics. EDF is achieved through a combination of point spread function engineering and digital image processing. A linear Wiener filter has been conventionally used to deconvolve the image, but it suffers from high frequency noise amplification and processing artifacts. A nonlinear processing scheme is proposed which extends the depth of field while minimizing background noise. The nonlinear filter is generated via a training algorithm and an iterative optimizer. Biological microscope images processed with the nonlinear filter show a significant improvement in image quality and signal-to-noise ratio over the conventional linear filter.

  16. Surface features on Sahara soil dust particles made visible by atomic force microscope (AFM) phase images

    OpenAIRE

    G. Helas; M. O. Andreae

    2008-01-01

    We show that atomic force microscopy (AFM) phase images can reveal surface features of soil dust particles, which are not evident using other microscopic methods. The non-contact AFM method is able to resolve topographical structures in the nanometer range as well as to uncover repulsive atomic forces and attractive van der Waals' forces, and thus gives insight to surface properties. Though the method does not allow quantitative assignment in terms of chemical compound description, it clearly...

  17. Pancam and microscopic imager observations of dust on the Spirit Rovers

    DEFF Research Database (Denmark)

    Vaughan....[], Alicia F.; Johnson, Jeffrey R.; Walter, Goetz

    2010-01-01

    This work describes dust deposits on the Spirit Rover over 2000 sols through examination of Pancam and Microscopic Imager observations of specific locations on the rover body, including portions of the solar array, Pancam and Mini-TES calibration targets, and the magnets. This data set reveals...... the three "cleaning events" experienced by Spirit to date, the spectral properties of dust, and the tendency of dust particles to form aggregates 100 um and larger...

  18. Algorithm for detection of overlapped red blood cells in microscopic images of blood smears

    OpenAIRE

    Romero-Rondón, Miguel Fabián; Sanabria-Rosas, Laura Melissa; Bautista-Rozo, Lola Xiomara; Mendoza-Castellanos, Alfonso

    2016-01-01

    The hemogram is one of the most requested medical tests as it presents details about the three cell series in the blood: red series, white series and platelet series. To make some diagnostics, the specialist must undertake the test manually, observing the blood cells under the microscope, which implies a great physical effort. In order to facilitate this work, different digital image processing techniques to detect and classify red blood cells have been proposed. However, a common problem is ...

  19. Fine-grained leukocyte classification with deep residual learning for microscopic images.

    Science.gov (United States)

    Qin, Feiwei; Gao, Nannan; Peng, Yong; Wu, Zizhao; Shen, Shuying; Grudtsin, Artur

    2018-08-01

    Leukocyte classification and cytometry have wide applications in medical domain, previous researches usually exploit machine learning techniques to classify leukocytes automatically. However, constrained by the past development of machine learning techniques, for example, extracting distinctive features from raw microscopic images are difficult, the widely used SVM classifier only has relative few parameters to tune, these methods cannot efficiently handle fine-grained classification cases when the white blood cells have up to 40 categories. Based on deep learning theory, a systematic study is conducted on finer leukocyte classification in this paper. A deep residual neural network based leukocyte classifier is constructed at first, which can imitate the domain expert's cell recognition process, and extract salient features robustly and automatically. Then the deep neural network classifier's topology is adjusted according to the prior knowledge of white blood cell test. After that the microscopic image dataset with almost one hundred thousand labeled leukocytes belonging to 40 categories is built, and combined training strategies are adopted to make the designed classifier has good generalization ability. The proposed deep residual neural network based classifier was tested on microscopic image dataset with 40 leukocyte categories. It achieves top-1 accuracy of 77.80%, top-5 accuracy of 98.75% during the training procedure. The average accuracy on the test set is nearly 76.84%. This paper presents a fine-grained leukocyte classification method for microscopic images, based on deep residual learning theory and medical domain knowledge. Experimental results validate the feasibility and effectiveness of our approach. Extended experiments support that the fine-grained leukocyte classifier could be used in real medical applications, assist doctors in diagnosing diseases, reduce human power significantly. Copyright © 2018 Elsevier B.V. All rights reserved.

  20. Microscope image based fully automated stomata detection and pore measurement method for grapevines

    Directory of Open Access Journals (Sweden)

    Hiranya Jayakody

    2017-11-01

    Full Text Available Abstract Background Stomatal behavior in grapevines has been identified as a good indicator of the water stress level and overall health of the plant. Microscope images are often used to analyze stomatal behavior in plants. However, most of the current approaches involve manual measurement of stomatal features. The main aim of this research is to develop a fully automated stomata detection and pore measurement method for grapevines, taking microscope images as the input. The proposed approach, which employs machine learning and image processing techniques, can outperform available manual and semi-automatic methods used to identify and estimate stomatal morphological features. Results First, a cascade object detection learning algorithm is developed to correctly identify multiple stomata in a large microscopic image. Once the regions of interest which contain stomata are identified and extracted, a combination of image processing techniques are applied to estimate the pore dimensions of the stomata. The stomata detection approach was compared with an existing fully automated template matching technique and a semi-automatic maximum stable extremal regions approach, with the proposed method clearly surpassing the performance of the existing techniques with a precision of 91.68% and an F1-score of 0.85. Next, the morphological features of the detected stomata were measured. Contrary to existing approaches, the proposed image segmentation and skeletonization method allows us to estimate the pore dimensions even in cases where the stomatal pore boundary is only partially visible in the microscope image. A test conducted using 1267 images of stomata showed that the segmentation and skeletonization approach was able to correctly identify the stoma opening 86.27% of the time. Further comparisons made with manually traced stoma openings indicated that the proposed method is able to estimate stomata morphological features with accuracies of 89.03% for area

  1. Towards native-state imaging in biological context in the electron microscope

    Science.gov (United States)

    Weston, Anne E.; Armer, Hannah E. J.

    2009-01-01

    Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

  2. Quantum theory of two-photon wavepacket interference in a beamsplitter

    International Nuclear Information System (INIS)

    Wang, Kaige

    2006-01-01

    A general theory is derived for the interference of a two-photon wavepacket in a beamsplitter. The theory is presented in the Schroedinger picture so that the quantum nature of the two-photon interference is explicitly revealed. We find that the topological symmetry of the probability-amplitude spectrum of the two-photon wavepacket dominates the nature of the two-photon interference, which may be distinguished by the increase or decrease of the coincidence probability in the absence of interference. However, two-photon entanglement can be identified by the nature of the interference. We demonstrate the necessary and sufficient conditions for perfect two-photon interference. It is shown that a two-photon entangled state with an anti-symmetric spectrum passes through a 50/50 beamsplitter with perfect transparency. The theory provides us with a unified understanding of the various two-photon interference effects. (topical review)

  3. A hybrid scanning force and light microscope for surface imaging and three-dimensional optical sectioning in differential interference contrast.

    Science.gov (United States)

    Stemmer, A

    1995-04-01

    The design of a scanned-cantilever-type force microscope is presented which is fully integrated into an inverted high-resolution video-enhanced light microscope. This set-up allows us to acquire thin optical sections in differential interference contrast (DIC) or polarization while the force microscope is in place. Such a hybrid microscope provides a unique platform to study how cell surface properties determine, or are affected by, the three-dimensional dynamic organization inside the living cell. The hybrid microscope presented in this paper has proven reliable and versatile for biological applications. It is the only instrument that can image a specimen by force microscopy and high-power DIC without having either to translate the specimen or to remove the force microscope. Adaptation of the design features could greatly enhance the suitability of other force microscopes for biological work.

  4. Automatic analysis of digitized TV-images by a computer-driven optical microscope

    International Nuclear Information System (INIS)

    Rosa, G.; Di Bartolomeo, A.; Grella, G.; Romano, G.

    1997-01-01

    New methods of image analysis and three-dimensional pattern recognition were developed in order to perform the automatic scan of nuclear emulsion pellicles. An optical microscope, with a motorized stage, was equipped with a CCD camera and an image digitizer, and interfaced to a personal computer. Selected software routines inspired the design of a dedicated hardware processor. Fast operation, high efficiency and accuracy were achieved. First applications to high-energy physics experiments are reported. Further improvements are in progress, based on a high-resolution fast CCD camera and on programmable digital signal processors. Applications to other research fields are envisaged. (orig.)

  5. Computer Aided Solution for Automatic Segmenting and Measurements of Blood Leucocytes Using Static Microscope Images.

    Science.gov (United States)

    Abdulhay, Enas; Mohammed, Mazin Abed; Ibrahim, Dheyaa Ahmed; Arunkumar, N; Venkatraman, V

    2018-02-17

    Blood leucocytes segmentation in medical images is viewed as difficult process due to the variability of blood cells concerning their shape and size and the difficulty towards determining location of Blood Leucocytes. Physical analysis of blood tests to recognize leukocytes is tedious, time-consuming and liable to error because of the various morphological components of the cells. Segmentation of medical imagery has been considered as a difficult task because of complexity of images, and also due to the non-availability of leucocytes models which entirely captures the probable shapes in each structures and also incorporate cell overlapping, the expansive variety of the blood cells concerning their shape and size, various elements influencing the outer appearance of the blood leucocytes, and low Static Microscope Image disparity from extra issues outcoming about because of noise. We suggest a strategy towards segmentation of blood leucocytes using static microscope images which is a resultant of three prevailing systems of computer vision fiction: enhancing the image, Support vector machine for segmenting the image, and filtering out non ROI (region of interest) on the basis of Local binary patterns and texture features. Every one of these strategies are modified for blood leucocytes division issue, in this manner the subsequent techniques are very vigorous when compared with its individual segments. Eventually, we assess framework based by compare the outcome and manual division. The findings outcome from this study have shown a new approach that automatically segments the blood leucocytes and identify it from a static microscope images. Initially, the method uses a trainable segmentation procedure and trained support vector machine classifier to accurately identify the position of the ROI. After that, filtering out non ROI have proposed based on histogram analysis to avoid the non ROI and chose the right object. Finally, identify the blood leucocytes type using

  6. Cancer cell imaging by stable wet near-field scanning optical microscope with resonance tracking method

    International Nuclear Information System (INIS)

    Park, Kyoung-Duck; Park, Doo-Jae; Jeong, Mun-Seok; Choi, Geun-Chang; Lee, Seung-Gol; Byeon, Clare-Chisu; Choi, Soo-Bong

    2014-01-01

    We report on a successful topographical and optical imaging of various cancer cells in liquid and in air by using a stable wet near-field scanning optical microscope that utilizes a resonance tracking method. We observed a clear dehydration which gives rise to a decrease in the cell volume down to 51%. In addition, a micro-ball lens effect due to the round-shaped young cancer cells was observed from near-field imaging, where the refractive index of young cancer cells was deduced.

  7. Cancer cell imaging by stable wet near-field scanning optical microscope with resonance tracking method

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kyoung-Duck [Sungkyunkwan University, Suwon (Korea, Republic of); Inha University, Incheon (Korea, Republic of); Park, Doo-Jae; Jeong, Mun-Seok [Sungkyunkwan University, Suwon (Korea, Republic of); Choi, Geun-Chang [Seoul National University, Seoul (Korea, Republic of); Lee, Seung-Gol [Inha University, Incheon (Korea, Republic of); Byeon, Clare-Chisu [Kyungpook National University, Daegu (Korea, Republic of); Choi, Soo-Bong [Incheon National University, Incheon (Korea, Republic of)

    2014-05-15

    We report on a successful topographical and optical imaging of various cancer cells in liquid and in air by using a stable wet near-field scanning optical microscope that utilizes a resonance tracking method. We observed a clear dehydration which gives rise to a decrease in the cell volume down to 51%. In addition, a micro-ball lens effect due to the round-shaped young cancer cells was observed from near-field imaging, where the refractive index of young cancer cells was deduced.

  8. Adaptive and automatic red blood cell counting method based on microscopic hyperspectral imaging technology

    Science.gov (United States)

    Liu, Xi; Zhou, Mei; Qiu, Song; Sun, Li; Liu, Hongying; Li, Qingli; Wang, Yiting

    2017-12-01

    Red blood cell counting, as a routine examination, plays an important role in medical diagnoses. Although automated hematology analyzers are widely used, manual microscopic examination by a hematologist or pathologist is still unavoidable, which is time-consuming and error-prone. This paper proposes a full-automatic red blood cell counting method which is based on microscopic hyperspectral imaging of blood smears and combines spatial and spectral information to achieve high precision. The acquired hyperspectral image data of the blood smear in the visible and near-infrared spectral range are firstly preprocessed, and then a quadratic blind linear unmixing algorithm is used to get endmember abundance images. Based on mathematical morphological operation and an adaptive Otsu’s method, a binaryzation process is performed on the abundance images. Finally, the connected component labeling algorithm with magnification-based parameter setting is applied to automatically select the binary images of red blood cell cytoplasm. Experimental results show that the proposed method can perform well and has potential for clinical applications.

  9. Ultrawidefield microscope for high-speed fluorescence imaging and targeted optogenetic stimulation.

    Science.gov (United States)

    Werley, Christopher A; Chien, Miao-Ping; Cohen, Adam E

    2017-12-01

    The rapid increase in the number and quality of fluorescent reporters and optogenetic actuators has yielded a powerful set of tools for recording and controlling cellular state and function. To achieve the full benefit of these tools requires improved optical systems with high light collection efficiency, high spatial and temporal resolution, and patterned optical stimulation, in a wide field of view (FOV). Here we describe our 'Firefly' microscope, which achieves these goals in a Ø6 mm FOV. The Firefly optical system is optimized for simultaneous photostimulation and fluorescence imaging in cultured cells. All but one of the optical elements are commercially available, yet the microscope achieves 10-fold higher light collection efficiency at its design magnification than the comparable commercially available microscope using the same objective. The Firefly microscope enables all-optical electrophysiology ('Optopatch') in cultured neurons with a throughput and information content unmatched by other neuronal phenotyping systems. This capability opens possibilities in disease modeling and phenotypic drug screening. We also demonstrate applications of the system to voltage and calcium recordings in human induced pluripotent stem cell derived cardiomyocytes.

  10. The system of digital-image optical microscope in semiconductor particle detector development

    International Nuclear Information System (INIS)

    Han Lixiang; Li Zhankui; Jin Genming; Wang Zhusheng; Xiao Guoqing

    2009-01-01

    Optical microscopic detection is very important in the process of semiconductor particle detector development. A system of digital-image optical microscope has been constructed with rather low price, which performance is comparable with the moderate-level imports. The system mounts powerful dry objective, and a 2μm resolution could be achieved. Observations with bright and dark field, polarized light,and interference light can be carried out on it. The system have large area on-line monitor,and the photographic device can be controlled by PC. It can be used in the control of defects and contaminations, pattern test, identification of crystal backing, inspection of the smoothness and the flatness of the crystal surface. It can also be used in some precise procedures, such as test, assembly, packaging and repairing. The quality of the bond could be examined by observing the appearance of the bond point and the microscopic structure of the solder. The surface fluctuation can be precisely measured under the microscope with the technology of multi-beam interference. In the article, the application of this system for semiconductor particle detector development has been illustrated, and the construction information has been described in detail. (authors)

  11. Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope

    International Nuclear Information System (INIS)

    De, Tathagata; Chettoor, Antony M.; Agarwal, Pranav; Salapaka, Murti V.; Nettikadan, Saju

    2010-01-01

    The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells (Kasas and Ikai, 1995), (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.

  12. Hyperspectral microscope for in vivo imaging of microstructures and cells in tissues

    Science.gov (United States)

    Demos,; Stavros, G [Livermore, CA

    2011-05-17

    An optical hyperspectral/multimodal imaging method and apparatus is utilized to provide high signal sensitivity for implementation of various optical imaging approaches. Such a system utilizes long working distance microscope objectives so as to enable off-axis illumination of predetermined tissue thereby allowing for excitation at any optical wavelength, simplifies design, reduces required optical elements, significantly reduces spectral noise from the optical elements and allows for fast image acquisition enabling high quality imaging in-vivo. Such a technology provides a means of detecting disease at the single cell level such as cancer, precancer, ischemic, traumatic or other type of injury, infection, or other diseases or conditions causing alterations in cells and tissue micro structures.

  13. Two-photon Absorption In Quantum Dots,quantum Dashes And Related Materials

    Energy Technology Data Exchange (ETDEWEB)

    Jain, Ravinder

    2009-08-31

    We have proposed the use of USQDs for various deep-tissue biological imaging applications, notably wavelength-multiplexed multicolor imaging and intra-nuclear studies such as those involving cell apoptosis, and have studied the issue of maximizing two-photon absorption-induced fluorescence (TPAF) signals from CdSe/ZnS USQDs to be used for this application. In particular, using 2 nm USQDs, we have shown that the TPAF signal at 780 nm is ~ 8 times that at 850 nm and 68 times that at 900 nm, two wavelengths that have been used in previous studies using CdSe/ZnS SQDs for deep-tissue imaging of biological studies via TPAF .

  14. Two-photon-based photoactivation in live zebrafish embryos.

    Science.gov (United States)

    Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

    2010-12-24

    Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

  15. Two-photon resonant, stimulated processes in krypton and xenon

    International Nuclear Information System (INIS)

    Miller, J.C.

    1988-11-01

    Both on-axis and conical emissions have been observed following two-photon pumping of the 5p states of krypton and the 6p', 7p, 8p, and 4f states of xenon. In the former case, coherent emissions from the 5p states to the 5s are observed, and in the latter case, many p→s, d→p, and f→d cascade emissions are observed. By analogy to the well-studied alkali and alkaline earth examples, the emissions are discussed in terms of amplified spontaneous emission (ASE), stimulated hyper-Raman scattering, and parametric four-wave mixing. The physical processes responsible for the conical emission and for intensity anomalies in the xenon p→s emissions are not understood at present. Interference effects due to coherent cancellation between competing excitation pathways may be occurring. 4 refs., 3 figs

  16. N-dimensional integrability from two-photon coalgebra symmetry

    International Nuclear Information System (INIS)

    Ballesteros, Angel; Blasco, Alfonso; Herranz, Francisco J

    2009-01-01

    A wide class of Hamiltonian systems with N degrees of freedom and endowed with, at least, (N - 2) functionally independent integrals of motion in involution is constructed by making use of the two-photon Lie-Poisson coalgebra (h 6 , Δ). The set of (N - 2) constants of the motion is shown to be a universal one for all these Hamiltonians, irrespective of the dependence of the latter on several arbitrary functions and N free parameters. Within this large class of quasi-integrable N-dimensional Hamiltonians, new families of completely integrable systems are identified by finding explicitly a new independent integral I through the analysis of the sub-coalgebra structure of h 6 . In particular, new completely integrable N-dimensional Hamiltonians describing natural systems, geodesic flows and static electromagnetic Hamiltonians are presented

  17. Phonon-assisted two-photon exciton transitions in semiconductors

    International Nuclear Information System (INIS)

    Hassan, A.R.

    1987-08-01

    The theory of phonon-assisted two-photon transitions to excitonic states in semiconductors has been theoretically investigated. The effects of both the nonparabolicity of the band and the degeneracy of the valence band have been taken into account. Expressions for the absorption coefficient through different band models are calculated. The numerical applications to CdI 2 and GaP show that the 4-band model gives the dominant contribution which leads to a final s-exciton state. An exciton peak appears at an energy which is close to that recently observed in CdI 2 . The non-parabolic effect enhances the absorption coefficient by a two-order of magnitude. (author). 6 refs, 1 fig., 1 tab

  18. Nanosecond Time-Resolved Microscopic Gate-Modulation Imaging of Polycrystalline Organic Thin-Film Transistors

    Science.gov (United States)

    Matsuoka, Satoshi; Tsutsumi, Jun'ya; Matsui, Hiroyuki; Kamata, Toshihide; Hasegawa, Tatsuo

    2018-02-01

    We develop a time-resolved microscopic gate-modulation (μ GM ) imaging technique to investigate the temporal evolution of the channel current and accumulated charges in polycrystalline pentacene thin-film transistors (TFTs). A time resolution of as high as 50 ns is achieved by using a fast image-intensifier system that could amplify a series of instantaneous optical microscopic images acquired at various time intervals after the stepped gate bias is switched on. The differential images obtained by subtracting the gate-off image allows us to acquire a series of temporal μ GM images that clearly show the gradual propagation of both channel charges and leaked gate fields within the polycrystalline channel layers. The frontal positions for the propagations of both channel charges and leaked gate fields coincide at all the time intervals, demonstrating that the layered gate dielectric capacitors are successively transversely charged up along the direction of current propagation. The initial μ GM images also indicate that the electric field effect is originally concentrated around a limited area with a width of a few micrometers bordering the channel-electrode interface, and that the field intensity reaches a maximum after 200 ns and then decays. The time required for charge propagation over the whole channel region with a length of 100 μ m is estimated at about 900 ns, which is consistent with the measured field-effect mobility and the temporal-response model for organic TFTs. The effect of grain boundaries can be also visualized by comparison of the μ GM images for the transient and the steady states, which confirms that the potential barriers at the grain boundaries cause the transient shift in the accumulated charges or the transient accumulation of additional charges around the grain boundaries.

  19. Helium Ion Microscope: A New Tool for Sub-nanometer Imaging of Soft Materials

    Science.gov (United States)

    Shutthanandan, V.; Arey, B.; Smallwood, C. R.; Evans, J. E.

    2017-12-01

    High-resolution inspection of surface details is needed in many biological and environmental researches to understand the Soil organic material (SOM)-mineral interactions along with identifying microbial communities and their interactions. SOM shares many imaging characteristics with biological samples and getting true surface details from these materials are challenging since they consist of low atomic number materials. FE-SEM imaging is the main imagining technique used to image these materials in the past. These SEM images often show loss of resolution and increase noise due to beam damage and charging issues. Newly developed Helium Ion Microscope (HIM), on the other hand can overcome these difficulties and give very fine details. HIM is very similar to scanning electron microscopy (SEM) but instead of using electrons as a probe beam, HIM uses helium ions with energy ranges from 5 to 40 keV. HIM offers a series of advantages compared to SEM such as nanometer and sub-nanometer image resolutions (about 0.35 nm), detailed surface topography, high surface sensitivity, low Z material imaging (especially for polymers and biological samples), high image contrast, and large depth of field. In addition, HIM also has the ability to image insulating materials without any conductive coatings so that surface details are not modified. In this presentation, several scientific applications across biology and geochemistry will be presented to highlight the effectiveness of this powerful microscope. Acknowledgements: Research was performed using the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at PNNL. Work was supported by DOE-BER Mesoscale to Molecules Bioimaging Project FWP# 66382.

  20. A preliminary investigation: the impact of microscopic condenser on depth of field in cytogenetic imaging

    Science.gov (United States)

    Ren, Liqiang; Qiu, Yuchen; Li, Zheng; Li, Yuhua; Zheng, Bin; Li, Shibo; Chen, Wei R.; Liu, Hong

    2013-02-01

    As one of the important components of optical microscopes, the condenser has a considerable impact on system performance, especially on the depth of field (DOF). DOF is a critical technical feature in cytogenetic imaging that may affect the efficiency and accuracy of clinical diagnosis. The purpose of this study is to investigate the influence of microscopic condenser on DOF using a prototype of transmitted optical microscope, based on objective and subjective evaluations. After the description of the relationship between condenser and objective lens and the theoretical analysis of the condenser impact on system numerical aperture and DOF, a standard resolution pattern and several cytogenetic samples are adopted to assess the condenser impact on DOF, respectively. The experimental results of these objective and subjective evaluations are in agreement with the theoretical analysis and show that, under the specific intermediate range of condenser numerical aperture ( NAcond ), the DOF value decreases with the increase of NAcond . Although the above qualitative results are obtained under the experimental conditions with a specific prototype system, the methods presented in this preliminary investigation could offer useful guidelines for optimizing operational parameters in cytogenetic imaging.

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

    Directory of Open Access Journals (Sweden)

    Philipp Steven

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

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

    Directory of Open Access Journals (Sweden)

    Denis Soulet

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

  3. Adaptive Scanning Optical Microscope (ASOM): A multidisciplinary optical microscope design for large field of view and high resolution imaging

    NARCIS (Netherlands)

    Potsaid, B.; Bellouard, Y.J.; Wen, J.T.

    2005-01-01

    From micro-assembly to biological observation, the optical microscope remains one of the most important tools for observing below the threshold of the naked human eye. However, in its conventional form, it suffers from a trade-off between resolution and field of view. This paper presents a new

  4. Characterization of platelet adhesion under flow using microscopic image sequence analysis.

    Science.gov (United States)

    Machin, M; Santomaso, A; Cozzi, M R; Battiston, M; Mazzuccato, M; De Marco, L; Canu, P

    2005-07-01

    A method for quantitative analysis of platelet deposition under flow is discussed here. The model system is based upon perfusion of blood platelets over an adhesive substrate immobilized on a glass coverslip acting as the lower surface of a rectangular flow chamber. The perfusion apparatus is mounted onto an inverted microscope equipped with epifluorescent illumination and intensified CCD video camera. Characterization is based on information obtained from a specific image analysis method applied to continuous sequences of microscopical images. Platelet recognition across the sequence of images is based on a time-dependent, bidimensional, gaussian-like pdf. Once a platelet is located,the variation of its position and shape as a function of time (i.e., the platelet history) can be determined. Analyzing the history we can establish if the platelet is moving on the surface, the frequency of this movement and the distance traveled before its resumes the velocity of a non-interacting cell. Therefore, we can determine how long the adhesion would last which is correlated to the resistance of the platelet-substrate bond. This algorithm enables the dynamic quantification of trajectories, as well as residence times, arrest and release frequencies for a high numbers of platelets at the same time. Statistically significant conclusions on platelet-surface interactions can then be obtained. An image analysis tool of this kind can dramatically help the investigation and characterization of the thrombogenic properties of artificial surfaces such as those used in artificial organs and biomedical devices.

  5. Open-dish incubator for live cell imaging with an inverted microscope.

    Science.gov (United States)

    Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

    2003-10-01

    Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

  6. Imaging C. elegans embryos using an epifluorescent microscope and open source software.

    Science.gov (United States)

    Verbrugghe, Koen J C; Chan, Raymond C

    2011-03-24

    Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples(1,2). Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage(3), thus providing an ideal experiment model for studying questions in cell biology(4,5)and development(6-9). C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis(10,11)) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis(12-15)). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters(16,17). These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo(18-21). In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

  7. Tissue imaging with a stigmatic mass microscope using laser desorption/ionization

    Science.gov (United States)

    Awazu, Kunio; Hazama, Hisanao; Hamanaka, Tomonori; Aoki, Jun; Toyoda, Michisato; Naito, Yasuhide

    2012-03-01

    A novel stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch were clearly observed, and the estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Tissue sections of a brain and eyes of a mouse stained with crystal violet and methylene blue were observed in the linear mode, and the stigmatic total ion images of crystal violet and methylene blue agreed well with the optical photomicrograph of the same sections. Especially, the fine structure in the cornea tissue was clearly observed with a spatial resolution in the range of micrometers. Although the total measurement time of the stigmatic ion image for the whole-eye section was about 59 minutes using a laser with a 10 Hz repetition rate, the measurement time could be reduced to about 35 s using a laser with a 1 kHz repetition rate and automation of measurements. The stigmatic mass microscope developed in this research should be suitable for high-spatial resolution and high-throughput imaging mass spectrometry for pathology, pharmacokinetics, and so on.

  8. The virtual microscopy database-sharing digital microscope images for research and education.

    Science.gov (United States)

    Lee, Lisa M J; Goldman, Haviva M; Hortsch, Michael

    2018-02-14

    Over the last 20 years, virtual microscopy has become the predominant modus of teaching the structural organization of cells, tissues, and organs, replacing the use of optical microscopes and glass slides in a traditional histology or pathology laboratory setting. Although virtual microscopy image files can easily be duplicated, creating them requires not only quality histological glass slides but also an expensive whole slide microscopic scanner and massive data storage devices. These resources are not available to all educators and researchers, especially at new institutions in developing countries. This leaves many schools without access to virtual microscopy resources. The Virtual Microscopy Database (VMD) is a new resource established to address this problem. It is a virtual image file-sharing website that allows researchers and educators easy access to a large repository of virtual histology and pathology image files. With the support from the American Association of Anatomists (Bethesda, MD) and MBF Bioscience Inc. (Williston, VT), registration and use of the VMD are currently free of charge. However, the VMD site is restricted to faculty and staff of research and educational institutions. Virtual Microscopy Database users can upload their own collection of virtual slide files, as well as view and download image files for their own non-profit educational and research purposes that have been deposited by other VMD clients. Anat Sci Educ. © 2018 American Association of Anatomists. © 2018 American Association of Anatomists.

  9. Performance limitations of imaging microscopes for soft x-ray applications

    International Nuclear Information System (INIS)

    Lewotsky, K.L.; Kotha, A.; Harvey, J.E.

    1993-01-01

    Recent advances in the fabrication of nanometer-scale multilayer structures have yielded high-reflectance mirrors operating at near-normal incidence for soft X-ray wavelengths. These developments have stimulated renewed interest in high-resolution soft X-ray microscopy. The design of a Schwarzschild imaging microscope for soft X-ray applications has been reported by Hoover and Shealy. Based upon a geometrical ray-trace analysis of the residual design errors, diffraction-limited performance at a wavelength of 100 angstrom was predicted over an object size (diameter) of 0.4 mm. In this paper the authors expand upon the previous analysis of the Schwarzschild X-ray microscope design by determining the total image degradation due to diffraction, geometrical aberrations, alignment errors, and realistic assumptions concerning optical fabrication errors. NASA's Optical Surface Analysis Code (OSAC) is used to model the image degradation effects of residual surface irregularities over the entire range of relevant spatial frequencies. This includes small angle scattering effects due to mid spatial frequency surface errors falling between the traditional figure and finish specifications. Performance predictions are presented parametrically to provide some insight into the optical fabrication and alignment tolerances necessary to meet a particular image quality requirement

  10. Imaging the p-n junction in a gallium nitride nanowire with a scanning microwave microscope

    Energy Technology Data Exchange (ETDEWEB)

    Imtiaz, Atif [Physical Measurement Laboratory, National Institute of Standards and Technology, Boulder, Colorado 80305 (United States); Department of Electrical, Computer, and Energy Engineering, University of Colorado, Boulder, Colorado 80309 (United States); Wallis, Thomas M.; Brubaker, Matt D.; Blanchard, Paul T.; Bertness, Kris A.; Sanford, Norman A.; Kabos, Pavel, E-mail: kabos@boulder.nist.gov [Physical Measurement Laboratory, National Institute of Standards and Technology, Boulder, Colorado 80305 (United States); Weber, Joel C. [Physical Measurement Laboratory, National Institute of Standards and Technology, Boulder, Colorado 80305 (United States); Department of Mechanical Engineering, University of Colorado, Boulder, Colorado 80309 (United States); Coakley, Kevin J. [Information Technology Laboratory, National Institute of Standards and Technology, Boulder, Colorado 80305 (United States)

    2014-06-30

    We used a broadband, atomic-force-microscope-based, scanning microwave microscope (SMM) to probe the axial dependence of the charge depletion in a p-n junction within a gallium nitride nanowire (NW). SMM enables the visualization of the p-n junction location without the need to make patterned electrical contacts to the NW. Spatially resolved measurements of S{sub 11}{sup ′}, which is the derivative of the RF reflection coefficient S{sub 11} with respect to voltage, varied strongly when probing axially along the NW and across the p-n junction. The axial variation in S{sub 11}{sup ′}  effectively mapped the asymmetric depletion arising from the doping concentrations on either side of the junction. Furthermore, variation of the probe tip voltage altered the apparent extent of features associated with the p-n junction in S{sub 11}{sup ′} images.

  11. Scanning Electron Microscope Calibration Using a Multi-Image Non-Linear Minimization Process

    Science.gov (United States)

    Cui, Le; Marchand, Éric

    2015-04-01

    A scanning electron microscope (SEM) calibrating approach based on non-linear minimization procedure is presented in this article. A part of this article has been published in IEEE International Conference on Robotics and Automation (ICRA), 2014. . Both the intrinsic parameters and the extrinsic parameters estimations are achieved simultaneously by minimizing the registration error. The proposed approach considers multi-images of a multi-scale calibration pattern view from different positions and orientations. Since the projection geometry of the scanning electron microscope is different from that of a classical optical sensor, the perspective projection model and the parallel projection model are considered and compared with distortion models. Experiments are realized by varying the position and the orientation of a multi-scale chessboard calibration pattern from 300× to 10,000×. The experimental results show the efficiency and the accuracy of this approach.

  12. Imaging of current density distributions with a Nb weak-link scanning nano-SQUID microscope

    Science.gov (United States)

    Shibata, Yusuke; Nomura, Shintaro; Kashiwaya, Hiromi; Kashiwaya, Satoshi; Ishiguro, Ryosuke; Takayanagi, Hideaki

    2015-10-01

    Superconducting quantum interference devices (SQUIDs) are accepted as one of the highest magnetic field sensitive probes. There are increasing demands to image local magnetic fields to explore spin properties and current density distributions in a two-dimensional layer of semiconductors or superconductors. Nano-SQUIDs have recently attracting much interest for high spatial resolution measurements in nanometer-scale samples. Whereas weak-link Dayem Josephson junction nano-SQUIDs are suitable to miniaturization, hysteresis in current-voltage (I-V) characteristics that is often observed in Dayem Josephson junction is not desirable for a scanning microscope. Here we report on our development of a weak-link nano-SQUIDs scanning microscope with small hysteresis in I-V curve and on reconstructions of two-dimensional current density vector in two-dimensional electron gas from measured magnetic field.

  13. Microscope-integrated optical coherence tomography for image-aided positioning of glaucoma surgery

    Science.gov (United States)

    Li, Xiqi; Wei, Ling; Dong, Xuechuan; Huang, Ping; Zhang, Chun; He, Yi; Shi, Guohua; Zhang, Yudong

    2015-07-01

    Most glaucoma surgeries involve creating new aqueous outflow pathways with the use of a small surgical instrument. This article reported a microscope-integrated, real-time, high-speed, swept-source optical coherence tomography system (SS-OCT) with a 1310-nm light source for glaucoma surgery. A special mechanism was designed to produce an adjustable system suitable for use in surgery. A two-graphic processing unit architecture was used to speed up the data processing and real-time volumetric rendering. The position of the surgical instrument can be monitored and measured using the microscope and a grid-inserted image of the SS-OCT. Finally, experiments were simulated to assess the effectiveness of this integrated system. Experimental results show that this system is a suitable positioning tool for glaucoma surgery.

  14. MIDAS - an atomic force microscope for in-situ imaging of cometary dust particles

    International Nuclear Information System (INIS)

    Fehringer, H.M.; Ruedenauer, F.G.; Steiger, W.

    1997-02-01

    Comets are interesting bodies, since they are considered to consist of matter remaining in essentially unchanged chemistry from the presolar nebula. Investigation of cometary matter therefore permits to draw conclusion s with respect to the composition of presolar matter. The atomic force microscope MIDAS will be the first instrument to analyze, within ESA's ROSETTA-mission priestine cometary matter in the form of dust particles emitted by comet WIRTANEN during its perihelion in 2013. Within this project, a dust model has been developed, permitting estimation of dust collection times required for statistically significant imaging of cometary particles. The dynamics of dust collection has been developed and experimental dust collection surfaces have been produced making use of modem nanostructuring techniques. Mechanical properties of 3-dimensional piezo-control elements, which are an essential part of the MIDAS microscope, have been determined. (author)

  15. Microscope-integrated optical coherence tomography for image-aided positioning of glaucoma surgery.

    Science.gov (United States)

    Li, Xiqi; Wei, Ling; Dong, Xuechuan; Huang, Ping; Zhang, Chun; He, Yi; Shi, Guohua; Zhang, Yudong

    2015-07-01

    Most glaucoma surgeries involve creating new aqueous outflow pathways with the use of a small surgical instrument. This article reported a microscope-integrated, real-time, high-speed, swept-source optical coherence tomography system (SS-OCT) with a 1310-nm light source for glaucoma surgery. A special mechanism was designed to produce an adjustable system suitable for use in surgery. A two-graphic processing unit architecture was used to speed up the data processing and real-time volumetric rendering. The position of the surgical instrument can be monitored and measured using the microscope and a grid-inserted image of the SS-OCT. Finally, experiments were simulated to assess the effectiveness of this integrated system. Experimental results show that this system is a suitable positioning tool for glaucoma surgery.

  16. Novel low-dose imaging technique for characterizing atomic structures through scanning transmission electron microscope

    Science.gov (United States)

    Su, Chia-Ping; Syu, Wei-Jhe; Hsiao, Chien-Nan; Lai, Ping-Shan; Chen, Chien-Chun

    2017-08-01

    To investigate dislocations or heterostructures across interfaces is now of great interest to condensed matter and materials scientists. With the advances in aberration-corrected electron optics, the scanning transmission electron microscope has demonstrated its excellent capability of characterizing atomic structures within nanomaterials, and well-resolved atomic-resolution images can be obtained through long-exposure data acquisition. However, the sample drifting, carbon contamination, and radiation damage hinder further analysis, such as deriving three-dimensional (3D) structures from a series of images. In this study, a method for obtaining atomic-resolution images with significantly reduced exposure time was developed, using which an original high-resolution image with approximately one tenth the electron dose can be obtained by combining a fast-scan high-magnification image and a slow-scan low-magnification image. The feasibility of obtaining 3D atomic structures using the proposed approach was demonstrated through multislice simulation. Finally, the feasibility and accuracy of image restoration were experimentally verified. This general method cannot only apply to electron microscopy but also benefit to image radiation-sensitive materials using various light sources.

  17. Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms.

    Science.gov (United States)

    Inada, H; Su, D; Egerton, R F; Konno, M; Wu, L; Ciston, J; Wall, J; Zhu, Y

    2011-06-01

    We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2011-03-14

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

  19. eSIP: A Novel Solution-Based Sectioned Image Property Approach for Microscope Calibration.

    Directory of Open Access Journals (Sweden)

    Malte Butzlaff

    Full Text Available Fluorescence confocal microscopy represents one of the central tools in modern sciences. Correspondingly, a growing amount of research relies on the development of novel microscopic methods. During the last decade numerous microscopic approaches were developed for the investigation of various scientific questions. Thereby, the former qualitative imaging methods became replaced by advanced quantitative methods to gain more and more information from a given sample. However, modern microscope systems being as complex as they are, require very precise and appropriate calibration routines, in particular when quantitative measurements should be compared over longer time scales or between different setups. Multispectral beads with sub-resolution size are often used to describe the point spread function and thus the optical properties of the microscope. More recently, a fluorescent layer was utilized to describe the axial profile for each pixel, which allows a spatially resolved characterization. However, fabrication of a thin fluorescent layer with matching refractive index is technically not solved yet. Therefore, we propose a novel type of calibration concept for sectioned image property (SIP measurements which is based on fluorescent solution and makes the calibration concept available for a broader number of users. Compared to the previous approach, additional information can be obtained by application of this extended SIP chart approach, including penetration depth, detected number of photons, and illumination profile shape. Furthermore, due to the fit of the complete profile, our method is less susceptible to noise. Generally, the extended SIP approach represents a simple and highly reproducible method, allowing setup independent calibration and alignment procedures, which is mandatory for advanced quantitative microscopy.

  20. Neurite density imaging versus imaging of microscopic anisotropy in diffusion MRI: A model comparison using spherical tensor encoding.

    Science.gov (United States)

    Lampinen, Björn; Szczepankiewicz, Filip; Mårtensson, Johan; van Westen, Danielle; Sundgren, Pia C; Nilsson, Markus

    2017-02-15

    In diffusion MRI (dMRI), microscopic diffusion anisotropy can be obscured by orientation dispersion. Separation of these properties is of high importance, since it could allow dMRI to non-invasively probe elongated structures such as neurites (axons and dendrites). However, conventional dMRI, based on single diffusion encoding (SDE), entangles microscopic anisotropy and orientation dispersion with intra-voxel variance in isotropic diffusivity. SDE-based methods for estimating microscopic anisotropy, such as the neurite orientation dispersion and density imaging (NODDI) method, must thus rely on model assumptions to disentangle these features. An alternative approach is to directly quantify microscopic anisotropy by the use of variable shape of the b-tensor. Along those lines, we here present the 'constrained diffusional variance decomposition' (CODIVIDE) method, which jointly analyzes data acquired with diffusion encoding applied in a single direction at a time (linear tensor encoding, LTE) and in all directions (spherical tensor encoding, STE). We then contrast the two approaches by comparing neurite density estimated using NODDI with microscopic anisotropy estimated using CODIVIDE. Data were acquired in healthy volunteers and in glioma patients. NODDI and CODIVIDE differed the most in gray matter and in gliomas, where NODDI detected a neurite fraction higher than expected from the level of microscopic diffusion anisotropy found with CODIVIDE. The discrepancies could be explained by the NODDI tortuosity assumption, which enforces a connection between the neurite density and the mean diffusivity of tissue. Our results suggest that this assumption is invalid, which leads to a NODDI neurite density that is inconsistent between LTE and STE data. Using simulations, we demonstrate that the NODDI assumptions result in parameter bias that precludes the use of NODDI to map neurite density. With CODIVIDE, we found high levels of microscopic anisotropy in white matter

  1. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue

    Science.gov (United States)

    Sakadžić, Sava; Roussakis, Emmanuel; Yaseen, Mohammad A.; Mandeville, Emiri T.; Srinivasan, Vivek J.; Arai, Ken; Ruvinskaya, Svetlana; Devor, Anna; Lo, Eng H.; Vinogradov, Sergei A.; Boas, David A.

    2010-01-01

    The ability to measure oxygen partial pressure (pO2) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO2 measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here, we report the first practical in vivo two-photon high-resolution pO2 measurements in small rodents’ cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 µm, sub-second temporal resolution and requires low probe concentration. Most importantly, the properties of the probe allowed for the first direct high-resolution measurement of cortical extravascular (tissue) pO2, opening numerous possibilities for functional metabolic brain studies. PMID:20693997

  2. Porous Porphyrin-Based Organosilica Nanoparticles for NIR Two-Photon Photodynamic Therapy and Gene Delivery in Zebrafish

    KAUST Repository

    Mauriello Jimenez, Chiara

    2018-03-30

    Periodic mesoporous organosilica nanoparticles emerge as promising vectors for nanomedicine applications. Their properties are very different from those of well-known mesoporous silica nanoparticles as there is no silica source for their synthesis. So far, they have only been synthesized from small bis-silylated organic precursors. However, no studies employing large stimuli-responsive precursors have been reported on such hybrid systems yet. Here, the synthesis of porphyrin-based organosilica nanoparticles from a large octasilylated metalated porphyrin precursor is described for applications in near-infrared two-photon-triggered spatiotemporal theranostics. The nanoparticles display unique interconnected large cavities of 10-80 nm. The framework of the nanoparticles is constituted with J-aggregates of porphyrins, which endows them with two-photon sensitivity. The nanoparticle efficiency for intracellular tracking is first demonstrated by the in vitro near-infrared imaging of breast cancer cells. After functionalization of the nanoparticles with aminopropyltriethoxysilane, two-photon-excited photodynamic therapy in zebrafish is successfully achieved. Two-photon photochemical internalization in cancer cells of the nanoparticles loaded with siRNA is also performed for the first time. Furthermore, siRNA targeting green fluorescent protein complexed with the nanoparticles is delivered in vivo in zebrafish embryos, which demonstrates the versatility of the nanovectors for biomedical applications.

  3. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.

    Science.gov (United States)

    Sakadzić, Sava; Roussakis, Emmanuel; Yaseen, Mohammad A; Mandeville, Emiri T; Srinivasan, Vivek J; Arai, Ken; Ruvinskaya, Svetlana; Devor, Anna; Lo, Eng H; Vinogradov, Sergei A; Boas, David A

    2010-09-01

    Measurements of oxygen partial pressure (pO(2)) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO(2) measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here we report to our knowledge the first practical in vivo two-photon high-resolution pO(2) measurements in small rodents' cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 microm, sub-second temporal resolution and requires low probe concentration. The properties of the probe allowed for direct high-resolution measurement of cortical extravascular (tissue) pO(2), opening many possibilities for functional metabolic brain studies.

  4. Development of Scanning-Imaging X-Ray Microscope for Quantitative Three-Dimensional Phase Contrast Microimaging

    International Nuclear Information System (INIS)

    Takeuchi, Akihisa; Suzuki, Yoshio; Uesugi, Kentaro

    2013-01-01

    A novel x-ray microscope system has been developed for the purpose of quantitative and sensitive three-dimensional (3D) phase-contrast x-ray microimaging. The optical system is a hybrid that consists of a scanning microscope optics with a one-dimensional (1D) focusing (line-focusing) device and an imaging microscope optics with a 1D objective. These two optics are orthogonally arranged regarding their common optical axis. Each is used for forming each dimension of two-dimensional (2D) image. The same data acquisition process as that of the scanning microscope system enables quantitative and sensitive x-ray imaging such as phase contrast and absorption contrast. Because a 2D image is measured with only 1D translation scan, much shorter measurement time than that of conventional scanning optics has been realized. By combining a computed tomography (CT) technique, some 3D CT application examples are demonstrated

  5. Targeted two-photon photodynamic therapy for the treatment of subcutaneous tumors

    Science.gov (United States)

    Spangler, Charles W.; Starkey, Jean R.; Meng, Fanqing; Gong, Aijun; Drobizhev, Mikhail; Rebane, Aleksander; Moss, B.

    2005-04-01

    Photodynamic therapy (PDT) has developed into a mature technology over the past several years, and is currently being exploited for the treatment of a variety of cancerous tumors, and more recently for age-related wet macular degeneration of the eye. However, there are still some unresolved problems with PDT that are retarding a more general acceptance in clinical settings, and thus, for the most part, the treatment of most cancerous rumors still involves some combination of invasive surgery, chemotherapy and radiation treatment, particularly subcutaneous tumors. Currently approved PDT agents are activated in the Visible portion of the spectrum below 700 nm, Laser light in this spectral region cannot penetrate the skin more than a few millimeters, and it would be more desirable if PDT could be initiated deep in the Near-infrared (NIR) in the tissue transparency window (700-1000 nm). MPA Technologies, Inc. and Rasiris, Inc. have been co-developing new porphyrin PDT designed to have greatly enhanced intrinsic two-photon cross-sections (>800 GM units) whose two-photon absorption maxima lie deep in the tissue transparency window (ca. 780-850 nm), and have solubility characteristics that would allow for direct IV injection into animal models. Classical PDT also suffers from the lengthy time necessary for accumulation at the tumor site, a relative lack of discrimination between healthy and diseased tissue, particularly at the tumor margins, and difficulty in clearing from the system in a reasonable amount of time post-PDT. We have recently discovered a new design paradigm for the delivery of our two-photon activated PDT agents by incorporating the porphyrins into a triad ensemble that includes a small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR one-photon imaging agent that allows the tracking of the triad in terms of accumulation and clearance rates. We are currently using these new two-photon PDT triads in efficacy

  6. High-speed imaging upgrade for a standard sample scanning atomic force microscope using small cantilevers

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Jonathan D.; Nievergelt, Adrian; Erickson, Blake W.; Yang, Chen; Dukic, Maja; Fantner, Georg E., E-mail: georg.fantner@epfl.ch [Ecole Polytechnique Fédérale de Lausanne, Lausanne (Switzerland)

    2014-09-15

    We present an atomic force microscope (AFM) head for optical beam deflection on small cantilevers. Our AFM head is designed to be small in size, easily integrated into a commercial AFM system, and has a modular architecture facilitating exchange of the optical and electronic assemblies. We present two different designs for both the optical beam deflection and the electronic readout systems, and evaluate their performance. Using small cantilevers with our AFM head on an otherwise unmodified commercial AFM system, we are able to take tapping mode images approximately 5–10 times faster compared to the same AFM system using large cantilevers. By using additional scanner turnaround resonance compensation and a controller designed for high-speed AFM imaging, we show tapping mode imaging of lipid bilayers at line scan rates of 100–500 Hz for scan areas of several micrometers in size.

  7. Advances in imaging and electron physics the scanning transmission electron microscope

    CERN Document Server

    Hawkes, Peter W

    2009-01-01

    Advances in Imaging and Electron Physics merges two long-running serials--Advances in Electronics and Electron Physics and Advances in Optical and Electron Microscopy. This series features extended articles on the physics of electron devices (especially semiconductor devices), particle optics at high and low energies, microlithography, image science and digital image processing, electromagnetic wave propagation, electron microscopy, and the computing methods used in all these domains.  This particular volume presents several timely articles on the scanning transmission electron microscope. Updated with contributions from leading international scholars and industry experts Discusses hot topic areas and presents current and future research trends Provides an invaluable reference and guide for physicists, engineers and mathematicians.

  8. High-speed multi-frame dynamic transmission electron microscope image acquisition system with arbitrary timing

    Science.gov (United States)

    Reed, Bryan W.; DeHope, William J.; Huete, Glenn; LaGrange, Thomas B.; Shuttlesworth, Richard M.

    2016-02-23

    An electron microscope is disclosed which has a laser-driven photocathode and an arbitrary waveform generator (AWG) laser system ("laser"). The laser produces a train of temporally-shaped laser pulses each being of a programmable pulse duration, and directs the laser pulses to the laser-driven photocathode to produce a train of electron pulses. An image sensor is used along with a deflector subsystem. The deflector subsystem is arranged downstream of the target but upstream of the image sensor, and has a plurality of plates. A control system having a digital sequencer controls the laser and a plurality of switching components, synchronized with the laser, to independently control excitation of each one of the deflector plates. This allows each electron pulse to be directed to a different portion of the image sensor, as well as to enable programmable pulse durations and programmable inter-pulse spacings.

  9. High-speed multiframe dynamic transmission electron microscope image acquisition system with arbitrary timing

    Science.gov (United States)

    Reed, Bryan W.; DeHope, William J.; Huete, Glenn; LaGrange, Thomas B.; Shuttlesworth, Richard M.

    2015-10-20

    An electron microscope is disclosed which has a laser-driven photocathode and an arbitrary waveform generator (AWG) laser system ("laser"). The laser produces a train of temporally-shaped laser pulses of a predefined pulse duration and waveform, and directs the laser pulses to the laser-driven photocathode to produce a train of electron pulses. An image sensor is used along with a deflector subsystem. The deflector subsystem is arranged downstream of the target but upstream of the image sensor, and has two pairs of plates arranged perpendicular to one another. A control system controls the laser and a plurality of switching components synchronized with the laser, to independently control excitation of each one of the deflector plates. This allows each electron pulse to be directed to a different portion of the image sensor, as well as to be provided with an independently set duration and independently set inter-pulse spacings.

  10. Image processing of muscle striations below the resolution limit of the light microscope

    International Nuclear Information System (INIS)

    Burns, D.H.; Holdren, D.N.; Periasamy, A.; Everts, W.C.; Pollack, G.H.

    1985-01-01

    We describe the use of digital deconvolution in the study of muscle striations below the resolution imposed by the optical diffraction of our video light microscope. To use deconvolution procedures on muscle images, the transfer function of the optical system is first characterized. This is accomplished by imaging a step object and fitting the image with the combination of a first order Bessel and Guassian function using a non-linear least squares approach. Due to the ill-conditioned nature of deconvolution, however, ambiguity in the reconstruction is sometimes found. To allow better estimation of the true object, separate deconvolution approaches are used and the reconstructions compared. In this manner, the fine structure of muscle striations is determined

  11. First test model of the optical microscope which images the whole vertical particle tracks without any depth scanning

    International Nuclear Information System (INIS)

    Soroko, L.M.

    2001-01-01

    The first test model of the optical microscope which produces the in focus image of the whole vertical particle track without depth scanning is described. The in focus image of the object consisting of the linear array of the point-like elements was obtained. A comparison with primary out of focus image of such an object has been made

  12. Two-Photon Absorbing Molecules as Potential Materials for 3D Optical Memory

    Directory of Open Access Journals (Sweden)

    Kazuya Ogawa

    2014-01-01

    Full Text Available In this review, recent advances in two-photon absorbing photochromic molecules, as potential materials for 3D optical memory, are presented. The investigations introduced in this review indicate that 3D data storage processing at the molecular level is possible. As 3D memory using two-photon absorption allows advantages over existing systems, the use of two-photon absorbing photochromic molecules is preferable. Although there are some photochromic molecules with good properties for memory, in most cases, the two-photon absorption efficiency is not high. Photochromic molecules with high two-photon absorption efficiency are desired. Recently, molecules having much larger two-photon absorption cross sections over 10,000 GM (GM= 10−50 cm4 s molecule−1 photon−1 have been discovered and are expected to open the way to realize two-photon absorption 3D data storage.

  13. Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing.

    Science.gov (United States)

    Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

    2015-01-01

    Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection.

  14. Science applications of a multispectral microscopic imager for the astrobiological exploration of Mars.

    Science.gov (United States)

    Núñez, Jorge I; Farmer, Jack D; Sellar, R Glenn; Swayze, Gregg A; Blaney, Diana L

    2014-02-01

    Future astrobiological missions to Mars are likely to emphasize the use of rovers with in situ petrologic capabilities for selecting the best samples at a site for in situ analysis with onboard lab instruments or for caching for potential return to Earth. Such observations are central to an understanding of the potential for past habitable conditions at a site and for identifying samples most likely to harbor fossil biosignatures. The Multispectral Microscopic Imager (MMI) provides multispectral reflectance images of geological samples at the microscale, where each image pixel is composed of a visible/shortwave infrared spectrum ranging from 0.46 to 1.73 μm. This spectral range enables the discrimination of a wide variety of rock-forming minerals, especially Fe-bearing phases, and the detection of hydrated minerals. The MMI advances beyond the capabilities of current microimagers on Mars by extending the spectral range into the infrared and increasing the number of spectral bands. The design employs multispectral light-emitting diodes and an uncooled indium gallium arsenide focal plane array to achieve a very low mass and high reliability. To better understand and demonstrate the capabilities of the MMI for future surface missions to Mars, we analyzed samples from Mars-relevant analog environments with the MMI. Results indicate that the MMI images faithfully resolve the fine-scale microtextural features of samples and provide important information to help constrain mineral composition. The use of spectral endmember mapping reveals the distribution of Fe-bearing minerals (including silicates and oxides) with high fidelity, along with the presence of hydrated minerals. MMI-based petrogenetic interpretations compare favorably with laboratory-based analyses, revealing the value of the MMI for future in situ rover-mediated astrobiological exploration of Mars. Mars-Microscopic imager-Multispectral imaging-Spectroscopy-Habitability-Arm instrument.

  15. Enhanced Emission from Single Isolated Gold Quantum Dots Investigated Using Two-Photon-Excited Fluorescence Near-Field Scanning Optical Microscopy.

    Science.gov (United States)

    Abeyasinghe, Neranga; Kumar, Santosh; Sun, Kai; Mansfield, John F; Jin, Rongchao; Goodson, Theodore

    2016-12-21

    New approaches in molecular nanoscopy are greatly desired for interrogation of biological, organic, and inorganic objects with sizes below the diffraction limit. Our current work investigates emergent monolayer-protected gold quantum dots (nanoclusters, NCs) composed of 25 Au atoms by utilizing two-photon-excited fluorescence (TPEF) near-field scanning optical microscopy (NSOM) at single NC concentrations. Here, we demonstrate an approach to synthesize and isolate single NCs on solid glass substrates. Subsequent investigation of the NCs using TPEF NSOM reveals that, even when they are separated by distances of several tens of nanometers, we can excite and interrogate single NCs individually. Interestingly, we observe an enhanced two-photon absorption (TPA) cross section for single Au 25 NCs that can be attributed to few-atom local field effects and to local field-induced microscopic cascading, indicating their potential for use in ultrasensitive sensing, disease diagnostics, cancer cell therapy, and molecular computers. Finally, we report room-temperature aperture-based TPEF NSOM imaging of these NCs for the first time at 30 nm point resolution, which is a ∼5-fold improvement compared to the previous best result for the same technique. This report unveils the unique combination of an unusually large TPA cross section and the high photostability of Au NCs to (non-destructively) investigate stable isolated single NCs using TPEF NSOM. This is the first reported optical study of monolayer-protected single quantum clusters, opening some very promising opportunities in spectroscopy of nanosized objects, bioimaging, ultrasensitive sensing, molecular computers, and high-density data storage.

  16. Garnet film rotator applied in polarizing microscope for domain image modulation (abstract)

    Science.gov (United States)

    Wakabayashi, K.; Numata, T.; Inokuchi, S.

    1991-04-01

    A garnet film polarization rotator placed before the analyzer in a polarizing microscope was investigated to obtain the difference image of a positive and a negative one of magnetic domain in real time along with an image processor. In the difference image, a nonmagnetic image can be reduced and hence the weak magnetic contrast enhanced. Theoretical calculation of S/N and contrast C of the domain image as a function of the rotation shows they take maxima at the rotation angle of 2.6° and 0.1°, respectively, with the extinction ratio of e=4×10-6 of a polarizing microscope. Thus, since the thickness of the garnet film required is 1 μm or so, the absorption by the garnet rotator does not bring a serious problem even in a visible region for the domain observation. The optimum rotation of the rotator for a high quality observation was obtained by a quantitative study of images obtained experimentally as well as by a visual evaluation. A magnetically unsaturated garnet film with perpendicular magnetization (i.e., multidomain) was employed as a rotator, in which the polarization rotation angle θm of the undeflected beam with respect to the light diffraction could be continuously varied by an applied magnetic field. The dependences of S/N and C on θm were measured, resulting in a well agreement between the measured and the calculated. The visually best image was obtained at θm=0.5° which made the product of S/N and C maximum. The domain image of the Kerr rotation angle of θk=0.22° was observed in S/N=47 dB and C=0.4 when Ar+ laser (λ=515 nm) of tenths of a watt was employed as a light source. Since the domain image with 47 dB S/N does not need an image summation for a noise reduction, a garnet film rotator makes it possible to invert the contrast of a domain image in a real time for an improved domain observation.

  17. High-resolution electron microscope image analysis approach for superconductor YBa2Cu3O7-x

    International Nuclear Information System (INIS)

    Xu, J.; Lu, F.; Jia, C.; Hua, Z.

    1991-01-01

    In this paper, an HREM (High-resolution electron microscope) image analysis approach has been developed. The image filtering, segmentation and particles extraction based on gray-scale mathematical morphological operations, are performed on the original HREM image. The final image is a pseudocolor image, with the background removed, relatively uniform brightness, filtered slanting elongation, regular shape for every kind of particle, and particle boundaries that no longer touch each other so that the superconducting material structure can be shown clearly

  18. Microspot two-photon photoemission spectroscopy for CuPc film on HOPG

    International Nuclear Information System (INIS)

    Yamada, T.; Yamamoto, R.; Munakata, T.

    2015-01-01

    Highlights: • Unoccupied levels of CuPc/HOPG are assigned by using 2PPE microspectroscopy. • Lateral distribution of unoccupied energy levels is imaged. • Modified IPS stabilized by the hole localized in the 2nd layer molecule is identified. - Abstract: Microspot two-photon photoemission (micro-2PPE) spectroscopy has been applied to measure the lateral distribution of unoccupied levels on copper phthalocyanine (CuPc) film on HOPG. In addition to the LUMO-derived level and the image potential state (IPS) on the film, we identified the modified IPS which is stabilized by the hole localized in a molecule. We show that modified IPS is observed only on bilayer area, reflecting the localization of the hole in a molecule. The modified IPS is absent on monolayer area, because the hole strongly interacts with substrate.

  19. In vivo microscopic imaging of the bronchial mucosa using an endo-cytoscopy system.

    Science.gov (United States)

    Shibuya, Kiyoshi; Fujiwara, Taiki; Yasufuku, Kazuhiro; Alaa, Mohamed; Chiyo, Masako; Nakajima, Takahiro; Hoshino, Hidehisa; Hiroshima, Kenzo; Nakatani, Yukio; Yoshino, Ichiro

    2011-05-01

    We investigated the capabilities of an endo-cytoscopy system (ECS) that enables microscopic imaging of the tracheobronchial tree during bronchoscopy, including normal bronchial epithelium, dysplastic mucosa and squamous cell carcinoma. The newly developed ECS has a 3.2 mm diameter that can be passed through the 4.2 mm working channel of a mother endoscope for insertion of the ECS. It has a high magnification of 570× on a 17 in. video monitor. Twenty-two patients (7 squamous cell carcinoma, 11 squamous dysplasia and 4 after PDT therapies) were underwent white light, NBI light and AFI bronchoscopy. Both abnormal areas of interest and normal bronchial mucosa were stained with 0.5% methylene blue and examined with ECS at high magnification (570×). Histological examinations using haematoxylin and eosin staining were made of biopsied specimens. Analyzed ECS images were compared with the corresponding histological examinations. In normal bronchial mucosa, ciliated columnar epithelial cells were visible. In bronchial squamous dysplasia, superficial cells with abundant cytoplasm were arranged regularly. In squamous cell carcinoma, large, polymorphic tumor cells showed increased cellular densities with irregular stratified patterns. These ECS images corresponded well with the light-microscopic examination of conventional histology. ECS was useful for the discrimination between normal bronchial epithelial cells and dysplastic cells or malignant cells during bronchoscopy in real time. This novel technology has an excellent potential to provide in vivo diagnosis during bronchoscopic examinations. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  20. A framed, 16-image Kirkpatrick-Baez x-ray microscope

    Science.gov (United States)

    Marshall, F. J.; Bahr, R. E.; Goncharov, V. N.; Glebov, V. Yu.; Peng, B.; Regan, S. P.; Sangster, T. C.; Stoeckl, C.

    2017-09-01

    A 16-image Kirkpatrick-Baez (KB)-type x-ray microscope consisting of compact KB mirrors [F. J. Marshall, Rev. Sci. Instrum. 83, 10E518 (2012)] has been assembled for the first time with mirrors aligned to allow it to be coupled to a high-speed framing camera. The high-speed framing camera has four independently gated strips whose emission sampling interval is ˜30 ps. Images are arranged four to a strip with ˜60-ps temporal spacing between frames on a strip. By spacing the timing of the strips, a frame spacing of ˜15 ps is achieved. A framed resolution of ˜6-μm is achieved with this combination in a 400-μm region of laser-plasma x-ray emission in the 2- to 8-keV energy range. A principal use of the microscope is to measure the evolution of the implosion stagnation region of cryogenic DT target implosions on the University of Rochester's OMEGA Laser System [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)]. The unprecedented time and spatial resolutions achieved with this framed, multi-image KB microscope have made it possible to accurately determine the cryogenic implosion core emission size and shape at the peak of stagnation. These core size measurements, taken in combination with those of ion temperature, neutron-production temporal width, and neutron yield allow for inference of core pressures, currently exceeding 50 Gbar in OMEGA cryogenic target implosions [Regan et al., Phys. Rev. Lett. 117, 025001 (2016)].