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Sample records for imaging microscopy phosphorescence

  1. Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

    PubMed Central

    Marriott, G; Clegg, R M; Arndt-Jovin, D J; Jovin, T M

    1991-01-01

    An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 PMID:1723311

  2. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  3. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells.

    PubMed

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  4. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  5. Combined fluorescence and phosphorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Shcheslavskiy, V. I.; Neubauer, A.; Bukowiecki, R.; Dinter, F.; Becker, W.

    2016-02-01

    We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

  6. Oxygen Microscopy by Two-Photon-Excited Phosphorescence

    PubMed Central

    Finikova, Olga S.; Lebedev, Artem Y.; Aprelev, Alexey; Troxler, Thomas; Gao, Feng; Garnacho, Carmen; Muro, Silvia; Hochstrasser, Robin M.; Vinogradov, Sergei A.

    2009-01-01

    High-resolution images of oxygen distributions in microheterogeneous samples are obtained by two-photon laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO2 in cells. PMID:18663708

  7. Phosphorescent metalloporphyrins as labels in time-resolved luminescence microscopy: effect of mounting on emission intensity.

    PubMed

    Soini, Aleksi E; Seveus, Lahja; Meltola, Niko J; Papkovsky, Dmitri B; Soini, Erkki

    2002-07-15

    In this study, we present an investigation of the effects of mounting media on the phosphorescence of metalloporphyrin stained microscopy samples. The samples were: (1) Platinum(II) coproporphyrin (=PtCP) stained porous Sephadex beads; (2) compact polystyrene microspheres coated with IgG-PtCP conjugate; and (3) immunocytochemically labeled human peripheral blood neutrophils. The human neutrophils in a mixed leukocyte population were fixed, permeabilized, and then immunolabeled with PtCP conjugate of monoclonal mouse IgG directed to the intracellular antigen myeloperoxidase. The samples were mounted in twelve different mounting media and studied with quantitative time-resolved luminescence imaging microscopy with respect to the intensity and stability of the phosphorescence signal. The results indicate that microscopy samples stained with PtCP exhibit the brightest phosphorescence emission in non-mounted form or when mounted in non-aqueous permanent mounting media. PMID:12203714

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems

    PubMed Central

    Lebedev, Artem Y.; Cheprakov, Andrei V.; Sakadžić, Sava; Boas, David A.; Wilson, David F.; Vinogradov, Sergei A.

    2009-01-01

    Oxygen levels in biological systems can be measured by the phosphorescence quenching method using probes with controllable quenching parameters and defined biodistributions. We describe a general approach to the construction of phosphorescent nanosensors with tunable spectral characteristics, variable degrees of quenching, and a high selectivity for oxygen. The probes are based on bright phosphorescent Pt and Pd complexes of porphyrins and symmetrically π-extended porphyrins (tetrabenzoporphyrins and tetranaphthoporphyrins). π-Extension of the core macrocycle allows tuning of the spectral parameters of the probes in order to meet the requirements of a particular imaging application (e.g., oxygen tomography versus planar microscopic imaging). Metalloporphyrins are encapsulated into poly(arylglycine) dendrimers, which fold in aqueous environments and create diffusion barriers for oxygen, making it possible to regulate the sensitivity and the dynamic range of the method. The periphery of the dendrimers is modified with poly(ethylene glycol) residues, which enhance the probe’s solubility, diminish toxicity, and help prevent interactions of the probes with the biological environment. The probe’s parameters were measured under physiological conditions and shown to be unaffected by the presence of biomacromolecules. The performance of the probes was demonstrated in applications, including in vivo microscopy of vascular pO2 in the rat brain. PMID:20072726

  10. Phosphorescent imaging of oxygen gradients in tissues

    NASA Astrophysics Data System (ADS)

    Swanson, Curtis J.; Kitakis, F.

    1995-08-01

    Until recently, the ability to measure the changing oxygen gradients in perfused tissues in response to metabolic demand, has been limited to point-measurements and/or averaged A-V oxygen differences during perfusion using oxygen electrodes. With the recent introduction of novel phosphorescent probes specifically quenched by oxygen, the ability to spacially map oxygen gradients in real-time may offer new insights into the dynamics of microvascular design and supply. Accordingly, this paper provides initial image data on Langendorff perfused rat hearts wherein the relative change in phosphorescent intensity of Pd-meso-tetra(4- carboxyphenyl)phorphine (2micrometers ) as the reporter probe, is quantitatively related to spacial oxygen gradients as seen on the left-ventricle during changing gassing conditions. Digital image analysis (frame advance), after proper calibration and alignment, provides images which can be usefully interpreted. Clinical applications of such emerging technologies could have wide-spread diagnostic applications not only as applied to the coronary bed, but other tissue surfaces displaying various degrees of aschemia and/or hypoxia.

  11. High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime

    PubMed Central

    Kurokawa, Hiromi; Ito, Hidehiro; Inoue, Mai; Tabata, Kenji; Sato, Yoshifumi; Yamagata, Kazuya; Kizaka-Kondoh, Shinae; Kadonosono, Tetsuya; Yano, Shigenobu; Inoue, Masahiro; Kamachi, Toshiaki

    2015-01-01

    Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism. PMID:26065366

  12. 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

    PubMed Central

    Choi, Heejin; Tzeranis, Dimitrios S.; Cha, Jae Won; Clémenceau, Philippe; de Jong, Sander J. G.; van Geest, Lambertus K.; Moon, Joong Ho; Yannas, Ioannis V.; So, Peter T. C.

    2012-01-01

    Fluorescence and phosphorescence lifetime imaging are powerful techniques for studying intracellular protein interactions and for diagnosing tissue pathophysiology. While lifetime-resolved microscopy has long been in the repertoire of the biophotonics community, current implementations fall short in terms of simultaneously providing 3D resolution, high throughput, and good tissue penetration. This report describes a new highly efficient lifetime-resolved imaging method that combines temporal focusing wide-field multiphoton excitation and simultaneous acquisition of lifetime information in frequency domain using a nanosecond gated imager from a 3D-resolved plane. This approach is scalable allowing fast volumetric imaging limited only by the available laser peak power. The accuracy and performance of the proposed method is demonstrated in several imaging studies important for understanding peripheral nerve regeneration processes. Most importantly, the parallelism of this approach may enhance the imaging speed of long lifetime processes such as phosphorescence by several orders of magnitude. PMID:23187477

  13. Pinhole shifting lifetime imaging microscopy.

    PubMed

    Ramshesh, Venkat K; Lemasters, John J

    2008-01-01

    Lifetime imaging microscopy is a powerful tool to probe biological phenomena independent of luminescence intensity and fluorophore concentration. We describe time-resolved imaging of long-lifetime luminescence with an unmodified commercial laser scanning confocal/multiphoton microscope. The principle of the measurement is displacement of the detection pinhole to collect delayed luminescence from a position lagging the rasting laser beam. As proof of principle, luminescence from microspheres containing europium (Eu(3+)), a red emitting probe, was compared to that of short-lifetime green-fluorescing microspheres and/or fluorescein and rhodamine in solution. Using 720-nm two-photon excitation and a pinhole diameter of 1 Airy unit, the short-lifetime fluorescence of fluorescein, rhodamine and green microspheres disappeared much more rapidly than the long-lifetime phosphorescence of Eu(3+) microspheres as the pinhole was repositioned in the lagging direction. In contrast, repositioning of the pinhole in the leading and orthogonal directions caused equal loss of short- and long-lifetime luminescence. From measurements at different lag pinhole positions, a lifetime of 270 micros was estimated for the Eu(3+) microspheres, consistent with independent measurements. This simple adaptation is the basis for quantitative 3-D lifetime imaging microscopy. PMID:19123648

  14. Phosphorescent Imaging of Living Cells Using a Cyclometalated Iridium(III) Complex

    PubMed Central

    Ma, Dik-Lung; Zhong, Hai-Jing; Fu, Wai-Chung; Chan, Daniel Shiu-Hin; Kwan, Hiu-Yee; Fong, Wang-Fun; Chung, Lai-Hon; Wong, Chun-Yuen; Leung, Chung-Hang

    2013-01-01

    A cell permeable cyclometalated iridium(III) complex has been developed as a phosphorescent probe for cell imaging. The iridium(III) solvato complex [Ir(phq)2(H2O]2)] preferentially stains the cytoplasm of both live and dead cells with a bright luminescence. PMID:23457478

  15. Measurement of Local Partial Pressure of Oxygen in the Brain Tissue under Normoxia and Epilepsy with Phosphorescence Lifetime Microscopy

    PubMed Central

    Zhang, Cong; Bélanger, Samuel; Pouliot, Philippe; Lesage, Frédéric

    2015-01-01

    In this work a method for measuring brain oxygen partial pressure with confocal phosphorescence lifetime microscopy system is reported. When used in conjunction with a dendritic phosphorescent probe, Oxyphor G4, this system enabled minimally invasive measurements of oxygen partial pressure (pO2) in cerebral tissue with high spatial and temporal resolution during 4-AP induced epileptic seizures. Investigating epileptic events, we characterized the spatio-temporal distribution of the "initial dip" in pO2 near the probe injection site and along nearby arterioles. Our results reveal a correlation between the percent change in the pO2 signal during the "initial dip" and the duration of seizure-like activity, which can help localize the epileptic focus and predict the length of seizure. PMID:26305777

  16. Imaging interferometric microscopy.

    PubMed

    Schwarz, Christian J; Kuznetsova, Yuliya; Brueck, S R J

    2003-08-15

    We introduce and demonstrate a new microscopy concept: imaging interferometric microscopy (IIM), which is related to holography, synthetic-aperture imaging, and off-axis-dark-field illumination techniques. IIM is a wavelength-division multiplex approach to image formation that combines multiple images covering different spatial-frequency regions to form a composite image with a resolution much greater than that permitted by the same optical system using conventional techniques. This new type of microscopy involves both off-axis coherent illumination and reinjection of appropriate zero-order reference beams. Images demonstrate high resolution, comparable with that of a high-numerical-aperture (NA) objective, while they retain the long working distance, the large depth of field, and the large field of view of a low-NA objective. A Fourier-optics model of IIM is in good agreement with the experiment. PMID:12943079

  17. Imaging of oxygenation in 3D tissue models with multi-modal phosphorescent probes

    NASA Astrophysics Data System (ADS)

    Papkovsky, Dmitri B.; Dmitriev, Ruslan I.; Borisov, Sergei

    2015-03-01

    Cell-penetrating phosphorescence based probes allow real-time, high-resolution imaging of O2 concentration in respiring cells and 3D tissue models. We have developed a panel of such probes, small molecule and nanoparticle structures, which have different spectral characteristics, cell penetrating and tissue staining behavior. The probes are compatible with conventional live cell imaging platforms and can be used in different detection modalities, including ratiometric intensity and PLIM (Phosphorescence Lifetime IMaging) under one- or two-photon excitation. Analytical performance of these probes and utility of the O2 imaging method have been demonstrated with different types of samples: 2D cell cultures, multi-cellular spheroids from cancer cell lines and primary neurons, excised slices from mouse brain, colon and bladder tissue, and live animals. They are particularly useful for hypoxia research, ex-vivo studies of tissue physiology, cell metabolism, cancer, inflammation, and multiplexing with many conventional fluorophors and markers of cellular function.

  18. Fourier plane imaging microscopy

    SciTech Connect

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

    2014-09-14

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

  19. Protein Labelling with Versatile Phosphorescent Metal Complexes for Live Cell Luminescence Imaging.

    PubMed

    Connell, Timothy U; James, Janine L; White, Anthony R; Donnelly, Paul S

    2015-09-28

    To take advantage of the luminescent properties of d(6) transition metal complexes to label proteins, versatile bifunctional ligands were prepared. Ligands that contain a 1,2,3-triazole heterocycle were synthesised using Cu(I) catalysed azide-alkyne cycloaddition "click" chemistry and were used to form phosphorescent Ir(III) and Ru(II) complexes. Their emission properties were readily tuned, by changing either the metal ion or the co-ligands. The complexes were tethered to the metalloprotein transferrin using several conjugation strategies. The Ir(III)/Ru(II)-protein conjugates could be visualised in cancer cells using live cell imaging for extended periods without significant photobleaching. These versatile phosphorescent protein-labelling agents could be widely applied to other proteins and biomolecules and are useful alternatives to conventional organic fluorophores for several applications. PMID:26264214

  20. Singlet oxygen phosphorescence lifetime imaging based on a fluorescence lifetime imaging microscope.

    PubMed

    Tian, Wenming; Deng, Liezheng; Jin, Shengye; Yang, Heping; Cui, Rongrong; Zhang, Qing; Shi, Wenbo; Zhang, Chunlei; Yuan, Xiaolin; Sha, Guohe

    2015-04-01

    The feasibility of singlet oxygen phosphorescence (SOP) lifetime imaging microscope was studied on a modified fluorescence lifetime imaging microscope (FLIM). SOP results from the infrared radiative transition of O2(a(1)Δg → X(3)Σg(-)) and O2(a(1)Δg) was produced in a C60 powder sample via photosensitization process. To capture the very weak SOP signal, a dichroic mirror was placed between the objective and tube lens of the FLIM and used to divide the luminescence returning from the sample into two beams: the reflected SOP beam and the transmitted photoluminescence of C60 (C60-PL) beam. The C60-PL beam entered the scanner of the FLIM and followed the normal optical path of the FLIM, while the SOP steered clear of the scanner and directly entered a finely designed SOP detection channel. Confocal C60-PL images and nonconfocal SOP images were then simultaneously obtained by using laser-scanning mode. Experimental results show that (1) under laser-scanning mode, the obstacle to confocal SOP imaging is the infrared-incompatible scanner, which can be solved by using an infrared-compatible scanner. Confocal SOP imaging is also expected to be realized under stage-scanning mode when the laser beam is parked and meanwhile a pinhole is added into the SOP detection channel. (2) A great challenge to SOP imaging is its extraordinarily long imaging time, and selecting only a few interesting points from fluorescence images to measure their SOP time-dependent traces may be a correct compromise. PMID:25781060

  1. Applications of phosphorescent materials for in-vivo imaging of brain structure and function

    NASA Astrophysics Data System (ADS)

    Boverman, Gregory; Shi, Xiaolei; Cotero, Victoria E.; Filkins, Robert J.; Srivastava, Alok M.; Lorraine, Peter W.; Neculaes, Vasile B.; Ishaque, A. N.

    2016-03-01

    A number of approaches have been developed for in-vivo imaging of neural function at the time scale of action potentials and at the spatial resolution of individual neurons. Remarkable results have been obtained with optogenetics, although the need for genetic modification is an important limitation of these approaches. Similarly, voltage and ion-sensitive dyes allow for optical imaging of action potentials but toxicity remains a problem. Additionally, optical techniques are often only able to be used up to a limited depth. Our preliminary work has shown that nanoparticles of common phosphorescent materials, believed to be generally non-toxic, specifically lutetium oxide and strontium aluminate, can be utilized for cellular imaging, for tomographic imaging, and that the particles can be designed to adhere to neurons. Additionally, lutetium oxide has been shown to be highly X-ray luminescent, potentially allowing for imaging deep within the brain, if the particles can be targeted properly. In ex vivo experiments, we have shown that the phosphorescence of strontium aluminate particles is significantly affected by electric fields similar in strength to those found in the vicinity of the cellular membrane of a neuron. This phenomenon is consistent with early published reports in the electroluminescence literature, namely the Gudden-Pohl effect. We will show results of the ex vivo imaging and dynamic electrical stimulation experiments. We will also show some preliminary ex vivo cell culture results, and will describe plans for future research, focusing on potential in both cell cultures and in vivo for animal models.

  2. Time-resolved luminescence imaging of intracellular oxygen levels based on long-lived phosphorescent iridium(III) complex.

    PubMed

    Liu, Shujuan; Zhang, Yangliu; Liang, Hua; Chen, Zejing; Liu, Ziyu; Zhao, Qiang

    2016-07-11

    Time-resolved luminescence imaging of intracellular oxygen levels has been demonstrated based on long-lived phosphorescent signal. A phosphorescent dinuclear iridium(III) complex Ir1 has been designed and synthesized, which exhibits excellent optical properties, such as high quantum yields, large Stokes shift, high photostability and long emission lifetime. The phosphorescent intensity and lifetime of complex are very sensitive to oxygen levels. Thus, the application of Ir1 for monitoring intracellular oxygen levels has been realized successfully. Especially, utilizing the advantageous long emission lifetime of Ir1, the background fluorescence interference could be eliminated effectively by using the photoluminescence lifetime imaging and time-gated luminescence imaging techniques, improving the signal-to-noise ratios in bioimaging. PMID:27410847

  3. NMR imaging microscopy

    SciTech Connect

    Not Available

    1986-10-01

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

  4. In vivo imaging of brain metabolism activity using a phosphorescent oxygen-sensitive probe

    PubMed Central

    Tsytsarev, Vassiliy; Arakawa, Hiroyuki; Borisov, Sergei; Pumbo, Elena; Erzurumlu, Reha S.; Papkovsky, Dmitri B.

    2013-01-01

    Several approaches have been adopted for real-time imaging of neural activity in vivo. We tested a new cell-penetrating phosphorescent oxygen-sensitive probe, NanO2-IR, to monitor temporal and spatial dynamics of oxygen metabolism in the neocortex following peripheral sensory stimulation. Probe solution was applied to the surface of anesthetized mouse brain; optical imaging was performed using a MiCAM-02 system. Trains of whisker stimuli were delivered and associated changes in phosphorescent signal were recorded in the contralateral somatosensory (“barrel”) cortex. Sensory stimulation led to changes in oxygenation of activated areas of the barrel cortex. The oxygen imaging results were compared to those produced by the voltage-sensitive dye RH-1691. While the signals emitted by the two probes differed in shape and amplitude, they both faithfully indicated specific whisker evoked cortical activity. Thus, NanO2-IR probe can be used as a tool in visualization and realtime analysis of sensory- evoked neural activity in vivo. PMID:23624034

  5. Role of manganese in red long-lasting phosphorescence of manganese-doped diopside for in vivo imaging

    SciTech Connect

    Lecointre, A.; Bessière, A.; Priolkar, K.R.; Gourier, D.; Wallez, G.; Viana, B.

    2013-05-15

    Highlights: ► Long-lasting phosphorescence of CaMgSi{sub 2}O{sub 6}:Mn is studied for bioimaging application. ► CaMgSi{sub 2}O{sub 6}:Mn yields orange and red luminescence of Mn{sup II}{sub Ca} and Mn{sup II}{sub Mg}, respectively. ► Red Mn{sup II}{sub Mg} emission dominates long-lasting phosphorescence spectra. ► Mn mainly substitutes Mg. ► Mn{sup II}{sub Mg} plays the role of hole trap in the persistent luminescence mechanism. - Abstract: Materials with red long-lasting phosphorescence, such as Mn{sup II}-doped diopsides, can be used for small animal in vivo imaging. CaMgSi{sub 2}O{sub 6}:Mn powders with various amounts of Mn were prepared by sol–gel to investigate their long-lasting phosphorescence mechanism. X-ray diffraction, X-ray absorption fine and near-edge structure and electron paramagnetic resonance showed that manganese is quantitatively introduced in the structure as Mn{sup II}. Most of the Mn doping ions substitute Mg and possess a highly elongated octahedral environment. While photoluminescence and X-ray excited optical luminescence spectra show both orange (585 nm) and red (685 nm) {sup 4}T{sub 1} ({sup 4}G) → {sup 6}A{sub 1} ({sup 6}S) emission of Mn{sup II}{sub Ca} and Mn{sup II}{sub Mg}, respectively, Mn{sup II}{sub Mg} red emission dominates long-lasting phosphorescence and thermally stimulated luminescence spectra. These results point to Mn{sup II}{sub Mg} as the preferential hole trap and recombination center in the long-lasting phosphorescence mechanism. An intense persistent red emission suitable for in vivo imaging probes is obtained for the highest nominal Mn content (7.5%)

  6. Iridium(III) Anthraquinone Complexes as Two-Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia.

    PubMed

    Sun, Lingli; Chen, Yu; Kuang, Shi; Li, Guanying; Guan, Ruilin; Liu, Jiangping; Ji, Liangnian; Chao, Hui

    2016-06-20

    In the present study, four mitochondria-specific and two-photon phosphorescence iridium(III) complexes, Ir1-Ir4, were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia-sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two-electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one- (λ=405 nm) and two-photon (λ=720 nm) excitation, with a two-photon absorption cross section of 76-153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two-photon phosphorescence imaging. PMID:27145442

  7. Phosphorescent Sensor for Biological Mobile Zinc

    PubMed Central

    You, Youngmin; Lee, Sumin; Kim, Taehee; Ohkubo, Kei; Chae, Weon-Sik; Fukuzumi, Shunichi; Jhon, Gil-Ja; Nam, Wonwoo; Lippard, Stephen J.

    2011-01-01

    A new phosphorescent zinc sensor (ZIrF) was constructed based on an Ir(III) complex bearing two 2-(2,4-difluorophenyl)pyridine (dfppy) cyclometalating ligands and a neutral 1,10-phenanthroline (phen) ligand. A zinc-specific di(2-picolyl)amino (DPA) receptor was introduced at the 4-position of the phen ligand via a methylene linker. The cationic Ir(III) complex exhibited dual phosphorescence bands in CH3CN solutions originating from blue and yellow emission of the dfppy and phen ligands, respectively. Zinc coordination selectively enhanced the latter, affording a phosphorescence ratiometric response. Electrochemical techniques, quantum chemical calculations, and steady-state and femtosecond spectroscopy were employed to establish a photophysical mechanism for this phosphorescence response. The studies revealed that zinc coordination perturbs nonemissive processes of photoinduced electron transfer (PeT) and intraligand charge transfer (ILCT) transition occurring between DPA and phen. ZIrF can detect zinc ions in a reversible and selective manner in buffered solution (pH 7.0, 25 mM PIPES) with Kd = 11 nM and pKa = 4.16. Enhanced signal-to-noise ratios were achieved by time-gated acquisition of long-lived phosphorescence signals. The sensor was applied to image biological free zinc ions in live A549 cells by confocal laser scanning microscopy. A fluorescence lifetime imaging microscope (FLIM) detected an increase in photoluminescence lifetime for zinc-treated A549 cells as compared to controls. ZIrF is the first successful phosphorescent sensor that detects zinc ions in biological samples. PMID:22023085

  8. Hypoxia-sensitive bis(2-(2'-benzothienyl)pyridinato-N,C3')iridium[poly(n-butyl cyanoacrylate]/chitosan nanoparticles and their phosphorescence tumor imaging in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Zeng, Yun; Zhang, Shaojuan; Jia, Menghui; Liu, Yang; Shang, Jin; Guo, Youmin; Xu, Jianhua; Wu, Daocheng

    2013-11-01

    A new hypoxia-sensitive coordination compound, bis(2-(2'-benzothienyl)pyridinato-N,C3')iridium[poly(n-butyl cyanoacrylate)], hereafter denoted as (btp)2Ir(PBCA), is synthesized and characterized by 13C nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR). (btp)2Ir(PBCA)/chitosan [(btp)2Ir(PBCA)/CS] nanoparticles (NPs) with a core-shell structure are prepared by a two-step fabrication process. The size distributions of these NPs are measured with a Malvern size analyzer, and their morphology is observed by transmission electron microscopy (TEM). The functional groups on the surface are confirmed by FTIR. Phosphorescence spectra are obtained and lifetimes are determined with a spectrophotofluorometer and a time-correlated single photon counting (TCSPC) apparatus, respectively. HeLa and CT26 cell lines are used to examine the cytotoxicity by the MTT assay, as well as to determine the imaging capability of the samples in air and nitrogen atmospheres, respectively. Tumor-bearing mouse models of colon adenocarcinoma are used for tumor imaging in vivo, and the imaging effect is evaluated with a Maestro 2 fluorescence imaging system. Compared with the hypoxia-associated probe bis(2-(2'-benzothienyl)pyridinato-N,C3')iridium(acetylacetonate) (BTP), the phosphorescence lifetime of (btp)2Ir(PBCA)/CS NPs significantly decreases, but the hypoxia-sensitivity increases after preparation of NPs. Apart from the significantly lower cytotoxicity, (btp)2Ir(PBCA)/CS NPs also enhance the tumor imaging effect by more than 10 times, maintaining the phosphorescence signal in tumor tissue for over 24 h and significantly decreasing the phosphorescence signal in normal tissue in vivo compared with the BTP probe.A new hypoxia-sensitive coordination compound, bis(2-(2'-benzothienyl)pyridinato-N,C3')iridium[poly(n-butyl cyanoacrylate)], hereafter denoted as (btp)2Ir(PBCA), is synthesized and characterized by 13C nuclear magnetic resonance (NMR) and Fourier transform

  9. Digital image inpainting and microscopy imaging.

    PubMed

    Stanciu, Stefan G; Hristu, Radu; Stanciu, George A

    2011-11-01

    A considerable amount of image processing techniques known as inpainting techniques have been recently developed aiming to provide solutions for filling in missing or damaged regions in a digital image. Typical such techniques reconstruct a defined area by using information from its neighborhood, for example, by completing inside the missing region the isophote lines arriving at its boundaries. In this article, we show that inpainting techniques have considerable potential usefulness in microscopy imaging, even though experimenting and using them in this domain has been almost entirely neglected up until now. In this purpose, we experiment the "curvature-preserving" partial differential equations as a solution to inpainting regions in images collected by several optical and scanning probe microscopy techniques. The results achieved are presented along with a discussion on typical problematic scenarios of microscopy imaging for which this type of techniques can provide a viable solution. PMID:21563264

  10. TH-C-17A-05: Cherenkov Excited Phosphorescence Oxygen (CEPhOx) Imaging During Multi-Beam Radiation Therapy

    SciTech Connect

    Zhang, R; Pogue, B; Holt, R; Esipova, T; Vinogradov, S; Gladstone, D

    2014-06-15

    Purpose: Cherenkov radiation is created during external beam radiation therapy that can excite phosphorescence in tissue from oxygen-sensitive, bio-compatible probes. Utilizing the known spatial information of the treatment plan with directed multiple beam angles, Cherenkov Excited Phosphorescence Oxygen (CEPhOx) imaging was realized from the reconstructions of Cherenkov excited phosphorescence lifetime. Methods: Platinum(II)-G4 (PtG4) was used as the oxygen-sensitive phosphorescent probe and added to a oxygenated cylindrical liquid phantom with a oxygenated/deoxygenated cylindrical anomaly. Cherenkov excited phosphorescence was imaged using a time-gated ICCD camera temporallysynchronized to the LINAC pulse output. Lifetime reconstruction was carried out in NIRFAST software. Multiple angles of the incident radiation beam was combined with the location of the prescribed treatment volume (PTV) to improve the tomographic recovery as a function of location. The tissue partial pressure of oxygen (pO2) in the background and PTV was calculated based on the recovered lifetime distribution and Stern-Volmer equation. Additionally a simulation study was performed to examine the accuracy of this technique in the setting of a human brain tumor. Results: Region-based pO2 values in the oxygenated background and oxygenated/deoxygenated PTV were correctly recovered, with the deoxygenated anomaly (15.4 mmHg) easily distinguished from the oxygenated background (143 mmHg). The data acquisition time could be achieved within the normal irradiation time for a human fractionated plan. The simulations indicated that CEPhOx would be a sufficient to sample tumor pO2 sensing from tumors which are larger than 2cm in diameter or within 23mm depth from the surface. Conclusion: CEPhOx could be a novel imaging tool for pO2 assessment during external radiation beam therapy. It is minimally invasive and should work within the established treatment plan of radiation therapy with multiple beams in

  11. Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

    PubMed Central

    Sinks, Louise E.; Roussakis, Emmanuel; Esipova, Tatiana V.; Vinogradov, Sergei A.

    2010-01-01

    Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (Förster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols. PMID:20200497

  12. Synthesis and calibration of phosphorescent nanoprobes for oxygen imaging in biological systems.

    PubMed

    Sinks, Louise E; Roussakis, Emmanuel; Esipova, Tatiana V; Vinogradov, Sergei A

    2010-01-01

    Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO(2;)). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (Förster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols. PMID:20200497

  13. Domain adaptation for microscopy imaging.

    PubMed

    Becker, Carlos; Christoudias, C Mario; Fua, Pascal

    2015-05-01

    Electron and light microscopy imaging can now deliver high-quality image stacks of neural structures. However, the amount of human annotation effort required to analyze them remains a major bottleneck. While machine learning algorithms can be used to help automate this process, they require training data, which is time-consuming to obtain manually, especially in image stacks. Furthermore, due to changing experimental conditions, successive stacks often exhibit differences that are severe enough to make it difficult to use a classifier trained for a specific one on another. This means that this tedious annotation process has to be repeated for each new stack. In this paper, we present a domain adaptation algorithm that addresses this issue by effectively leveraging labeled examples across different acquisitions and significantly reducing the annotation requirements. Our approach can handle complex, nonlinear image feature transformations and scales to large microscopy datasets that often involve high-dimensional feature spaces and large 3D data volumes. We evaluate our approach on four challenging electron and light microscopy applications that exhibit very different image modalities and where annotation is very costly. Across all applications we achieve a significant improvement over the state-of-the-art machine learning methods and demonstrate our ability to greatly reduce human annotation effort. PMID:25474809

  14. Dynamic imaging with electron microscopy

    SciTech Connect

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-02-20

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

  15. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-05-30

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

  16. Near-infrared phosphorescent metalloporphyrins

    NASA Astrophysics Data System (ADS)

    Savitsky, Alexander P.; Savitskaja, Anna V.; Lukyanets, Eugeny A.; Dashkevich, Svetlana N.; Makarova, Elena A.

    1997-05-01

    In the near infrared range fluorescent background signals are very small and it is possible to reach high sensitivity in the detection of labeled compounds. With phosphorescent compounds as labels, it is possible, firstly, to add microsecond temporal resolution for background rejection for NIR labels and thus to improve sensitivity. Secondly, compounds that are phosphorescent in NIR are very promising for oxygen life-time imaging of living tissue. Several different groups of palladium and zinc porphyrins and phthalocyanins (meso-tetraphenyl)-(tetrabezo)-porphyrin, meso-tetraphenyl-(tetranaphtho)-porphyrin, tetraazaporphyrins, phthalocyanines) which possess strong absorbance in NIR range were synthesized and analyzed for room temperature phosphorescent properties in organic solvents and in water solution. Among them only Pd- tetrabenzo-(tetraphenyl) porphyrins have high quantum efficiency (10%) with the life-time 328 us and excitation 630 nm, emission 800 nm. In the NIR spectral range water strongly quenches the long-lived phosphorescence of metalloporphyrins. Metalloporphyrins can form inclusion complex with cyclodextrines in which water quenching is almost eliminated. Quantum efficiency and life-time in cyclodextrin solutions are the same as in organic solvents. We analyzed the influence of three different cyclodextrines (alfa, beta and gamma) on the phosphorescent properties of Pd-porphyrins and highest enhancement of the phosphorescence signal occurred for hydroxypropilated (Beta) -cyclodextrin.

  17. Spectroscopic imaging in electron microscopy

    SciTech Connect

    Pennycook, Stephen J; Colliex, C.

    2012-01-01

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

  18. Fidelity imaging for atomic force microscopy

    SciTech Connect

    Ghosal, Sayan Salapaka, Murti

    2015-01-05

    Atomic force microscopy is widely employed for imaging material at the nanoscale. However, real-time measures on image reliability are lacking in contemporary atomic force microscopy literature. In this article, we present a real-time technique that provides an image of fidelity for a high bandwidth dynamic mode imaging scheme. The fidelity images define channels that allow the user to have additional authority over the choice of decision threshold that facilitates where the emphasis is desired, on discovering most true features on the sample with the possible detection of high number of false features, or emphasizing minimizing instances of false detections. Simulation and experimental results demonstrate the effectiveness of fidelity imaging.

  19. Phosphorescence imaging of homocysteine and cysteine in living cells based on a cationic iridium(III) complex.

    PubMed

    Xiong, Liqin; Zhao, Qiang; Chen, Huili; Wu, Yanbo; Dong, Zesheng; Zhou, Zhiguo; Li, Fuyou

    2010-07-19

    Homocysteine (Hcy) and cysteine (Cys) are crucial to the physiological balance in living systems. Specific detection of intracellular Hcy and Cys is of growing importance. Herein, we demonstrated phosphorescence imaging of intracellular Hcy and Cys using a cationic iridium(III) complex Ir(pba)(2)(bpy)(+).PF(6)(-) [pba = 4-(2-pyridyl)benzaldehyde, bpy = bipyridine] containing aldehyde groups as a luminescent probe. Upon addition of Hcy or Cys to a semiaqueous solution of Ir(pba)(2)(bpy)(+), a change in luminescence from yellow to red was visible to the naked eye. The successful chemical reaction of the aldehyde in Ir(pba)(2)(bpy)(+) with Hcy and Cys to form thiazinane and thiazolidine was confirmed by (1)H NMR. Moreover, complexation with Hcy and Cys disturbed the p-pi conjugation between the aldehyde group and the bpy moiety, and led to the excited states switching to [dpi(Ir)-pi(N(wedge)N)*] (3)MLCT and [pi(C(wedge)N)-pi(N(wedge)N)*] (3)LLCT from (pi-pi*)(pba(-)) (3)IL. Furthermore, the MTT assay was used to determine that the probe has low cytotoxicity. Importantly, cell imaging experiments demonstrated that the probe is membrane permeable and can monitor the changes of Hcy/Cys within living cells in a ratiometric mode. PMID:20565069

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

    PubMed

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

    2008-06-01

    Three-dimensional image formation in microscopy is greatly enhanced by the use of computed imaging techniques. In particular, Interferometric Synthetic Aperture Microscopy (ISAM) allows the removal of out-of-focus blur in broadband, coherent microscopy. Earlier methods, such as optical coherence tomography (OCT), utilize interferometric ranging, but do not apply computed imaging methods and therefore must scan the focal depth to acquire extended volumetric images. ISAM removes the need to scan the focus by allowing volumetric image reconstruction from data collected at a single focal depth. ISAM signal processing techniques are similar to the Fourier migration methods of seismology and the Fourier reconstruction methods of Synthetic Aperture Radar (SAR). In this article ISAM is described and the close ties between ISAM and SAR are explored. ISAM and a simple strip-map SAR system are placed in a common mathematical framework and compared to OCT and radar respectively. This article is intended to serve as a review of ISAM, and will be especially useful to readers with a background in SAR. PMID:20948975

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

    PubMed Central

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

    2008-01-01

    Three-dimensional image formation in microscopy is greatly enhanced by the use of computed imaging techniques. In particular, Interferometric Synthetic Aperture Microscopy (ISAM) allows the removal of out-of-focus blur in broadband, coherent microscopy. Earlier methods, such as optical coherence tomography (OCT), utilize interferometric ranging, but do not apply computed imaging methods and therefore must scan the focal depth to acquire extended volumetric images. ISAM removes the need to scan the focus by allowing volumetric image reconstruction from data collected at a single focal depth. ISAM signal processing techniques are similar to the Fourier migration methods of seismology and the Fourier reconstruction methods of Synthetic Aperture Radar (SAR). In this article ISAM is described and the close ties between ISAM and SAR are explored. ISAM and a simple strip-map SAR system are placed in a common mathematical framework and compared to OCT and radar respectively. This article is intended to serve as a review of ISAM, and will be especially useful to readers with a background in SAR. PMID:20948975

  2. Biomolecular Imaging with Coherent Nonlinear Vibrational Microscopy

    PubMed Central

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

    2014-01-01

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

  3. Microscopy image segmentation tool: Robust image data analysis

    SciTech Connect

    Valmianski, Ilya Monton, Carlos; Schuller, Ivan K.

    2014-03-15

    We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

  4. Coherent nonlinear optical imaging: beyond fluorescence microscopy.

    PubMed

    Min, Wei; Freudiger, Christian W; Lu, Sijia; Xie, X Sunney

    2011-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  5. Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy

    PubMed Central

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; Xie, X. Sunney

    2012-01-01

    The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy, including stimulated Raman scattering and two photon absorption, and pump-probe microscopy, including stimulated emission, excited state absorption and ground state depletion, provide distinct and powerful image contrasts for non-fluorescent species. Thanks to high-frequency modulation transfer scheme, they exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles, excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques. PMID:21453061

  6. Image Quality Ranking Method for Microscopy

    PubMed Central

    Koho, Sami; Fazeli, Elnaz; Eriksson, John E.; Hänninen, Pekka E.

    2016-01-01

    Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics. PMID:27364703

  7. Image Quality Ranking Method for Microscopy.

    PubMed

    Koho, Sami; Fazeli, Elnaz; Eriksson, John E; Hänninen, Pekka E

    2016-01-01

    Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics. PMID:27364703

  8. Image Quality Ranking Method for Microscopy

    NASA Astrophysics Data System (ADS)

    Koho, Sami; Fazeli, Elnaz; Eriksson, John E.; Hänninen, Pekka E.

    2016-07-01

    Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics.

  9. Microscopy imaging device with advanced imaging properties

    SciTech Connect

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2015-11-24

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  10. Multidepth imaging by chromatic dispersion confocal microscopy

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Shelton, Ryan L.; Saldua, Meagan A.; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

    2012-03-01

    Confocal microscopy has shown potential as an imaging technique to detect precancer. Imaging cellular features throughout the depth of epithelial tissue may provide useful information for diagnosis. However, the current in vivo axial scanning techniques for confocal microscopy are cumbersome, time-consuming, and restrictive when attempting to reconstruct volumetric images acquired in breathing patients. Chromatic dispersion confocal microscopy (CDCM) exploits severe longitudinal chromatic aberration in the system to axially disperse light from a broadband source and, ultimately, spectrally encode high resolution images along the depth of the object. Hyperchromat lenses are designed to have severe and linear longitudinal chromatic aberration, but have not yet been used in confocal microscopy. We use a hyperchromat lens in a stage scanning confocal microscope to demonstrate the capability to simultaneously capture information at multiple depths without mechanical scanning. A photonic crystal fiber pumped with a 830nm wavelength Ti:Sapphire laser was used as a supercontinuum source, and a spectrometer was used as the detector. The chromatic aberration and magnification in the system give a focal shift of 140μm after the objective lens and an axial resolution of 5.2-7.6μm over the wavelength range from 585nm to 830nm. A 400x400x140μm3 volume of pig cheek epithelium was imaged in a single X-Y scan. Nuclei can be seen at several depths within the epithelium. The capability of this technique to achieve simultaneous high resolution confocal imaging at multiple depths may reduce imaging time and motion artifacts and enable volumetric reconstruction of in vivo confocal images of the epithelium.

  11. Image Correlation Microscopy for Uniform Illumination

    PubMed Central

    Gaborski, Thomas R.; Sealander, Michael N.; Ehrenberg, Morton; Waugh, Richard E.; McGrath, James L.

    2011-01-01

    Image cross-correlation microscopy (ICM) is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. ICM has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy (FCS). In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy (UI-ICM). Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning ICM, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function (SACF). Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function (TACF) depends strongly on particle size and not particle shape. In this report, we establish the relationships between the SACF feature size, TACF characteristic time and the diffusion coefficient for UI-ICM using analytical, Monte-Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate UI-ICM analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils. PMID:20055917

  12. Fluorescence Microscopy Imaging in Biomedical Sciences

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Periasamy, Ammasi

    Fluorescence microscopy is an important tool in biological sciences which provides excellent sensitivity for detecting very low concentrations of molecules over broad spatial and temporal dimensions. With fast developments of new fluorescent probes, advanced electronic and optical devices, and sophisticated data acquisition and analysis software, fluorescence microscopy resides on the central stage of life-sciences research. This chapter covers several commonly used and advanced fluorescence microscopy techniques and focuses on fluorescence lifetime imaging microscopy (FLIM). A number of FLIM systems and their applications are reviewed. As an example, we describe how we built and calibrated a two-photon excitation time-correlated single-photon counting (TPE-TCSPC) FLIM system and employed the system to investigate protein-protein interactions in living cells.

  13. Confocal microscopy imaging of solid tissue

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning ...

  14. Orientation imaging microscopy of polycrystalline sodium chloride

    SciTech Connect

    Staiger, M.P.; Kolbeinsson, I.; Newman, J.; Woodfield, T.; Sato, T.

    2010-04-15

    A novel preparation technique is described that makes possible grain size analysis of polycrystalline NaCl using orientation imaging microscopy via electron backscatter diffraction (EBSD). The preparation methodology is specifically developed to overcome difficulties in preparing microporous NaCl for microscopy. The grain size and crystallographic texture of polycrystalline NaCl samples, prepared via solution pressure and sintered in the range of 650-780 deg. C, were able to be measured successfully with EBSD. The limitations of the preparation technique for EBSD analysis of NaCl are also discussed.

  15. Image simulation for biological microscopy: microlith

    PubMed Central

    Mehta, Shalin B.; Oldenbourg, Rudolf

    2014-01-01

    Image simulation remains under-exploited for the most widely used biological phase microscopy methods, because of difficulties in simulating partially coherent illumination. We describe an open-source toolbox, microlith (https://code.google.com/p/microlith), which accurately predicts three-dimensional images of a thin specimen observed with any partially coherent imaging system, as well as images of coherently illuminated and self-luminous incoherent specimens. Its accuracy is demonstrated by comparing simulated and experimental bright-field and dark-field images of well-characterized amplitude and phase targets, respectively. The comparison provides new insights about the sensitivity of the dark-field microscope to mass distributions in isolated or periodic specimens at the length-scale of 10nm. Based on predictions using microlith, we propose a novel approach for detecting nanoscale structural changes in a beating axoneme using a dark-field microscope. PMID:24940543

  16. From Quantitative Microscopy to Automated Image Understanding

    PubMed Central

    Huang, Kai; Murphy, Robert F.

    2005-01-01

    Quantitative microscopy has been extensively used in biomedical research and has provided significant insights into structure and dynamics at the cell and tissue level. The entire procedure of quantitative microscopy is comprised of specimen preparation, light absorption/reflection/emission from the specimen, microscope optical processing, optical/electrical conversion by a camera or detector, and computational processing of digitized images. Although many of the latest digital signal processing techniques have been successfully applied to compress, restore, and register digital microscope images, automated approaches for recognition and understanding of complex subcellular patterns in light microscope images have been far less widely used. In this review, we describe a systematic approach for interpreting protein subcellular distributions using various sets of Subcellular Location Features (SLF) in combination with supervised classification and unsupervised clustering methods. These methods can handle complex patterns in digital microscope images and the features can be applied for other purposes such as objectively choosing a representative image from a collection and performing statistical comparison of image sets. PMID:15447010

  17. Classification of microscopy images of Langerhans islets

    NASA Astrophysics Data System (ADS)

    Å vihlík, Jan; Kybic, Jan; Habart, David; Berková, Zuzana; Girman, Peter; Kříž, Jan; Zacharovová, Klára

    2014-03-01

    Evaluation of images of Langerhans islets is a crucial procedure for planning an islet transplantation, which is a promising diabetes treatment. This paper deals with segmentation of microscopy images of Langerhans islets and evaluation of islet parameters such as area, diameter, or volume (IE). For all the available images, the ground truth and the islet parameters were independently evaluated by four medical experts. We use a pixelwise linear classifier (perceptron algorithm) and SVM (support vector machine) for image segmentation. The volume is estimated based on circle or ellipse fitting to individual islets. The segmentations were compared with the corresponding ground truth. Quantitative islet parameters were also evaluated and compared with parameters given by medical experts. We can conclude that accuracy of the presented fully automatic algorithm is fully comparable with medical experts.

  18. Edge detection in microscopy images using curvelets

    PubMed Central

    Gebäck, Tobias; Koumoutsakos, Petros

    2009-01-01

    Background Despite significant progress in imaging technologies, the efficient detection of edges and elongated features in images of intracellular and multicellular structures acquired using light or electron microscopy is a challenging and time consuming task in many laboratories. Results We present a novel method, based on the discrete curvelet transform, to extract a directional field from the image that indicates the location and direction of the edges. This directional field is then processed using the non-maximal suppression and thresholding steps of the Canny algorithm to trace along the edges and mark them. Optionally, the edges may then be extended along the directions given by the curvelets to provide a more connected edge map. We compare our scheme to the Canny edge detector and an edge detector based on Gabor filters, and show that our scheme performs better in detecting larger, elongated structures possibly composed of several step or ridge edges. Conclusion The proposed curvelet based edge detection is a novel and competitive approach for imaging problems. We expect that the methodology and the accompanying software will facilitate and improve edge detection in images available using light or electron microscopy. PMID:19257905

  19. Transcutaneous pO2 measurement during tourniquet-induced venous occlusion using dynamic phosphorescence imaging.

    PubMed

    Geis, S; Babilas, P; Schreml, S; Angele, P; Nerlich, M; Jung, E M; Prantl, L

    2008-01-01

    A sufficient oxygen supply in skin grafts requires a functioning microcirculation. Venous occlusion impairs the microcirculation and is therefore a major threat of healing. Luminescence life time imaging (LLI) enables the non-invasive and two-dimensional assessment of the transcutaneous oxygen partial pressure (p(tc)O2). In the current trial this new device was applied for monitoring of venous congestion. A tourniquet on the upper arm was inflated up to 40-50 mmHg and released after 10 min in eight healthy volunteers. The p(tc)O2 was measured at the lower arm every minute prior to, during and up to 10 min after cuff occlusion (40 degrees C applied skin temperature) using LLI of platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix. For validation the polarographic Clark electrode technique was applied in close proximity and measurement was performed simultaneously. p(tc)O2 measurements prior to (Clark: 50.68+/-5.69 mmHg vs. LLI: 50.89+/-4.96 mmHg) and at the end of the venous congestion (Clark: 16.41+/-4.54 mmHg vs. LLI: 23.82+/-3.23 mmHg) did not differ significantly using the Clark electrode vs. LLI. At the initial congestion respectively reperfusion phase the Clark electrode measured faster decreases respectively increase of p(tc)O2 due to oxygen consumption of this method. This experimental trial demonstrates the applicability of LLI to quantify the p(tc)O2 under changing venous blood flow. The use of planar transparent sensors allows the non-invasive generation of two-dimensional maps of surface pO2 what makes this method particular suitable for monitoring of skin grafts. PMID:19126987

  20. Imaging Septum Formation by Fluorescence Microscopy.

    PubMed

    Ribas, Juan Carlos; Cortés, Juan Carlos G

    2016-01-01

    Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosin-based ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flanked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fission yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifically stained by the fluorochrome Calcofluor white. Recent findings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental approaches are needed. Here we describe the methods developed to image the septum structure by fluorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy. PMID:26519306

  1. Light Microscopy Module Imaging Tested and Demonstrated

    NASA Technical Reports Server (NTRS)

    Gati, Frank

    2004-01-01

    The Fluids Integrated Rack (FIR), a facility-class payload, and the Light Microscopy Module (LMM), a subrack payload, are integrated research facilities that will fly in the U.S. Laboratory module, Destiny, aboard the International Space Station. Both facilities are being engineered, designed, and developed at the NASA Glenn Research Center by Northrop Grumman Information Technology. The FIR is a modular, multiuser scientific research facility that is one of two racks that make up the Fluids and Combustion Facility (the other being the Combustion Integrated Rack). The FIR has a large volume dedicated for experimental hardware; easily reconfigurable diagnostics, power, and data systems that allow for unique experiment configurations; and customizable software. The FIR will also provide imagers, light sources, power management and control, command and data handling for facility and experiment hardware, and data processing and storage. The first payload in the FIR will be the LMM. The LMM integrated with the FIR is a remotely controllable, automated, on-orbit microscope subrack facility, with key diagnostic capabilities for meeting science requirements--including video microscopy to observe microscopic phenonema and dynamic interactions, interferometry to make thin-film measurements with nanometer resolution, laser tweezers to manipulate micrometer-sized particles, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure the photonic properties of materials. Vibration disturbances were identified early in the LMM development phase as a high risk for contaminating the science microgravity environment. An integrated FIR-LMM test was conducted in Glenn's Acoustics Test Laboratory to assess mechanical sources of vibration and their impact to microscopic imaging. The primary purpose of the test was to characterize the LMM response at the sample location, the x-y stage within the microscope, to vibration

  2. Image restoration in cryo-electron microscopy.

    PubMed

    Penczek, Pawel A

    2010-01-01

    Image restoration techniques are used to obtain, given experimental measurements, the best possible approximation of the original object within the limits imposed by instrumental conditions and noise level in the data. In molecular electron microscopy (EM), we are mainly interested in linear methods that preserve the respective relationships between mass densities within the restored map. Here, we describe the methodology of image restoration in structural EM, and more specifically, we will focus on the problem of the optimum recovery of Fourier amplitudes given electron microscope data collected under various defocus settings. We discuss in detail two classes of commonly used linear methods, the first of which consists of methods based on pseudoinverse restoration, and which is further subdivided into mean-square error, chi-square error, and constrained based restorations, where the methods in the latter two subclasses explicitly incorporates non-white distribution of noise in the data. The second class of methods is based on the Wiener filtration approach. We show that the Wiener filter-based methodology can be used to obtain a solution to the problem of amplitude correction (or "sharpening") of the EM map that makes it visually comparable to maps determined by X-ray crystallography, and thus amenable to comparative interpretation. Finally, we present a semiheuristic Wiener filter-based solution to the problem of image restoration given sets of heterogeneous solutions. We conclude the chapter with a discussion of image restoration protocols implemented in commonly used single particle software packages. PMID:20888957

  3. Nonlinear Polarimetric Microscopy for Biomedical Imaging

    NASA Astrophysics Data System (ADS)

    Samim, Masood

    A framework for the nonlinear optical polarimetry and polarimetric microscopy is developed. Mathematical equations are derived in terms of linear and nonlinear Stokes Mueller formalism, which comprehensively characterize the polarization properties of the incoming and outgoing radiations, and provide structural information about the organization of the investigated materials. The algebraic formalism developed in this thesis simplifies many predictions for a nonlinear polarimetry study and provides an intuitive understanding of various polarization properties for radiations and the intervening medium. For polarimetric microscopy experiments, a custom fast-scanning differential polarization microscope is developed, which is also capable of real-time three-dimensional imaging. The setup is equipped with a pair of high-speed resonant and galvanometric scanning mirrors, and supplemented by advanced adaptive optics and data acquisition modules. The scanning mirrors when combined with the adaptive optics deformable mirror enable fast 3D imaging. Deformable membrane mirrors and genetic algorithm optimization routines are employed to improve the imaging conditions including correcting the optical aberrations, maximizing signal intensities, and minimizing point-spread-functions of the focal volume. A field-programmable-gate array (FPGA) chip is exploited to rapidly acquire and process the multidimensional data. Using the nonlinear optical polarimetry framework and the home-built polarization microscope, a few biologically important tissues are measured and analyzed to gain insight as to their structure and dynamics. The structure and distribution of muscle sarcomere myosins, connective tissue collagen, carbohydrate-rich starch, and fruit fly eye retinal molecules are characterized with revealing polarization studies. In each case, using the theoretical framework, polarization sensitive data are analyzed to decipher the molecular orientations and nonlinear optical

  4. Nanoscale imaging of RNA with expansion microscopy.

    PubMed

    Chen, Fei; Wassie, Asmamaw T; Cote, Allison J; Sinha, Anubhav; Alon, Shahar; Asano, Shoh; Daugharthy, Evan R; Chang, Jae-Byum; Marblestone, Adam; Church, George M; Raj, Arjun; Boyden, Edward S

    2016-08-01

    The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy of RNA. We developed a small-molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, postexpansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity as well as single-molecule precision in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) separates RNAs and supports amplification of single-molecule signals (i.e., via hybridization chain reaction) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine. PMID:27376770

  5. FISH digital imaging microscopy in mosquito genomics.

    PubMed

    Ferguson, M L; Brown, S E; Knudson, D L

    1996-03-01

    The yellow fever mosquito, Aedes aegypti, transmits pathogens that affect both humans and livestock, and has been the focus of extensive research to identify genetic loci that may be useful in control strategies. Fluorescence in situ hybridization (FISH) and digital imaging microscopy have provided a rapid mechanism to populate the physical map with probes derived from genetic markers, cDNAs and recombinant genomic libraries. When the physical and genetic linkage maps are aligned, map-based cloning will allow the rapid isolation of target genomic sequences. The strategy of FISH mapping and the results of initial hybridization studies are reviewed here by Martin Ferguson, Susan Brown and Dennis Knudson. An Ae. aegypti-specific genomic database, which collates data from mapping studies, sequences, references and other relevant information, is also discussed. PMID:15275237

  6. Small Animal Imaging with Magnetic Resonance Microscopy

    PubMed Central

    Driehuys, Bastiaan; Nouls, John; Badea, Alexandra; Bucholz, Elizabeth; Ghaghada, Ketan; Petiet, Alexandra; Hedlund, Laurence W.

    2009-01-01

    Small animal magnetic resonance microscopy (MRM) has evolved significantly from testing the boundaries of imaging physics to its expanding use today as a tool in non-invasive biomedical investigations. This review is intended to capture the state-of-the-art in MRM for scientists who may be unfamiliar with this modality, but who want to apply its capabilities to their research. We therefore include a brief review of MR concepts and methods of animal handling and support before covering a range of MRM applications including the heart, lung, brain, and the emerging field of MR histology. High-resolution anatomical imaging reveals increasingly exquisite detail in healthy animals and subtle architectural aberrations that occur in genetically altered models. Resolution of 100 µm in all dimensions is now routinely attained in living animals, and 10 µm3 is feasible in fixed specimens. Such images almost rival conventional histology while allowing the object to be viewed interactively in any plane. MRM is now increasingly used to provide functional information in living animals. Images of the beating heart, breathing lung, and functioning brain can be recorded. While clinical MRI focuses on diagnosis, MRM is used to reveal fundamental biology or to non-invasively measure subtle changes in the structure or function of organs during disease progression or in response to experimental therapies. The ability of MRM to provide a detailed functional and anatomical picture in rats and mice, and to track this picture over time, makes it a promising platform with broad applications in biomedical research. PMID:18172332

  7. Imaging Cytoskeleton Components by Electron Microscopy

    PubMed Central

    Svitkina, Tatyana

    2016-01-01

    The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers—actin filaments, microtubules, and intermediate filaments—are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell. PMID:26498781

  8. Nanoscale chemical imaging by photoinduced force microscopy.

    PubMed

    Nowak, Derek; Morrison, William; Wickramasinghe, H Kumar; Jahng, Junghoon; Potma, Eric; Wan, Lei; Ruiz, Ricardo; Albrecht, Thomas R; Schmidt, Kristin; Frommer, Jane; Sanders, Daniel P; Park, Sung

    2016-03-01

    Correlating spatial chemical information with the morphology of closely packed nanostructures remains a challenge for the scientific community. For example, supramolecular self-assembly, which provides a powerful and low-cost way to create nanoscale patterns and engineered nanostructures, is not easily interrogated in real space via existing nondestructive techniques based on optics or electrons. A novel scanning probe technique called infrared photoinduced force microscopy (IR PiFM) directly measures the photoinduced polarizability of the sample in the near field by detecting the time-integrated force between the tip and the sample. By imaging at multiple IR wavelengths corresponding to absorption peaks of different chemical species, PiFM has demonstrated the ability to spatially map nm-scale patterns of the individual chemical components of two different types of self-assembled block copolymer films. With chemical-specific nanometer-scale imaging, PiFM provides a powerful new analytical method for deepening our understanding of nanomaterials. PMID:27051870

  9. Nanoscale chemical imaging by photoinduced force microscopy

    PubMed Central

    Nowak, Derek; Morrison, William; Wickramasinghe, H. Kumar; Jahng, Junghoon; Potma, Eric; Wan, Lei; Ruiz, Ricardo; Albrecht, Thomas R.; Schmidt, Kristin; Frommer, Jane; Sanders, Daniel P.; Park, Sung

    2016-01-01

    Correlating spatial chemical information with the morphology of closely packed nanostructures remains a challenge for the scientific community. For example, supramolecular self-assembly, which provides a powerful and low-cost way to create nanoscale patterns and engineered nanostructures, is not easily interrogated in real space via existing nondestructive techniques based on optics or electrons. A novel scanning probe technique called infrared photoinduced force microscopy (IR PiFM) directly measures the photoinduced polarizability of the sample in the near field by detecting the time-integrated force between the tip and the sample. By imaging at multiple IR wavelengths corresponding to absorption peaks of different chemical species, PiFM has demonstrated the ability to spatially map nm-scale patterns of the individual chemical components of two different types of self-assembled block copolymer films. With chemical-specific nanometer-scale imaging, PiFM provides a powerful new analytical method for deepening our understanding of nanomaterials. PMID:27051870

  10. Imaging Bioorthogonal Groups in Their Ultrastructural Context with Electron Microscopy.

    PubMed

    van Elsland, Daphne M; van Kasteren, Sander I

    2016-08-01

    Spitting image: Herein a recent paper on the imaging of bioorthogonal groups using three-dimensional electron microscopy is discussed. The work has demonstrated electron microscopy imaging as a technique suitable for gaining structural information on bioorthogonal groups in their cellular context. PMID:27346592

  11. Spectral imaging microscopy web sites and data.

    PubMed

    McNamara, George; Gupta, Amit; Reynaert, James; Coates, Thomas D; Boswell, Carl

    2006-08-01

    The Internet is enabling greater access to spectral imaging publications, spectral graphs, and data than that was available a generation ago. The spectral imaging systems discussed in this issue of Cytometry work because reagent and hardware spectra are reproducible, reusable, and provide input to spectral unmixing and spectral components recognition algorithms. These spectra need to be readily available in order to determine what to purchase, how to use it, and what the output means. We refer to several commercially sponsored and academic spectral web sites and discuss our spectral graphing and data sites. Sites include fluorescent dye graph servers from Invitrogen/Molecular Probes, BD Biosciences, Zeiss/Bio-Rad Cell Sciences, and filter set servers from Chroma Technology and Omega Optical. Several of these sites include data download capabilities. Recently, two microscope manufacturers have published on their web sites transmission curves for select objective lenses-crucial data for anyone doing multiphoton excitation microscopy. Notable among the academic sites, PhotoChemCAD 2.0 has over 200 dyes and a downloadable database/graphing program, and the USC-A Chemistry UV-vis Database displays absorption spectra of many dyes and indicators used in clinical histology and pathology. Our Fluorescent Spectra graphing/calculator site presents dyes, filters, and illumination data from many of these and additional sources. PubSpectra is our free download site which uses Microsoft Excel files as standardized human/machine readable format with over 2,000 biomedical spectra. The principle that data is not subject to copyright provides a framework in which all scientific data should be made freely accessible. PMID:16969821

  12. Functional cardiac imaging by random access microscopy

    PubMed Central

    Crocini, Claudia; Coppini, Raffaele; Ferrantini, Cecilia; Pavone, Francesco S.; Sacconi, Leonardo

    2014-01-01

    Advances in the development of voltage sensitive dyes and Ca2+ sensors in combination with innovative microscopy techniques allowed researchers to perform functional measurements with an unprecedented spatial and temporal resolution. At the moment, one of the shortcomings of available technologies is their incapability of imaging multiple fast phenomena while controlling the biological determinants involved. In the near future, ultrafast deflectors can be used to rapidly scan laser beams across the sample, performing optical measurements of action potential and Ca2+ release from multiple sites within cardiac cells and tissues. The same scanning modality could also be used to control local Ca2+ release and membrane electrical activity by activation of caged compounds and light-gated ion channels. With this approach, local Ca2+ or voltage perturbations could be induced, simulating arrhythmogenic events, and their impact on physiological cell activity could be explored. The development of this optical methodology will provide fundamental insights in cardiac disease, boosting new therapeutic strategies, and, more generally, it will represent a new approach for the investigation of the physiology of excitable cells. PMID:25368580

  13. Oxygen distributions within tissue by phosphorescence quenching

    NASA Astrophysics Data System (ADS)

    Wilson, David F.; Grosul, Pavel; Rozhkov, Vladimir; Dugan, Benjamin W.; Reitveld, Ivo; Vinogradov, Sergei A.

    2002-06-01

    Oxygen dependent quenching of phosphorescence is a powerful method for measuring oxygen. Phosphors are now available that absorb and emit in the near IR region of the spectrum, are nontoxic, and remain in the blood, allowing rapid measure of oxygen through out selected tissue volumes. In vivo measurements are non-invasive except for the need to inject phosphor into the blood, and phosphorescence lifetimes can be measured without interference by tissue pigments that absorb or fluorescence at the measurement wavelengths. Phosphorescence quenching is uniquely useful for: (1) imaging oxygen in optically clear media or in the surface layer of the tissue, such as in the retina of the eye; (2) determining the distribution of oxygen in media, such as tissue, which have heterogeneous distributions by deconvoluting phosphorescence decay dat. These can be used to calculate the corresponding oxygen histograms. Measurement in 2D grids can b used to construct contour maps of the fraction of the sampled tissue volume with any selected range of oxygen pressures. These maps accurately show the location and size of any regions of hypoxia within the sampled tissue.

  14. Restoration of uneven illumination in light sheet microscopy images.

    PubMed

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

    2011-08-01

    Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images. PMID:21682937

  15. VirtualMicroscopy: ultra-fast interactive microscopy of gigapixel/terapixel images over internet.

    PubMed

    Wang, Ching-Wei; Huang, Cheng-Ta; Hung, Chu-Mei

    2015-01-01

    As digital imaging technology advances, gigapixel or terapixel super resolution microscopic images become available. We have built a real time virtual microscopy technique for interactive analysis of super high resolution microscopic images over internet on standard desktops, laptops or mobile devices. The presented virtual microscopy technique is demonstrated to perform as fast as using a microscopy locally without any delay to assess gigapixel ultra high resolution image data through wired or wireless internet by a Tablet or a standard PC. More importantly, the presented technology enables analysis of super high resolution microscopic image across sites and time and allows multi-person analysis at the same time, which greatly speed up data analysis process and reduces miscommunication among scientists and doctors. A web site has been created for illustration purposes. (http://www-o.ntust.edu.tw/~cweiwang/VirtualMicroscopy). PMID:26360909

  16. VirtualMicroscopy: ultra-fast interactive microscopy of gigapixel/terapixel images over internet

    PubMed Central

    Wang, Ching-Wei; Huang, Cheng-Ta; Hung, Chu-Mei

    2015-01-01

    As digital imaging technology advances, gigapixel or terapixel super resolution microscopic images become available. We have built a real time virtual microscopy technique for interactive analysis of super high resolution microscopic images over internet on standard desktops, laptops or mobile devices. The presented virtual microscopy technique is demonstrated to perform as fast as using a microscopy locally without any delay to assess gigapixel ultra high resolution image data through wired or wireless internet by a Tablet or a standard PC. More importantly, the presented technology enables analysis of super high resolution microscopic image across sites and time and allows multi-person analysis at the same time, which greatly speed up data analysis process and reduces miscommunication among scientists and doctors. A web site has been created for illustration purposes. (http://www-o.ntust.edu.tw/~cweiwang/VirtualMicroscopy). PMID:26360909

  17. Bioluminescence microscopy using a short focal-length imaging lens.

    PubMed

    Ogoh, K; Akiyoshi, R; May-Maw-Thet; Sugiyama, T; Dosaka, S; Hatta-Ohashi, Y; Suzuki, H

    2014-03-01

    Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy. PMID:24386879

  18. Phosphorescent perylene imides.

    PubMed

    Ventura, Barbara; Langhals, Heinz; Böck, Bernd; Flamigni, Lucia

    2012-05-01

    Asymmetrically substituted perylene imide derivatives PIa and PIx display phosphorescence in glassy matrices at 77 K. The lifetime is 49.0 ms for PIa and 13.5 ms for PIx. The triplet energy is 1.79 eV for PIa and 1.68 eV for PIx as confirmed by sensitization experiments of the C(60) triplet. PMID:22436977

  19. Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.

    PubMed

    Ye, Rui-Rong; Tan, Cai-Ping; Chen, Mu-He; Hao, Liang; Ji, Liang-Nian; Mao, Zong-Wan

    2016-06-01

    Elucidation of relationship among chemical structure, cellular uptake, localization, and biological activity of anticancer metal complexes is important for the understanding of their mechanisms of action. Organometallic rhenium(I) tricarbonyl compounds have emerged as potential multifunctional anticancer drug candidates that can integrate therapeutic and imaging capabilities in a single molecule. Herein, two mononuclear phosphorescent rhenium(I) complexes (Re1 and Re2), along with their corresponding dinuclear complexes (Re3 and Re4), were designed and synthesized as potent anticancer agents. The subcellular accumulation of Re1-Re4 was conveniently analyzed by confocal microscopy in situ in live cells by utilizing their intrinsic phosphorescence. We found that increased lipophilicity of the bidentate ligands could enhance their cellular uptake, leading to improved anticancer efficacy. The dinuclear complexes were more potent than the mononuclear counterparts. The molecular anticancer mechanisms of action evoked by Re3 and Re4 were explored in detail. Re3 with a lower lipophilicity localizes to lysosomes and induces caspase-independent apoptosis, whereas Re4 with higher lipophilicity specially accumulates in mitochondria and induces caspase-independent paraptosis in cancer cells. Our study demonstrates that subcellular localization is crucial for the anticancer mechanisms of these phosphorescent rhenium(I) complexes. PMID:27106876

  20. Multimodal light-sheet microscopy for fluorescence live imaging

    NASA Astrophysics Data System (ADS)

    Oshima, Y.; Kajiura-Kobayashi, H.; Nonaka, S.

    2012-03-01

    Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

  1. Biological imaging with coherent Raman scattering microscopy: a tutorial

    PubMed Central

    Alfonso-García, Alba; Mittal, Richa; Lee, Eun Seong; Potma, Eric O.

    2014-01-01

    Abstract. Coherent Raman scattering (CRS) microscopy is gaining acceptance as a valuable addition to the imaging toolset of biological researchers. Optimal use of this label-free imaging technique benefits from a basic understanding of the physical principles and technical merits of the CRS microscope. This tutorial offers qualitative explanations of the principles behind CRS microscopy and provides information about the applicability of this nonlinear optical imaging approach for biological research. PMID:24615671

  2. Bacterial cell identification in differential interference contrast microscopy images

    PubMed Central

    2013-01-01

    Background Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. Results We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Conclusions Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins. PMID:23617824

  3. Image Resolution in Scanning Transmission Electron Microscopy

    SciTech Connect

    Pennycook, S. J.; Lupini, A.R.

    2008-06-26

    Digital images captured with electron microscopes are corrupted by two fundamental effects: shot noise resulting from electron counting statistics and blur resulting from the nonzero width of the focused electron beam. The generic problem of computationally undoing these effects is called image reconstruction and for decades has proved to be one of the most challenging and important problems in imaging science. This proposal concerned the application of the Pixon method, the highest-performance image-reconstruction algorithm yet devised, to the enhancement of images obtained from the highest-resolution electron microscopes in the world, now in operation at Oak Ridge National Laboratory.

  4. Translation Microscopy (TRAM) for super-resolution imaging

    PubMed Central

    Qiu, Zhen; Wilson, Rhodri S; Liu, Yuewei; R Dun, Alison; Saleeb, Rebecca S; Liu, Dongsheng; Rickman, Colin; Frame, Margaret; Duncan, Rory R; Lu, Weiping

    2016-01-01

    Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology. PMID:26822455

  5. Versatile Room-Temperature-Phosphorescent Materials Prepared from N-Substituted Naphthalimides: Emission Enhancement and Chemical Conjugation.

    PubMed

    Chen, Xiaofeng; Xu, Cheng; Wang, Tao; Zhou, Cao; Du, Jiajun; Wang, Zhongping; Xu, Hangxun; Xie, Tongqing; Bi, Guoqiang; Jiang, Jun; Zhang, Xuepeng; Demas, James N; Trindle, Carl O; Luo, Yi; Zhang, Guoqing

    2016-08-16

    Purely organic materials with room-temperature phosphorescence (RTP) are currently under intense investigation because of their potential applications in sensing, imaging, and displaying. Inspired by certain organometallic systems, where ligand-localized phosphorescence ((3) π-π*) is mediated by ligand-to-metal or metal-to-ligand charge transfer (CT) states, we now show that donor-to-acceptor CT states from the same organic molecule can also mediate π-localized RTP. In the model system of N-substituted naphthalimides (NNIs), the relatively large energy gap between the NNI-localized (1) π-π* and (3) π-π* states of the aromatic ring can be bridged by intramolecular CT states when the NNI is chemically modified with an electron donor. These NNI-based RTP materials can be easily conjugated to both synthetic and natural macromolecules, which can be used for RTP microscopy. PMID:27385550

  6. Classifying and segmenting microscopy images with deep multiple instance learning

    PubMed Central

    Kraus, Oren Z.; Ba, Jimmy Lei; Frey, Brendan J.

    2016-01-01

    Motivation: High-content screening (HCS) technologies have enabled large scale imaging experiments for studying cell biology and for drug screening. These systems produce hundreds of thousands of microscopy images per day and their utility depends on automated image analysis. Recently, deep learning approaches that learn feature representations directly from pixel intensity values have dominated object recognition challenges. These tasks typically have a single centered object per image and existing models are not directly applicable to microscopy datasets. Here we develop an approach that combines deep convolutional neural networks (CNNs) with multiple instance learning (MIL) in order to classify and segment microscopy images using only whole image level annotations. Results: We introduce a new neural network architecture that uses MIL to simultaneously classify and segment microscopy images with populations of cells. We base our approach on the similarity between the aggregation function used in MIL and pooling layers used in CNNs. To facilitate aggregating across large numbers of instances in CNN feature maps we present the Noisy-AND pooling function, a new MIL operator that is robust to outliers. Combining CNNs with MIL enables training CNNs using whole microscopy images with image level labels. We show that training end-to-end MIL CNNs outperforms several previous methods on both mammalian and yeast datasets without requiring any segmentation steps. Availability and implementation: Torch7 implementation available upon request. Contact: oren.kraus@mail.utoronto.ca PMID:27307644

  7. Estimating Geometric Dislocation Densities in Polycrystalline Materialsfrom Orientation Imaging Microscopy

    SciTech Connect

    Man, Chi-Sing; Gao, Xiang; Godefroy, Scott; Kenik, Edward A

    2010-01-01

    Herein we consider polycrystalline materials which can be taken as statistically homogeneous and whose grains can be adequately modeled as rigid-plastic. Our objective is to obtain, from orientation imaging microscopy (OIM), estimates of geometrically necessary dislocation (GND) densities.

  8. A multistaged automatic restoration of noisy microscopy cell images.

    PubMed

    Xu, Jinwei; Hu, Jiankun; Jia, Xiuping

    2015-01-01

    Automated cell segmentation for microscopy cell images has recently become an initial step for further image analysis in cell biology. However, microscopy cell images are easily degraded by noise during the readout procedure via optical-electronic imaging systems. Such noise degradations result in low signal-to-noise ratio (SNR) and poor image quality for cell identification. In order to improve SNR for subsequent segmentation and image-based quantitative analysis, the commonly used state-of-art restoration techniques are applied but few of them are suitable for corrupted microscopy cell images. In this paper, we propose a multistaged method based on a novel integration of trend surface analysis, quantile-quantile plot, bootstrapping, and the Gaussian spatial kernel for the restoration of noisy microscopy cell images. We show this multistaged approach achieves higher performance compared with other state-of-art restoration techniques in terms of peak signal-to-noise ratio and structure similarity in synthetic noise experiments. This paper also reports an experiment on real noisy microscopy data which demonstrated the advantages of the proposed restoration method for improving segmentation performance. PMID:25291801

  9. Nonlinear optical microscopy for imaging thin films and surfaces

    SciTech Connect

    Smilowitz, L.B.; McBranch, D.W.; Robinson, J.M.

    1995-03-01

    We have used the inherent surface sensitivity of second harmonic generation to develop an instrument for nonlinear optical microscopy of surfaces and interfaces. We have demonstrated the use of several nonlinear optical responses for imaging thin films. The second harmonic response of a thin film of C{sub 60} has been used to image patterned films. Two photon absorption light induced fluorescence has been used to image patterned thin films of Rhodamine 6G. Applications of nonlinear optical microscopy include the imaging of charge injection and photoinduced charge transfer between layers in semiconductor heterojunction devices as well as across membranes in biological systems.

  10. PSF engineering in multifocus microscopy for increased depth volumetric imaging.

    PubMed

    Hajj, Bassam; El Beheiry, Mohamed; Dahan, Maxime

    2016-03-01

    Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm. PMID:27231584

  11. PSF engineering in multifocus microscopy for increased depth volumetric imaging

    PubMed Central

    Hajj, Bassam; El Beheiry, Mohamed; Dahan, Maxime

    2016-01-01

    Imaging and localizing single molecules with high accuracy in a 3D volume is a challenging task. Here we combine multifocal microscopy, a recently developed volumetric imaging technique, with point spread function engineering to achieve an increased depth for single molecule imaging. Applications in 3D single molecule localization-based super-resolution imaging is shown over an axial depth of 4 µm as well as for the tracking of diffusing beads in a fluid environment over 8 µm. PMID:27231584

  12. Combined scanning electrochemical atomic force microscopy for tapping mode imaging

    NASA Astrophysics Data System (ADS)

    Kueng, A.; Kranz, C.; Mizaikoff, B.; Lugstein, A.; Bertagnolli, E.

    2003-03-01

    With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) tips using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy (SECM) with atomic force microscopy has recently been demonstrated. Simultaneous imaging of topography and electrochemistry at a sample surface in AFM tapping mode with integrated SECM-AFM cantilevers oscillated at or near their resonance frequency is shown. In contrast to contact mode AFM imaging frictional forces at the sample surface are minimized. Hence, topographical and electrochemical information of soft surfaces (e.g., biological species) can be obtained.

  13. TOPICAL REVIEW: Fluorescence lifetime imaging microscopy in life sciences

    NASA Astrophysics Data System (ADS)

    Willem Borst, Jan; Visser, Antonie J. W. G.

    2010-10-01

    Fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) are versatile tools for the investigation of the molecular environment of fluorophores in living cells. Owing to nanometre-scale interactions via Förster resonance energy transfer (FRET), FLIM and FAIM are powerful microscopy methods for the detection of conformational changes and protein-protein interactions reflecting the biochemical status of live cells. This review provides an overview of recent advances in photonics techniques, quantitative data analysis methods and applications in the life sciences.

  14. Image force microscopy of molecular resonance: A microscope principle

    PubMed Central

    Rajapaksa, I.; Uenal, K.; Wickramasinghe, H. Kumar

    2010-01-01

    We demonstrate a technique in microscopy which extends the domain of atomic force microscopy to optical spectroscopy at the nanometer scale. We show that molecular resonance of feature sizes down to the single molecular level can be detected and imaged purely by mechanical detection of the force gradient between the interaction of the optically driven molecular dipole and its mirror image in a platinum coated scanning probe tip. This microscopy and spectroscopy technique is extendable to frequencies ranging from radio to infrared and the ultraviolet. PMID:20859536

  15. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy.

    PubMed

    Fu, Dan; Lu, Fa-Ke; Zhang, Xu; Freudiger, Christian; Pernik, Douglas R; Holtom, Gary; Xie, Xiaoliang Sunney

    2012-02-29

    Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging. PMID:22316340

  16. Super-resolution Microscopy in Plant Cell Imaging.

    PubMed

    Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

    2015-12-01

    Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. PMID:26482957

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

    PubMed Central

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

    2015-01-01

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

  18. 3D fluorescence anisotropy imaging using selective plane illumination microscopy.

    PubMed

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-08-24

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  19. Simulating Realistic Imaging Conditions For In-Situ Liquid Microscopy

    PubMed Central

    Welch, David A.; Faller, Roland; Evans, James E.; Browning, Nigel D.

    2013-01-01

    In situ transmission electron microscopy enables the imaging of biological cells, macromolecular protein complexes, nanoparticles, and other systems in a near-native environment. In order to improve interpretation of image contrast features and also predict ideal imaging conditions ahead of time, new virtual electron microscopic techniques are needed. A technique for virtual fluid-stage high-angle annular dark-field scanning transmission electron microscopy with the multislice method is presented that enables the virtual imaging of model fluid-stage systems composed of millions of atoms. The virtual technique is exemplified by simulating images of PbS nanoparticles under different imaging conditions and the results agree with previous experimental findings. General insight is obtained on the influence of the effects of fluid path length, membrane thickness, nanoparticle position, defocus and other microscope parameters on attainable image quality. PMID:23872040

  20. Simulating realistic imaging conditions for in situ liquid microscopy

    SciTech Connect

    Welch, David A.; Faller, Roland; Evans, James E.; Browning, Nigel D.

    2013-12-01

    In situ transmission electron microscopy enables the imaging of biological cells, macromolecular protein complexes, nanoparticles, and other systems in a near-native environment. In order to improve interpretation of image contrast features and also predict ideal imaging conditions ahead of time, new virtual electron microscopic techniques are needed. A technique for virtual fluid-stage high-angle annular dark-field scanning transmission electron microscopy with the multislice method is presented that enables the virtual imaging of model fluid-stage systems composed of millions of atoms. The virtual technique is exemplified by simulating images of PbS nanoparticles under different imaging conditions and the results agree with previous experimental findings. General insight is obtained on the influence of the effects of fluid path length, membrane thickness, nanoparticle position, defocus and other microscope parameters on attainable image quality.

  1. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  2. Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy

    PubMed Central

    Hajj, Bassam; Wisniewski, Jan; El Beheiry, Mohamed; Chen, Jiji; Revyakin, Andrey; Wu, Carl; Dahan, Maxime

    2014-01-01

    Single molecule-based superresolution imaging has become an essential tool in modern cell biology. Because of the limited depth of field of optical imaging systems, one of the major challenges in superresolution imaging resides in capturing the 3D nanoscale morphology of the whole cell. Despite many previous attempts to extend the application of photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) techniques into three dimensions, effective localization depths do not typically exceed 1.2 µm. Thus, 3D imaging of whole cells (or even large organelles) still demands sequential acquisition at different axial positions and, therefore, suffers from the combined effects of out-of-focus molecule activation (increased background) and bleaching (loss of detections). Here, we present the use of multifocus microscopy for volumetric multicolor superresolution imaging. By simultaneously imaging nine different focal planes, the multifocus microscope instantaneously captures the distribution of single molecules (either fluorescent proteins or synthetic dyes) throughout an ∼4-µm-deep volume, with lateral and axial localization precisions of ∼20 and 50 nm, respectively. The capabilities of multifocus microscopy to rapidly image the 3D organization of intracellular structures are illustrated by superresolution imaging of the mammalian mitochondrial network and yeast microtubules during cell division. PMID:25422417

  3. Semitransparent nanostructured films for imaging mass spectrometry and optical microscopy.

    PubMed

    Forsythe, Jay G; Broussard, Joshua A; Lawrie, Jenifer L; Kliman, Michal; Jiao, Yang; Weiss, Sharon M; Webb, Donna J; McLean, John A

    2012-12-18

    Semitransparent porous silicon substrates have been developed for pairing nanostructure-initiator mass spectrometry (NIMS) imaging with traditional optical-based microscopy techniques. Substrates were optimized to generate the largest NIMS signal while maintaining sufficient transparency to allow visible light to pass through for optical microscopy. Using these substrates, both phase-contrast and NIMS images of phospholipids from a scratch-wounded cell monolayer were obtained. NIMS images were generated using a spatial resolution of 14 μm. Coupled with further improvements in spatial resolution, this approach may allow for the localization of intact biological molecules within cells without the need for labeling. PMID:23146026

  4. Unconventional methods of imaging: computational microscopy and compact implementations

    NASA Astrophysics Data System (ADS)

    McLeod, Euan; Ozcan, Aydogan

    2016-07-01

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

  5. Unconventional methods of imaging: computational microscopy and compact implementations.

    PubMed

    McLeod, Euan; Ozcan, Aydogan

    2016-07-01

    In the past two decades or so, there has been a renaissance of optical microscopy research and development. Much work has been done in an effort to improve the resolution and sensitivity of microscopes, while at the same time to introduce new imaging modalities, and make existing imaging systems more efficient and more accessible. In this review, we look at two particular aspects of this renaissance: computational imaging techniques and compact imaging platforms. In many cases, these aspects go hand-in-hand because the use of computational techniques can simplify the demands placed on optical hardware in obtaining a desired imaging performance. In the first main section, we cover lens-based computational imaging, in particular, light-field microscopy, structured illumination, synthetic aperture, Fourier ptychography, and compressive imaging. In the second main section, we review lensfree holographic on-chip imaging, including how images are reconstructed, phase recovery techniques, and integration with smart substrates for more advanced imaging tasks. In the third main section we describe how these and other microscopy modalities have been implemented in compact and field-portable devices, often based around smartphones. Finally, we conclude with some comments about opportunities and demand for better results, and where we believe the field is heading. PMID:27214407

  6. Imaging Extraterrestrial Rocks with Scanning Magnetic Microscopy

    NASA Astrophysics Data System (ADS)

    Andrade Lima, E.; Weiss, B. P.; Gattacceca, J.

    2013-05-01

    Scanning magnetic microscopes map the magnetic field produced by a geological sample at submillimeter scales. Such magnetic field maps reveal invaluable information about rocks with complex fine-scale structures. In particular, instruments based on high-sensitivity SQUID sensors can detect magnetic moments as weak as 10^-16 Am2, outperforming by four orders of magnitude the detection limit of the best commercial moment magnetometers. This unique combination of high spatial resolution and high moment sensitivity enables paleomagnetic analyses on samples that have not been accessible to standard moment magnetometry. Targets for scanning magnetic microscopy include extended samples (such as thin sections of meteorites, lunar rocks, and earth rocks) and individual particles of small size (< 500 μm) comprising impact melt spherules, zircon and other silicate cristals, chondrules, and cosmic dust. Here we present applications of the technique focusing on extraterrestrial samples and discuss how it can be an important tool in investigating the effects of shock on the magnetic record in rocks.

  7. Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

    PubMed Central

    Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

    2008-01-01

    Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

  8. Comparison of image reconstruction methods for structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Lukeš, Tomas; Hagen, Guy M.; Křížek, Pavel; Švindrych, Zdeněk.; Fliegel, Karel; Klíma, Miloš

    2014-05-01

    Structured illumination microscopy (SIM) is a recent microscopy technique that enables one to go beyond the diffraction limit using patterned illumination. The high frequency information is encoded through aliasing into the observed image. By acquiring multiple images with different illumination patterns aliased components can be separated and a highresolution image reconstructed. Here we investigate image processing methods that perform the task of high-resolution image reconstruction, namely square-law detection, scaled subtraction, super-resolution SIM (SR-SIM), and Bayesian estimation. The optical sectioning and lateral resolution improvement abilities of these algorithms were tested under various noise level conditions on simulated data and on fluorescence microscopy images of a pollen grain test sample and of a cultured cell stained for the actin cytoskeleton. In order to compare the performance of the algorithms, the following objective criteria were evaluated: Signal to Noise Ratio (SNR), Signal to Background Ratio (SBR), circular average of the power spectral density and the S3 sharpness index. The results show that SR-SIM and Bayesian estimation combine illumination patterned images more effectively and provide better lateral resolution in exchange for more complex image processing. SR-SIM requires one to precisely shift the separated spectral components to their proper positions in reciprocal space. High noise levels in the raw data can cause inaccuracies in the shifts of the spectral components which degrade the super-resolved image. Bayesian estimation has proven to be more robust to changes in noise level and illumination pattern frequency.

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

    PubMed

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

    2015-12-01

    In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. PMID:26206941

  10. Time-encoded structured illumination microscopy: toward ultrafast superresolution imaging.

    PubMed

    Wang, Yuxi; Guo, Qiang; Chen, Hongwei; Chen, Minghua; Yang, Sigang; Xie, Shizhong

    2016-08-15

    An imaging strategy based on optical time-encoded structured illumination microscopy (TE-SIM) opens the way toward ultrafast superresolution imaging. A proof-of-principle experiment is conducted and the introduced TE-SIM accelerates the generation rate of sinusoidal fringe patterns to an unprecedented speed (dozens of megahertz). At such a high speed, superresolution imaging that surpasses the diffraction limit by a factor of 1.4 is demonstrated. This imaging strategy with high temporal and spatial resolution has great potential in many exciting applications, such as dynamic live cell imaging or high-throughput screening. PMID:27519081

  11. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy

    SciTech Connect

    Dohnalkova, A.C.; Marshall, M. J.; Arey, B. W.; Williams, K. H.; Buck, E. C.; Fredrickson, J. K.

    2011-01-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  12. Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy

    SciTech Connect

    Dohnalkova, Alice; Marshall, Matthew J.; Arey, Bruce W.; Williams, Kenneth H.; Buck, Edgar C.; Fredrickson, Jim K.

    2011-02-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryo-electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in the collapse of hydrated gel-like EPS into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  13. Deep insights: intravital imaging with two-photon microscopy.

    PubMed

    Schießl, Ina Maria; Castrop, Hayo

    2016-09-01

    Intravital multiphoton microscopy is widely used to assess the structure and function of organs in live animals. Although different tissues vary in their accessibility for intravital multiphoton imaging, considerable progress has been made in the imaging quality of all tissues due to substantial technical improvements in the relevant imaging components, such as optics, excitation laser, detectors, and signal analysis software. In this review, we provide an overview of the technical background of intravital multiphoton microscopy. Then, we note a few seminal findings that were made through the use of multiphoton microscopy. Finally, we address the technical limitations of the method and provide an outlook for how these limitations may be overcome through future technical developments. PMID:27352273

  14. Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy.

    PubMed

    Dohnalkova, Alice C; Marshall, Matthew J; Arey, Bruce W; Williams, Kenneth H; Buck, Edgar C; Fredrickson, James K

    2011-02-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment. PMID:21169451

  15. Segmentation and learning in the quantitative analysis of microscopy images

    NASA Astrophysics Data System (ADS)

    Ruggiero, Christy; Ross, Amy; Porter, Reid

    2015-02-01

    In material science and bio-medical domains the quantity and quality of microscopy images is rapidly increasing and there is a great need to automatically detect, delineate and quantify particles, grains, cells, neurons and other functional "objects" within these images. These are challenging problems for image processing because of the variability in object appearance that inevitably arises in real world image acquisition and analysis. One of the most promising (and practical) ways to address these challenges is interactive image segmentation. These algorithms are designed to incorporate input from a human operator to tailor the segmentation method to the image at hand. Interactive image segmentation is now a key tool in a wide range of applications in microscopy and elsewhere. Historically, interactive image segmentation algorithms have tailored segmentation on an image-by-image basis, and information derived from operator input is not transferred between images. But recently there has been increasing interest to use machine learning in segmentation to provide interactive tools that accumulate and learn from the operator input over longer periods of time. These new learning algorithms reduce the need for operator input over time, and can potentially provide a more dynamic balance between customization and automation for different applications. This paper reviews the state of the art in this area, provides a unified view of these algorithms, and compares the segmentation performance of various design choices.

  16. Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging.

    PubMed

    Seifert, Jan; Rheinlaender, Johannes; Novak, Pavel; Korchev, Yuri E; Schäffer, Tilman E

    2015-06-23

    Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell's elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM. PMID:26011471

  17. Detecting overlapping instances in microscopy images using extremal region trees.

    PubMed

    Arteta, Carlos; Lempitsky, Victor; Noble, J Alison; Zisserman, Andrew

    2016-01-01

    In many microscopy applications the images may contain both regions of low and high cell densities corresponding to different tissues or colonies at different stages of growth. This poses a challenge to most previously developed automated cell detection and counting methods, which are designed to handle either the low-density scenario (through cell detection) or the high-density scenario (through density estimation or texture analysis). The objective of this work is to detect all the instances of an object of interest in microscopy images. The instances may be partially overlapping and clustered. To this end we introduce a tree-structured discrete graphical model that is used to select and label a set of non-overlapping regions in the image by a global optimization of a classification score. Each region is labeled with the number of instances it contains - for example regions can be selected that contain two or three object instances, by defining separate classes for tuples of objects in the detection process. We show that this formulation can be learned within the structured output SVM framework and that the inference in such a model can be accomplished using dynamic programming on a tree structured region graph. Furthermore, the learning only requires weak annotations - a dot on each instance. The candidate regions for the selection are obtained as extremal region of a surface computed from the microscopy image, and we show that the performance of the model can be improved by considering a proxy problem for learning the surface that allows better selection of the extremal regions. Furthermore, we consider a number of variations for the loss function used in the structured output learning. The model is applied and evaluated over six quite disparate data sets of images covering: fluorescence microscopy, weak-fluorescence molecular images, phase contrast microscopy and histopathology images, and is shown to exceed the state of the art in performance. PMID:25980675

  18. Biological imaging by soft x-ray diffraction microscopy

    DOE PAGESBeta

    Shapiro, D.; Thibault, P.; Beetz, T.; Elser, V.; Howells, M.; Jacobsen, C.; Kirz, J.; Lima, E.; Miao, H.; Neiman, A. M.; et al

    2005-10-25

    We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffractionmore » microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.« less

  19. Biological imaging by soft x-ray diffraction microscopy

    SciTech Connect

    Shapiro, D.; Thibault, P.; Beetz, T.; Elser, V.; Howells, M.; Jacobsen, C.; Kirz, J.; Lima, E.; Miao, H.; Neiman, A. M.; Sayre, D.

    2005-10-25

    We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.

  20. Oxygen-sensitive phosphorescent nanomaterials produced from high-density polyethylene films by local solvent-crazing.

    PubMed

    Toncelli, Claudio; Arzhakova, Olga V; Dolgova, Alla; Volynskii, Aleksandr L; Bakeev, Nikolai F; Kerry, Joe P; Papkovsky, Dmitri B

    2014-02-01

    Discrete solid-state phosphorescent oxygen sensors produced by local solvent-crazing of high density polyethylene films are described. The simple spotting of dye solution followed by tensile drawing of the polymer substrate provides uniform nanostructures with good spatial control, effective encapsulation of dye molecules, and quenchability by O2. The dye-polymer composite sensors prepared using toluene as a solvent and stabilized by annealing at high temperature, show moderate optical signals, near-optimal sensitivity to O2 (RSD at 21 KPa 1.9%), and reproducible phosphorescence lifetime readings. Calibration experiments performed over 0-25 kPa O2 and 10-30 °C temperatures ranges reveal linear Stern-Volmer plots and temperature dependences and minimal effect of humidity on sensor calibration. The high degree of lateral and in-depth homogeneity of these O2-sensitive materials was confirmed by high-resolution atomic force and wide-field optical microscopy, including 2D and 3D phosphorescence lifetime imaging. PMID:24422456

  1. Intravital microscopy to image membrane trafficking in live rats

    PubMed Central

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

    2014-01-01

    Summary Intravital microscopy (IVM) is a powerful tool that enables imaging various biological processes in live animals. Here, we describe a series of procedures designed to image subcellular structures, such as endsosomes and secretory vesicles in the salivary glands (SGs) of live rats. To this aim, we used fluorescently labeled molecules and/or fluorescently-tagged proteins that were transiently transfected in the live animal. PMID:23027003

  2. Embryonic Heart Morphogenesis from Confocal Microscopy Imaging and Automatic Segmentation

    PubMed Central

    Gribble, Megan; Pertsov, Arkady M.; Shi, Pengcheng

    2013-01-01

    Embryonic heart morphogenesis (EHM) is a complex and dynamic process where the heart transforms from a single tube into a four-chambered pump. This process is of great biological and clinical interest but is still poorly understood for two main reasons. On the one hand, the existing imaging modalities for investigating EHM suffered from either limited penetration depth or limited spatial resolution. On the other hand, current works typically adopted manual segmentation, which was tedious, subjective, and time consuming considering the complexity of developing heart geometry and the large size of images. In this paper, we propose to utilize confocal microscopy imaging with tissue optical immersion clearing technique to image the heart at different stages of development for EHM study. The imaging method is able to produce high spatial resolution images and achieve large penetration depth at the same time. Furthermore, we propose a novel convex active contour model for automatic image segmentation. The model has the ability to deal with intensity fall-off in depth which is characterized by confocal microscopy images. We acquired the images of embryonic quail hearts from day 6 to day 14 of incubation for EHM study. The experimental results were promising and provided us with an insight view of early heart growth pattern and also paved the road for data-driven heart growth modeling. PMID:24454530

  3. Multidirectional Image Sensing for Microscopy Based on a Rotatable Robot

    PubMed Central

    Shen, Yajing; Wan, Wenfeng; Zhang, Lijun; Yong, Li; Lu, Haojian; Ding, Weili

    2015-01-01

    Image sensing at a small scale is essentially important in many fields, including microsample observation, defect inspection, material characterization and so on. However, nowadays, multi-directional micro object imaging is still very challenging due to the limited field of view (FOV) of microscopes. This paper reports a novel approach for multi-directional image sensing in microscopes by developing a rotatable robot. First, a robot with endless rotation ability is designed and integrated with the microscope. Then, the micro object is aligned to the rotation axis of the robot automatically based on the proposed forward-backward alignment strategy. After that, multi-directional images of the sample can be obtained by rotating the robot within one revolution under the microscope. To demonstrate the versatility of this approach, we view various types of micro samples from multiple directions in both optical microscopy and scanning electron microscopy, and panoramic images of the samples are processed as well. The proposed method paves a new way for the microscopy image sensing, and we believe it could have significant impact in many fields, especially for sample detection, manipulation and characterization at a small scale. PMID:26694391

  4. X-ray holographic microscopy: Improved images of zymogen granules

    SciTech Connect

    Jacobsen, C.; Howells, M.; Kirz, J.; McQuaid, K.; Rothman, S.

    1988-10-01

    Soft x-ray holography has long been considered as a technique for x-ray microscopy. It has been only recently, however, that sub-micron resolution has been obtained in x-ray holography. This paper will concentrate on recent progress we have made in obtaining reconstructed images of improved quality. 15 refs., 6 figs.

  5. Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

    PubMed Central

    Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

    2013-01-01

    In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

  6. Rapid analysis and exploration of fluorescence microscopy images.

    PubMed

    Pavie, Benjamin; Rajaram, Satwik; Ouyang, Austin; Altschuler, Jason M; Steininger, Robert J; Wu, Lani F; Altschuler, Steven J

    2014-01-01

    Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard. Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens. PMID:24686220

  7. Quantification of photoacoustic microscopy images for ovarian cancer detection

    NASA Astrophysics Data System (ADS)

    Wang, Tianheng; Yang, Yi; Alqasemi, Umar; Kumavor, Patrick D.; Wang, Xiaohong; Sanders, Melinda; Brewer, Molly; Zhu, Quing

    2014-03-01

    In this paper, human ovarian tissues with malignant and benign features were imaged ex vivo by using an opticalresolution photoacoustic microscopy (OR-PAM) system. Several features were quantitatively extracted from PAM images to describe photoacoustic signal distributions and fluctuations. 106 PAM images from 18 human ovaries were classified by applying those extracted features to a logistic prediction model. 57 images from 9 ovaries were used as a training set to train the logistic model, and 49 images from another 9 ovaries were used to test our prediction model. We assumed that if one image from one malignant ovary was classified as malignant, it is sufficient to classify this ovary as malignant. For the training set, we achieved 100% sensitivity and 83.3% specificity; for testing set, we achieved 100% sensitivity and 66.7% specificity. These preliminary results demonstrate that PAM could be extremely valuable in assisting and guiding surgeons for in vivo evaluation of ovarian tissue.

  8. In-line digital holographic imaging in volume holographic microscopy.

    PubMed

    Zhai, Xiaomin; Lin, Wei-Tang; Chen, Hsi-Hsun; Wang, Po-Hao; Yeh, Li-Hao; Tsai, Jui-Chang; Singh, Vijay Raj; Luo, Yuan

    2015-12-01

    A dual-plane in-line digital holographic imaging method incorporating volume holographic microscopy (VHM) is presented to reconstruct objects in a single shot while eliminating zero-order and twin-image diffracted waves. The proposed imaging method is configured such that information from different axial planes is acquired simultaneously using multiplexed volume holographic imaging gratings, as used in VHM, and recorded as in-line holograms where the corresponding reference beams are generated in the fashion of Gabor's in-line holography. Unlike conventional VHM, which can take axial intensity information only at focal depths, the proposed method digitally reconstructs objects at any axial position. Further, we demonstrate the proposed imaging technique's ability to effectively eliminate zero-order and twin images for single-shot three-dimensional object reconstruction. PMID:26625046

  9. Fractal descriptors for discrimination of microscopy images of plant leaves

    NASA Astrophysics Data System (ADS)

    Silva, N. R.; Florindo, J. B.; Gómez, M. C.; Kolb, R. M.; Bruno, O. M.

    2014-03-01

    This study proposes the application of fractal descriptors method to the discrimination of microscopy images of plant leaves. Fractal descriptors have demonstrated to be a powerful discriminative method in image analysis, mainly for the discrimination of natural objects. In fact, these descriptors express the spatial arrangement of pixels inside the texture under different scales and such arrangements are directly related to physical properties inherent to the material depicted in the image. Here, we employ the Bouligand-Minkowski descriptors. These are obtained by the dilation of a surface mapping the gray-level texture. The classification of the microscopy images is performed by the well-known Support Vector Machine (SVM) method and we compare the success rate with other literature texture analysis methods. The proposed method achieved a correctness rate of 89%, while the second best solution, the Co-occurrence descriptors, yielded only 78%. This clear advantage of fractal descriptors demonstrates the potential of such approach in the analysis of the plant microscopy images.

  10. Multimodal confocal hyperspectral imaging microscopy with wavelength sweeping source

    NASA Astrophysics Data System (ADS)

    Kim, Young-Duk; Do, Dukho; Yoo, Hongki; Gweon, DaeGab

    2015-02-01

    There exist microscopes that are able to obtain the chemical properties of a sample, because there are some cases in which it is difficult to find out causality of a phenomenon by using only the structural information of a sample. Obtaining the chemical properties of a sample is important in biomedical imaging, because most biological phenomena include changes in the chemical properties of the sample. Hyperspectral imaging (HSI) is one of the popular imaging methods for characterizing materials and biological samples by measuring the reflectance or emission spectrum of the sample. Because all materials have a unique reflectance spectrum, it is possible to analyze material properties and detect changes in the chemical properties of a sample by measuring the spectral changes with respect to the original spectrum. Because of its ability to measure the spectrum of a sample, HSI is widely used in materials identification applications such as aerial reconnaissance and is the subject of various studies in microscopy. Although there are many advantages to using the method, conventional HSI has some limitations because of its complex configuration and slow speed. In this research we propose a new type of multimodal confocal hyperspectral imaging microscopy with fast image acquisition and a simple configuration that is capable of both confocal and HSI microscopies.

  11. Imaging nonmelanoma skin cancers with combined ultrasound-photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Sunar, Ulas; Rohrbach, Daniel J.; Morgan, Janet; Zeitouni, Natalie

    2013-03-01

    PDT has become a treatment of choice especially for the cases with multiple sites and large areas. However, the efficacy of PDT is limited for thicker and deeper tumors. Depth and size information as well as vascularity can provide useful information to clinicians for planning and evaluating PDT. High-resolution ultrasound and photoacoustic imaging can provide information regarding skin structure and vascularity. We utilized combined ultrasound-photoacoustic microscopy for imaging a basal cell carcinoma (BCC) tumor pre-PDT and the results indicate that combined ultrasound-photoacoustic imaging can be useful tool for PDT planning by providing both structural and functional contrasts.

  12. Elemental imaging of cartilage by scanning x-ray microscopy

    SciTech Connect

    Buckley, C.J.; Foster, G.F.; Burge, R.E. ); Ali, S.Y.; Scotchford, C.A. , Royal National Orthopaedic Hospital, Stanmore, Middlesex ); Kirz, J. ); Rivers, M.L. )

    1992-01-01

    Elemental imaging via scanning transmission x-ray microscopy (STXM) and scanning fluorescence x-ray microscopy (SFXM) has been used to image calcium deposits in cartilage. In the case of STXM, 0.1 {mu}m thick sections were imaged to investigate the proximity of calcium deposits in relation to chondrocyte cells. The resolution available was 0.5 {mu}m, and field widths of up to 25 {mu}m were used at this resolution. The resolution available in SFXM was 10 {mu}m, and field widths of up to 2 mm were used at this resolution on 5-{mu}m thick specimens. Together these techniques were used to map calcium deposits at the cellular level, and at the full tissue size level.

  13. Imaging bacterial spores by soft-x-ray microscopy

    SciTech Connect

    Stead, A.D.; Ford, T.W.; Judge, J.

    1997-04-01

    Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.

  14. Bright-field quantitative phase microscopy (BFQPM) for accurate phase imaging using conventional microscopy hardware

    NASA Astrophysics Data System (ADS)

    Jenkins, Micah; Gaylord, Thomas K.

    2015-03-01

    Most quantitative phase microscopy methods require the use of custom-built or modified microscopic configurations which are not typically available to most bio/pathologists. There are, however, phase retrieval algorithms which utilize defocused bright-field images as input data and are therefore implementable in existing laboratory environments. Among these, deterministic methods such as those based on inverting the transport-of-intensity equation (TIE) or a phase contrast transfer function (PCTF) are particularly attractive due to their compatibility with Köhler illuminated systems and numerical simplicity. Recently, a new method has been proposed, called multi-filter phase imaging with partially coherent light (MFPI-PC), which alleviates the inherent noise/resolution trade-off in solving the TIE by utilizing a large number of defocused bright-field images spaced equally about the focal plane. Despite greatly improving the state-ofthe- art, the method has many shortcomings including the impracticality of high-speed acquisition, inefficient sampling, and attenuated response at high frequencies due to aperture effects. In this report, we present a new method, called bright-field quantitative phase microscopy (BFQPM), which efficiently utilizes a small number of defocused bright-field images and recovers frequencies out to the partially coherent diffraction limit. The method is based on a noiseminimized inversion of a PCTF derived for each finite defocus distance. We present simulation results which indicate nanoscale optical path length sensitivity and improved performance over MFPI-PC. We also provide experimental results imaging live bovine mesenchymal stem cells at sub-second temporal resolution. In all, BFQPM enables fast and accurate phase imaging with unprecedented spatial resolution using widely available bright-field microscopy hardware.

  15. Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy

    PubMed Central

    Schulz, Olaf; Pieper, Christoph; Clever, Michaela; Pfaff, Janine; Ruhlandt, Aike; Kehlenbach, Ralph H.; Wouters, Fred S.; Großhans, Jörg; Bunt, Gertrude; Enderlein, Jörg

    2013-01-01

    We demonstrate how a conventional confocal spinning-disk (CSD) microscope can be converted into a doubly resolving image scanning microscopy (ISM) system without changing any part of its optical or mechanical elements. Making use of the intrinsic properties of a CSD microscope, we illuminate stroboscopically, generating an array of excitation foci that are moved across the sample by varying the phase between stroboscopic excitation and rotation of the spinning disk. ISM then generates an image with nearly doubled resolution. Using conventional fluorophores, we have imaged single nuclear pore complexes in the nuclear membrane and aggregates of GFP-conjugated Tau protein in three dimensions. Multicolor ISM was shown on cytoskeletal-associated structural proteins and on 3D four-color images including MitoTracker and Hoechst staining. The simple adaptation of conventional CSD equipment allows superresolution investigations of a broad variety of cell biological questions. PMID:24324140

  16. Scanning tunneling microscopy on rough surfaces-quantitative image analysis

    NASA Astrophysics Data System (ADS)

    Reiss, G.; Brückl, H.; Vancea, J.; Lecheler, R.; Hastreiter, E.

    1991-07-01

    In this communication, the application of scanning tunneling microscopy (STM) for a quantitative evaluation of roughnesses and mean island sizes of polycrystalline thin films is discussed. Provided strong conditions concerning the resolution are satisfied, the results are in good agreement with standard techniques as, for example, transmission electron microscopy. Owing to its high resolution, STM can supply a better characterization of surfaces than established methods, especially concerning the roughness. Microscopic interpretations of surface dependent physical properties thus can be considerably improved by a quantitative analysis of STM images.

  17. Intravital Microscopy for Imaging the Tumor Microenvironment in Live Mice.

    PubMed

    Naumenko, Victor; Jenne, Craig; Mahoney, Douglas J

    2016-01-01

    The development of intravital microscopy has provided unprecedented capacity to study the tumor microenvironment in live mice. The dynamic behavior of cancer, stromal, vascular, and immune cells can be monitored in real time, in situ, in both primary tumors and metastatic lesions, allowing treatment responses to be observed at single cell resolution and therapies tracked in vivo. These features provide a unique opportunity to elucidate the cellular mechanisms underlying the biology and treatment of cancer. We describe here a method for imaging the microenvironment of subcutaneous tumors grown in mice using intravital microscopy. PMID:27581025

  18. Electromechanical Imaging of Biomaterials by Scanning Probe Microscopy

    SciTech Connect

    Rodriguez, Brian J; Kalinin, Sergei V; Shin, Junsoo; Jesse, Stephen; Grichko, V.; Thundat, Thomas George; Baddorf, Arthur P; Gruverman, A.

    2006-01-01

    The majority of calcified and connective tissues possess complex hierarchical structure spanning the length scales from nanometers to millimeters. Understanding the biological functionality of these materials requires reliable methods for structural imaging on the nanoscale. Here, we demonstrate an approach for electromechanical imaging of the structure of biological samples on the length scales from tens of microns to nanometers using piezoresponse force microscopy (PFM), which utilizes the intrinsic piezoelectricity of biopolymers such as proteins and polysaccharides as the basis for high-resolution imaging. Nanostructural imaging of a variety of protein-based materials, including tooth, antler, and cartilage, is demonstrated. Visualization of protein fibrils with sub-10 nm spatial resolution in a human tooth is achieved. Given the near-ubiquitous presence of piezoelectricity in biological systems, PFM is suggested as a versatile tool for micro- and nanostructural imaging in both connective and calcified tissues.

  19. [A tracking algorithm for live mitochondria in fluorescent microscopy images].

    PubMed

    Xu, Junmei; Li, Yang; Du, Sidan; Zhao, Kanglian

    2012-04-01

    Quantitative analysis of biological image data generally involves the detection of many pixel spots. In live mitochondria video image, for which fluorescent microscopy is often used, the signal-to-noise ratio (SNR) can be extremely low, making the detection and tracking of mitochondria particle difficult. It is especially not easy to get the movement curve when the movement of the mitochondria involves its self-move and the motion caused by the neuron. An tracking algorithm for live mitochondria is proposed in this paper. First the whole image sequence is frame-to-frame registered, in which the edge corners are chosen to be the feature points. Then the mitochondria particles are tracked by frame-to-frame displacement vector. The algorithm proposed has been applied to the dynamic image sequence including neuron and mitochondria, saving time without manually picking up the feature points. It provides an new method and reference for medical image processing and biotechnological research. PMID:22616189

  20. Imaging intracellular protein dynamics by spinning disk confocal microscopy

    PubMed Central

    Stehbens, Samantha; Pemble, Hayley; Murrow, Lindsay; Wittmann, Torsten

    2012-01-01

    The palette of fluorescent proteins has grown exponentially over the last decade, and as a result live imaging of cells expressing fluorescently tagged proteins is becoming more and more main stream. Spinning disk confocal microscopy (SDC) is a high speed optical sectioning technique, and a method of choice to observe and analyze intracellular fluorescent protein dynamics at high spatial and temporal resolution. In an SDC system, a rapidly rotating pinhole disk generates thousands of points of light that scan the specimen simultaneously, which allows direct capture of the confocal image with low noise scientific grade cooled charged-coupled device (CCD) cameras, and can achieve frame rates of up 1000 frames per second. In this chapter we describe important components of a state-of-the-art spinning disk system optimized for live cell microscopy, and provide a rationale for specific design choices. We also give guidelines how other imaging techniques such as total internal reflection (TIRF) microscopy or spatially controlled photoactivation can be coupled with SDC imaging, and provide a short protocol on how to generate cell lines stably expressing fluorescently tagged proteins by lentivirus-mediated transduction. PMID:22264541

  1. Size-Invariant Detection of Cell Nuclei in Microscopy Images.

    PubMed

    Ram, Sundaresh; Rodriguez, Jeffrey J

    2016-07-01

    Accurate detection of individual cell nuclei in microscopy images is an essential and fundamental task for many biological studies. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. Manual detection of individual cell nuclei by visual inspection is time consuming, and prone to induce subjective bias. This makes automatic detection of cell nuclei essential for large-scale, objective studies of cell cultures. Blur, clutter, bleed-through, imaging noise and touching and partially overlapping nuclei with varying sizes and shapes make automated detection of individual cell nuclei a challenging task using image analysis. In this paper we propose a new automated method for fast and robust detection of individual cell nuclei based on their radial symmetric nature in fluorescence in-situ hybridization (FISH) images obtained via confocal microscopy. The main contributions are two-fold. 1) This work presents a more accurate cell nucleus detection system using the fast radial symmetry transform (FRST). 2) The proposed cell nucleus detection system is robust against most occlusions and variations in size and moderate shape deformations. We evaluate the performance of the proposed algorithm using precision/recall rates, Fβ-score and root-mean-squared distance (RMSD) and show that our algorithm provides improved detection accuracy compared to existing algorithms. PMID:26886972

  2. Spatial compound imaging for fiber-bundle optic microscopy

    NASA Astrophysics Data System (ADS)

    Cheon, Gyeong Woo; Cha, Jaepyeong; Kang, Jin U.

    2014-02-01

    Coherent fiber bundles with high core density give both flexibility and high resolution to microscopy. Despite of these advantages, fiber bundles inevitably have uncovered region between adjacent cores. The region results in structural artifact known as pixelation effect. Many kinds of image processing techniques have been introduced to remove this pixelation artifact such as frequency domain filter and Gaussian filter. However, these methods fundamentally have limitation because they use the information of adjacent pixels to make up for these uncovered area; therefore, they cannot avoid blurring effect as a result. To overcome this problem, we introduce spatial compound imaging method to overcome this pixelation artifact. The method uses multiple frames taken with small deviation of position. Some parts of these images include information which is devoid of in other images. The total amount of information increase as more images are added up and we can expect the improvement of resolution in the final images. At the same time, the duplicated parts among these images can be averaged to improve SNR ratio. For these improvements, we essentially need sophisticated registration algorithm. The pixelation artifact is troublesome again in registration process because its structural artifacts are strong features shared with whole images. However, we can solve this problem by using reference image and divide the sample images into two parts: effective and ineffective regions. We used effective regions for registration. We used USAF target to evaluate our method and we could get a result that SNR and resolution are both critically increase.

  3. Raman Microscopy : A Versatile Approach to Bio-Imaging

    NASA Astrophysics Data System (ADS)

    McGarvey, J. J.; Renwick Beattie, J.

    Raman microscopy has become established as a key probe technique in biology and biomedicine. In combination with imaging and mapping it has been employed in the investigation of a diverse array of problems ranging from ex vivo and in vivo single cell studies to elucidation of the often complex, interacting structures which constitute human and animal tissues. This chapter emphasises the unique attributes of Raman microscopy as a bioimaging technique, including its non-invasive, spectral multiplexing ability, allied with high spatial resolution and underpinned by a range of multivariate data processing methods. A number of illustrative examples have been selected for discussion from the fields of molecular biology, ophthalmology, respiratory medicine as well as some non-medical examples. Recent advances and pointers to future activity in the uses of Raman microscopy as a structurally and functionally informative bioimaging technique are briefly considered.

  4. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging

    NASA Astrophysics Data System (ADS)

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering.

  5. Comparison between optical-resolution photoacoustic microscopy and confocal laser scanning microscopy for turbid sample imaging.

    PubMed

    U-Thainual, Paweena; Kim, Do-Hyun

    2015-12-01

    Optical-resolution photoacoustic microscopy (ORPAM) in theory provides lateral resolution equivalent to the optical diffraction limit. Scattering media, such as biological turbid media, attenuates the optical signal and also alters the diffraction-limited spot size of the focused beam. The ORPAM signal is generated only from a small voxel in scattering media with dimensions equivalent to the laser spot size after passing through scattering layers and is detected by an acoustic transducer, which is not affected by optical scattering. Thus, both ORPAM and confocal laser scanning microscopy (CLSM) reject scattered light. A multimodal optical microscopy platform that includes ORPAM and CLSM was constructed, and the lateral resolution of both modes was measured using patterned thin metal film with and without a scattering barrier. The effect of scattering media on the lateral resolution was studied using different scattering coefficients and was compared to computational results based on Monte Carlo simulations. It was found that degradation of lateral resolution due to optical scattering was not significant for either ORPAM or CLSM. The depth discrimination capability of ORPAM and CLSM was measured using microfiber embedded in a light scattering phantom material. ORPAM images demonstrated higher contrast compared to CLSM images partly due to reduced acoustic signal scattering. PMID:26256640

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

    PubMed

    Jones, Chris G

    2012-01-01

    Scanning electron microscopy (SEM) has been almost universally applied for the surface examination and characterization of both natural and man-made objects. Although an invasive technique, developments in electron microscopy over the years has given the microscopist a much clearer choice in how invasive the technique will be. With the advent of low vacuum SEM in the 1970s (The environmental cold stage, 1970) and environmental SEM in the late 1980s (J Microsc 160(pt. 1):9-19, 1989), it is now possible in some circumstances to examine samples without preparation. However, for the examination of biological tissue and cells it is still advisable to chemically fix, dehydrate, and coat samples for SEM imaging and analysis. This chapter aims to provide an overview of SEM as an imaging tool, and a general introduction to some of the methods applied for the preparation of samples. PMID:22907399

  7. Intermodulation electrostatic force microscopy for imaging surface photo-voltage

    SciTech Connect

    Borgani, Riccardo Forchheimer, Daniel; Thorén, Per-Anders; Haviland, David B.; Bergqvist, Jonas; Inganäs, Olle

    2014-10-06

    We demonstrate an alternative to Kelvin Probe Force Microscopy for imaging surface potential. The open-loop, single-pass technique applies a low-frequency AC voltage to the atomic force microscopy tip while driving the cantilever near its resonance frequency. Frequency mixing due to the nonlinear capacitance gives intermodulation products of the two drive frequencies near the cantilever resonance, where they are measured with high signal to noise ratio. Analysis of this intermodulation response allows for quantitative reconstruction of the contact potential difference. We derive the theory of the method, validate it with numerical simulation and a control experiment, and we demonstrate its utility for fast imaging of the surface photo-voltage on an organic photo-voltaic material.

  8. Three-dimensional snow images by MR microscopy.

    PubMed

    Ozeki, Toshihiro; Kose, Katsumi; Haishi, Tomoyuki; Hashimoto, Seitarou; Nakatsubo, Shun-ichi; Nishimura, Kouichi

    2003-01-01

    MR microscopy technique was introduced to visualize and quantify the three-dimensional structure of snowpack. Since the NMR signal from the ice was week, we looked at the air space instead filling with dodecane or aniline doped with iron acetylacetonate. Four types of snow were tested: ice spheres, large rounded poly crystals, small rounded mono-crystals and depth hoar crystals. A specific specimen-cooling system was developed to keep the temperature below 0 degrees C. In the experiments 0.5 to 2 h were necessary to accumulate the signals enough to obtain a 3D micro-image; the image matrix 128(3), voxel size (200 microm)3 or 256(3) (120 microm)3. Comparison with the 2D data using the conventional section plane method was also carried out and MR microscopy is proved to be a very useful method to visualize the microstructure of snowpack. PMID:12850731

  9. New filtering techniques to restore scanning tunneling microscopy images

    NASA Astrophysics Data System (ADS)

    Pancorbo, M.; Aguilar, M.; Anguiano, E.; Diaspro, A.

    1991-07-01

    An asymmetric transfer function — based on the symmetric one used in optical cases to correct blurring and defocusing effects in systems with circular aperture — is presented here to restore STM (scanning tunneling microscopy) images. A Wien filter is implemented that utilize this transfer function. In the STM case, the defocusing has two different origins depending on the scan direction that produce a set of two fitting parameters.

  10. Hybrid Imaging for Extended Depth of Field Microscopy

    NASA Astrophysics Data System (ADS)

    Zahreddine, Ramzi Nicholas

    An inverse relationship exists in optical systems between the depth of field (DOF) and the minimum resolvable feature size. This trade-off is especially detrimental in high numerical aperture microscopy systems where resolution is pushed to the diffraction limit resulting in a DOF on the order of 500 nm. Many biological structures and processes of interest span over micron scales resulting in significant blurring during imaging. This thesis explores a two-step computational imaging technique known as hybrid imaging to create extended DOF (EDF) microscopy systems with minimal sacrifice in resolution. In the first step a mask is inserted at the pupil plane of the microscope to create a focus invariant system over 10 times the traditional DOF, albeit with reduced contrast. In the second step the contrast is restored via deconvolution. Several EDF pupil masks from the literature are quantitatively compared in the context of biological microscopy. From this analysis a new mask is proposed, the incoherently partitioned pupil with binary phase modulation (IPP-BPM), that combines the most advantageous properties from the literature. Total variation regularized deconvolution models are derived for the various noise conditions and detectors commonly used in biological microscopy. State of the art algorithms for efficiently solving the deconvolution problem are analyzed for speed, accuracy, and ease of use. The IPP-BPM mask is compared with the literature and shown to have the highest signal-to-noise ratio and lowest mean square error post-processing. A prototype of the IPP-BPM mask is fabricated using a combination of 3D femtosecond glass etching and standard lithography techniques. The mask is compared against theory and demonstrated in biological imaging applications.

  11. Image contrast reversals in contact resonance atomic force microscopy

    SciTech Connect

    Ma, Chengfu; Chen, Yuhang Wang, Tian

    2015-02-15

    Multiple image contrast inversions are observed along with the increase of modulation frequency for contact resonance atomic force microscopy (CR-AFM) imaging of a highly oriented pyrolytic graphite (HOPG) specimen. Analysis of the contact vibrational spectra indicates that the inversions can be attributed to structure-induced variations of tip-sample contact mechanics. Contact stiffness and damping at HOPG step edges exhibit significant increases relative to those in the flat regions. For quantitative evaluation of mechanical properties in CR-AFM, coupling effects of the surface geometry must be considered.

  12. Validity criterion for the Born approximation convergence in microscopy imaging.

    PubMed

    Trattner, Sigal; Feigin, Micha; Greenspan, Hayit; Sochen, Nir

    2009-05-01

    The need for the reconstruction and quantification of visualized objects from light microscopy images requires an image formation model that adequately describes the interaction of light waves with biological matter. Differential interference contrast (DIC) microscopy, as well as light microscopy, uses the common model of the scalar Helmholtz equation. Its solution is frequently expressed via the Born approximation. A theoretical bound is known that limits the validity of such an approximation to very small objects. We present an analytic criterion for the validity region of the Born approximation. In contrast to the theoretical known bound, the suggested criterion considers the field at the lens, external to the object, that corresponds to microscopic imaging and extends the validity region of the approximation. An analytical proof of convergence is presented to support the derived criterion. The suggested criterion for the Born approximation validity region is described in the context of a DIC microscope, yet it is relevant for any light microscope with similar fundamental apparatus. PMID:19412231

  13. Comparative analysis of imaging configurations and objectives for Fourier microscopy.

    PubMed

    Kurvits, Jonathan A; Jiang, Mingming; Zia, Rashid

    2015-11-01

    Fourier microscopy is becoming an increasingly important tool for the analysis of optical nanostructures and quantum emitters. However, achieving quantitative Fourier space measurements requires a thorough understanding of the impact of aberrations introduced by optical microscopes that have been optimized for conventional real-space imaging. Here we present a detailed framework for analyzing the performance of microscope objectives for several common Fourier imaging configurations. To this end, we model objectives from Nikon, Olympus, and Zeiss using parameters that were inferred from patent literature and confirmed, where possible, by physical disassembly. We then examine the aberrations most relevant to Fourier microscopy, including the alignment tolerances of apodization factors for different objective classes, the effect of magnification on the modulation transfer function, and vignetting-induced reductions of the effective numerical aperture for wide-field measurements. Based on this analysis, we identify an optimal objective class and imaging configuration for Fourier microscopy. In addition, the Zemax files for the objectives and setups used in this analysis have been made publicly available as a resource for future studies. PMID:26560923

  14. Corneal imaging by second and third harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Brocas, Arnaud; Jay, Louis; Mottay, Eric; Brunette, Isabelle; Ozaki, Tsuneyuki

    2008-02-01

    Advanced imaging methods are essential tools for improved outcome of refractive surgery. Second harmonic generation (SHG) and third harmonic generation (THG) microscopy are noninvasive high-resolution imaging methods, which can discriminate the different layers of the cornea, thus having strong impact on the outcome of laser surgery. In this work, we use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering, and allows simultaneous SHG and THG imaging. We present SHG and THG images and profiles of pig corneas that clearly show the anterior surface of the cornea, the entry in the stroma and its end, and the posterior surface of the cornea. These observations allow localizing the epithelium, the stroma and the endothelium. Other experiments give information about the structure and cytology of the corneal layers.

  15. Color normalization for robust evaluation of microscopy images

    NASA Astrophysics Data System (ADS)

    Švihlík, Jan; Kybic, Jan; Habart, David

    2015-09-01

    This paper deals with color normalization of microscopy images of Langerhans islets in order to increase robustness of the islet segmentation to illumination changes. The main application is automatic quantitative evaluation of the islet parameters, useful for determining the feasibility of islet transplantation in diabetes. First, background illumination inhomogeneity is compensated and a preliminary foreground/background segmentation is performed. The color normalization itself is done in either lαβ or logarithmic RGB color spaces, by comparison with a reference image. The color-normalized images are segmented using color-based features and pixel-wise logistic regression, trained on manually labeled images. Finally, relevant statistics such as the total islet area are evaluated in order to determine the success likelihood of the transplantation.

  16. Combined FLIM and reflectance confocal microscopy for epithelial imaging

    NASA Astrophysics Data System (ADS)

    Jabbour, Joey M.; Cheng, Shuna; Shrestha, Sebina; Malik, Bilal; Jo, Javier A.; Applegate, Brian; Maitland, Kristen C.

    2012-03-01

    Current methods for detection of oral cancer lack the ability to delineate between normal and precancerous tissue with adequate sensitivity and specificity. The usual diagnostic mechanism involves visual inspection and palpation followed by tissue biopsy and histopathology, a process both invasive and time-intensive. A more sensitive and objective screening method can greatly facilitate the overall process of detection of early cancer. To this end, we present a multimodal imaging system with fluorescence lifetime imaging (FLIM) for wide field of view guidance and reflectance confocal microscopy for sub-cellular resolution imaging of epithelial tissue. Moving from a 12 x 12 mm2 field of view with 157 ìm lateral resolution using FLIM to 275 x 200 μm2 with lateral resolution of 2.2 μm using confocal microscopy, hamster cheek pouch model is imaged both in vivo and ex vivo. The results indicate that our dual modality imaging system can identify and distinguish between different tissue features, and, therefore, can potentially serve as a guide in early oral cancer detection..

  17. Superresolved multiphoton microscopy with spatial frequency-modulated imaging.

    PubMed

    Field, Jeffrey J; Wernsing, Keith A; Domingue, Scott R; Allende Motz, Alyssa M; DeLuca, Keith F; Levi, Dean H; DeLuca, Jennifer G; Young, Michael D; Squier, Jeff A; Bartels, Randy A

    2016-06-14

    Superresolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all superresolution imaging techniques reported to date rely on real energy states of fluorescent molecules to circumvent the diffraction limit, preventing superresolved imaging with contrast mechanisms that occur via virtual energy states, including harmonic generation (HG). We report a superresolution technique based on spatial frequency-modulated imaging (SPIFI) that permits superresolved nonlinear microscopy with any contrast mechanism and with single-pixel detection. We show multimodal superresolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2η below the diffraction limit, where η is the highest power of the nonlinear intensity response. MP-SPIFI can be used to provide enhanced resolution in optically thin media and may provide a solution for superresolved imaging deep in scattering media. PMID:27231219

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

    NASA Astrophysics Data System (ADS)

    Esipova, Tatiana V.; Vinogradov, Sergei A.

    2016-03-01

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

  19. EUV actinic brightfield mask microscopy for predicting printed defect images

    NASA Astrophysics Data System (ADS)

    Goldberg, Kenneth; Benk, Markus P.; Wojdyla, Antoine; Verduijn, Erik; Wood, Obert R.; Mangat, Pawitter

    2015-10-01

    Improving our collective understanding of extreme ultraviolet (EUV) photomask defects and the imaging properties of available defect imaging tools is essential for improving EUV mask defectivity, defect repair and mitigation, and for high-level strategic decision-making. In this work, we perform a qualitative comparison of twenty-five defects imaged with mask scanning electron microscopy (SEM), EUV actinic mask imaging, and wafer SEM imaging. All but two of the defect locations were first identified by non-actinic mask blank inspection, prior to patterning. The others were identified as repeating defects on the wafer. We find that actinic defect imaging is predictive of the wafer prints, with small-scale features clearly replicated. While some mask defect SEM images match the wafer prints, others print with a larger outline indicating the presence of sub-surface disruptions hidden from the SEM's view. Fourteen other defects were subjected to an aerial image phase measurement method called Fourier Ptychography (FP). Although phase shifts were observed in the larger defects, the smaller defects in the dataset showed no significant phase shifting. We attribute this discrepancy to non-actinic mask blank inspection's limited ability to detect small phase defects under normal operating conditions.

  20. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.

    PubMed

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  1. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

    PubMed Central

    Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  2. Registration and 3D visualization of large microscopy images

    NASA Astrophysics Data System (ADS)

    Mosaliganti, Kishore; Pan, Tony; Sharp, Richard; Ridgway, Randall; Iyengar, Srivathsan; Gulacy, Alexandra; Wenzel, Pamela; de Bruin, Alain; Machiraju, Raghu; Huang, Kun; Leone, Gustavo; Saltz, Joel

    2006-03-01

    Inactivation of the retinoblastoma gene in mouse embryos causes tissue infiltrations into critical sections of the placenta, which has been shown to affect fetal survivability. Our collaborators in cancer genetics are extremely interested in examining the three dimensional nature of these infiltrations given a stack of two dimensional light microscopy images. Three sets of wildtype and mutant placentas was sectioned serially and digitized using a commercial light microscopy scanner. Each individual placenta dataset consisted of approximately 1000 images totaling 700 GB in size, which were registered into a volumetric dataset using National Library of Medicine's (NIH/NLM) Insight Segmentation and Registration Toolkit (ITK). This paper describes our method for image registration to aid in volume visualization of tissue level intermixing for both wildtype and Rb - specimens. The registration process faces many challenges arising from the large image sizes, damages during sectioning, staining gradients both within and across sections, and background noise. These issues limit the direct application of standard registration techniques due to frequent convergence to local solutions. In this work, we develop a mixture of automated and semi-automated enhancements with ground-truth validation for the mutual information-based registration algorithm. Our final volume renderings clearly show tissue intermixing differences between both wildtype and Rb - specimens which are not obvious prior to registration.

  3. Nanocrystal size distribution analysis from transmission electron microscopy images

    NASA Astrophysics Data System (ADS)

    van Sebille, Martijn; van der Maaten, Laurens J. P.; Xie, Ling; Jarolimek, Karol; Santbergen, Rudi; van Swaaij, René A. C. M. M.; Leifer, Klaus; Zeman, Miro

    2015-12-01

    We propose a method, with minimal bias caused by user input, to quickly detect and measure the nanocrystal size distribution from transmission electron microscopy (TEM) images using a combination of Laplacian of Gaussian filters and non-maximum suppression. We demonstrate the proposed method on bright-field TEM images of an a-SiC:H sample containing embedded silicon nanocrystals with varying magnifications and we compare the accuracy and speed with size distributions obtained by manual measurements, a thresholding method and PEBBLES. Finally, we analytically consider the error induced by slicing nanocrystals during TEM sample preparation on the measured nanocrystal size distribution and formulate an equation to correct this effect.We propose a method, with minimal bias caused by user input, to quickly detect and measure the nanocrystal size distribution from transmission electron microscopy (TEM) images using a combination of Laplacian of Gaussian filters and non-maximum suppression. We demonstrate the proposed method on bright-field TEM images of an a-SiC:H sample containing embedded silicon nanocrystals with varying magnifications and we compare the accuracy and speed with size distributions obtained by manual measurements, a thresholding method and PEBBLES. Finally, we analytically consider the error induced by slicing nanocrystals during TEM sample preparation on the measured nanocrystal size distribution and formulate an equation to correct this effect. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06292f

  4. Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

    PubMed Central

    Mohan, Kavya; Purnapatra, Subhajit B.; Mondal, Partha Pratim

    2014-01-01

    We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated equi-intense light-sheets of thickness approximately with an inter-sheet separation of . Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics. PMID:24911061

  5. High resolution surface plasmon microscopy for cell imaging

    NASA Astrophysics Data System (ADS)

    Argoul, F.; Monier, K.; Roland, T.; Elezgaray, J.; Berguiga, L.

    2010-04-01

    We introduce a new non-labeling high resolution microscopy method for cellular imaging. This method called SSPM (Scanning Surface Plasmon Microscopy) pushes down the resolution limit of surface plasmon resonance imaging (SPRi) to sub-micronic scales. High resolution SPRi is obtained by the surface plasmon lauching with a high numerical aperture objective lens. The advantages of SPPM compared to other high resolution SPRi's rely on three aspects; (i) the interferometric detection of the back reflected light after plasmon excitation, (ii) the twodimensional scanning of the sample for image reconstruction, (iii) the radial polarization of light, enhancing both resolution and sensitivity. This microscope can afford a lateral resolution of - 150 nm in liquid environment and - 200 nm in air. We present in this paper images of IMR90 fibroblasts obtained with SSPM in dried environment. Internal compartments such as nucleus, nucleolus, mitochondria, cellular and nuclear membrane can be recognized without labelling. We propose an interpretation of the ability of SSPM to reveal high index contrast zones by a local decomposition of the V (Z) function describing the response of the SSPM.

  6. Airway surface liquid depth imaged by surface laser reflectance microscopy.

    PubMed

    Thiagarajah, Jay R; Song, Yuanlin; Derichs, Nico; Verkman, A S

    2010-09-01

    The thin layer of liquid at the surface of airway epithelium, the airway surface liquid (ASL), is important in normal airway physiology and in the pathophysiology of cystic fibrosis. At present, the best method to measure ASL depth involves scanning confocal microscopy after staining with an aqueous-phase fluorescent dye. We describe here a simple, noninvasive imaging method to measure ASL depth by reflectance imaging of an epithelial mucosa in which the surface is illuminated at a 45-degree angle by an elongated 13-microm wide rectangular beam produced by a 670-nm micro-focus laser. The principle of the method is that air-liquid, liquid-liquid, and liquid-cell interfaces produce distinct specular or diffuse reflections that can be imaged to give a micron-resolution replica of the mucosal surface. The method was validated using fluid layers of specified thicknesses and applied to measure ASL depth in cell cultures and ex vivo fragments of pig trachea. In addition, the method was adapted to measure transepithelial fluid transport from the dynamics of fluid layer depth. Compared with confocal imaging, ASL depth measurement by surface laser reflectance microscopy does not require dye staining or costly instrumentation, and can potentially be adapted for in vivo measurements using fiberoptics. PMID:20713545

  7. Imaging articular cartilage using second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Mansfield, Jessica C.; Winlove, C. Peter; Knapp, Karen; Matcher, Stephen J.

    2006-02-01

    Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.

  8. Compact Image Slicing Spectrometer (ISS) for hyperspectral fluorescence microscopy

    PubMed Central

    Gao, Liang; Kester, Robert T.; Tkaczyk, Tomasz S.

    2009-01-01

    An image slicing spectrometer (ISS) for microscopy applications is presented. Its principle is based on the redirecting of image zones by specially organized thin mirrors within a custom fabricated component termed an image slicer. The demonstrated prototype can simultaneously acquire a 140nm spectral range within its 2D field of view from a single image. The spectral resolution of the system is 5.6nm. The FOV and spatial resolution of the ISS depend on the selected microscope objective and for the results presented is 45×45μm2 and 0.45μm respectively. This proof-of-concept system can be easily improved in the future for higher (both spectral and spatial) resolution imaging. The system requires no scanning and minimal post data processing. In addition, the reflective nature of the image slicer and use of prisms for spectral dispersion make the system light efficient. Both of the above features are highly valuable for real time fluorescent-spectral imaging in biological and diagnostic applications. PMID:19654631

  9. Acoustic and photoacoustic microscopy imaging of single leukocytes

    NASA Astrophysics Data System (ADS)

    Strohm, Eric M.; Moore, Michael J.; Kolios, Michael C.

    2016-03-01

    An acoustic/photoacoustic microscope was used to create micrometer resolution images of stained cells from a blood smear. Pulse echo ultrasound images were made using a 1000 MHz transducer with 1 μm resolution. Photoacoustic images were made using a fiber coupled 532 nm laser, where energy losses through stimulated Raman scattering enabled output wavelengths from 532 nm to 620 nm. The laser was focused onto the sample using a 20x objective, and the laser spot co-aligned with the 1000 MHz transducer opposite the laser. The blood smear was stained with Wright-Giemsa, a common metachromatic dye that differentially stains the cellular components for visual identification. A neutrophil, lymphocyte and a monocyte were imaged using acoustic and photoacoustic microscopy at two different wavelengths, 532 nm and 600 nm. Unique features in each imaging modality enabled identification of the different cell types. This imaging method provides a new way of imaging stained leukocytes, with applications towards identifying and differentiating cell types, and detecting disease at the single cell level.

  10. In vivo imaging of small animal models by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Ye, Shuoqi; Yang, Ran; Xiong, Jingwei; Shung, K. Kirk; Zhou, Qifa; Li, Changhui; Ren, Qiushi

    2012-02-01

    Small animal models, such as zebrafish, drosophila, C. elegan, is considered to be important models in comparative biology and diseases researches. Traditional imaging methods primarily employ several optical microscopic imaging modalities that rely on fluorescence labeling, which may have potential to affect the natural physiological progress. Thus a label-free imaging method is desired. Photoacoustic (PA) microscopy (PAM) is an emerging biomedical imaging method that combines optical contrast with ultrasonic detection, which is highly sensitive to the optical absorption contrast of living tissues, such as pigments, the vasculature and other optically absorbing organs. In this work, we reported the whole body label-free imaging of zebrafish larvae and drosophila pupa by PAM. Based on intrinsic optical absorption contrast, high resolution images of pigments, microvasculature and several other major organs have been obtained in vivo and non-invasively, and compared with their optical counterparts. We demonstrated that PAM has the potential to be a powerful non-invasive imaging method for studying larvae and pupa of various animal models.

  11. Electron microscopy imaging of proteins on gallium phosphide semiconductor nanowires

    NASA Astrophysics Data System (ADS)

    Hjort, Martin; Bauer, Mikael; Gunnarsson, Stefan; Mårsell, Erik; Zakharov, Alexei A.; Karlsson, Gunnel; Sanfins, Elodie; Prinz, Christelle N.; Wallenberg, Reine; Cedervall, Tommy; Mikkelsen, Anders

    2016-02-01

    We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the laminin conformation on the NWs. In blood plasma, an intermediate sized corona around the NWs indicates a corona with a mixture of plasma proteins. The ability to directly visualize proteins on nanostructures in situ holds great promise for assessing the conformation and thickness of the protein corona, which is key to understanding and predicting the properties of engineered nanomaterials in a biological environment.We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the

  12. Dynamic oxygenation measurements using a phosphorescent coating within a mammary window chamber mouse model

    PubMed Central

    Schafer, Rachel; Gmitro, Arthur F.

    2015-01-01

    Phosphorescent lifetime imaging was employed to measure the spatial and temporal distribution of oxygen partial pressure in tissue under the coverslip of a mammary window chamber breast cancer mouse model. A thin platinum-porphyrin coating, whose phosphorescent lifetime varies monotonically with oxygen partial pressure, was applied to the coverslip surface. Dynamic temporal responses to induced modulations in oxygenation levels were measured using this approach. PMID:25780753

  13. Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

    NASA Astrophysics Data System (ADS)

    Dou, Ziwei; Morikawa, Sei; Wang, Shu-Wei; Smith, Charles; Watanabe, Kenji; Taniguchi, Takashi; Masubuchi, Satoru; Machida, Tomoki; Connolly, Malcolm

    Graphene layers encapsulated by hexagon boron-nitride enable charge carriers to travel ballistically over several microns and provide an opportunity to realise electron optics with Dirac fermions. Scanning gate microscopy is a valuable tool for directly imaging such effects and has recently been applied to investigate coherent scattering in graphene pnp junctions. In this work we use SGM to image magnetic focusing of ballistic carriers in a graphene device. By locally varying the carrier concentration and electrostatic potential with the tip we are able to image electrons bouncing from the graphene edges. Moreover, by refocusing misaligned electrons back to collector, our results show how scanning probe tips can be used as mobile lenses for manipulating Dirac fermions in novel device concepts Supported by EPSRC.

  14. CARS and non-linear microscopy imaging of brain tumors

    NASA Astrophysics Data System (ADS)

    Galli, Roberta; Uckermann, Ortrud; Tamosaityte, Sandra; Geiger, Kathrin; Schackert, Gabriele; Steiner, Gerald; Koch, Edmund; Kirsch, Matthias

    2013-06-01

    Nonlinear optical microscopy offers a series of techniques that have the potential to be applied in vivo, for intraoperative identification of tumor border and in situ pathology. By addressing the different content of lipids that characterize the tumors with respect to the normal brain tissue, CARS microscopy enables to discern primary and secondary brain tumors from healthy tissue. A study performed in mouse models shows that the reduction of the CARS signal is a reliable quantity to identify brain tumors, irrespective from the tumor type. Moreover it enables to identify tumor borders and infiltrations at a cellular resolution. Integration of CARS with autogenous TPEF and SHG adds morphological and compositional details about the tissue. Examples of multimodal CARS imaging of different human tumor biopsies demonstrate the ability of the technique to retrieve information useful for histopathological diagnosis.

  15. Drive frequency dependent phase imaging in piezoresponse force microscopy

    SciTech Connect

    Bo Huifeng; Kan Yi; Lu Xiaomei; Liu Yunfei; Peng Song; Wang Xiaofei; Cai Wei; Xue Ruoshi; Zhu Jinsong

    2010-08-15

    The drive frequency dependent piezoresponse (PR) phase signal in near-stoichiometric lithium niobate crystals is studied by piezoresponse force microscopy. It is clearly shown that the local and nonlocal electrostatic forces have a great contribution to the PR phase signal. The significant PR phase difference of the antiparallel domains are observed at the contact resonances, which is related to the electrostatic dominated electromechanical interactions of the cantilever and tip-sample system. Moreover, the modulation voltage induced frequency shift at higher eigenmodes could be attributed to the change of indention force depending on the modulation amplitude with a piezoelectric origin. The PR phase of the silicon wafer is also measured for comparison. It is certificated that the electrostatic interactions are universal in voltage modulated scanning probe microscopy and could be extended to other phase imaging techniques.

  16. Managing multiple image stacks from confocal laser scanning microscopy

    NASA Astrophysics Data System (ADS)

    Zerbe, Joerg; Goetze, Christian H.; Zuschratter, Werner

    1999-05-01

    A major goal in neuroanatomy is to obtain precise information about the functional organization of neuronal assemblies and their interconnections. Therefore, the analysis of histological sections frequently requires high resolution images in combination with an overview about the structure. To overcome this conflict we have previously introduced a software for the automatic acquisition of multiple image stacks (3D-MISA) in confocal laser scanning microscopy. Here, we describe a Windows NT based software for fast and easy navigation through the multiple images stacks (MIS-browser), the visualization of individual channels and layers and the selection of user defined subregions. In addition, the MIS browser provides useful tools for the visualization and evaluation of the datavolume, as for instance brightness and contrast corrections of individual layers and channels. Moreover, it includes a maximum intensity projection, panning and zoom in/out functions within selected channels or focal planes (x/y) and tracking along the z-axis. The import module accepts any tiff-format and reconstructs the original image arrangement after the user has defined the sequence of images in x/y and z and the number of channels. The implemented export module allows storage of user defined subregions (new single image stacks) for further 3D-reconstruction and evaluation.

  17. 3D Cell Culture Imaging with Digital Holographic Microscopy

    NASA Astrophysics Data System (ADS)

    Dimiduk, Thomas; Nyberg, Kendra; Almeda, Dariela; Koshelva, Ekaterina; McGorty, Ryan; Kaz, David; Gardel, Emily; Auguste, Debra; Manoharan, Vinothan

    2011-03-01

    Cells in higher organisms naturally exist in a three dimensional (3D) structure, a fact sometimes ignored by in vitro biological research. Confinement to a two dimensional culture imposes significant deviations from the native 3D state. One of the biggest obstacles to wider use of 3D cultures is the difficulty of 3D imaging. The confocal microscope, the dominant 3D imaging instrument, is expensive, bulky, and light-intensive; live cells can be observed for only a short time before they suffer photodamage. We present an alternative 3D imaging techinque, digital holographic microscopy, which can capture 3D information with axial resolution better than 2 μm in a 100 μm deep volume. Capturing a 3D image requires only a single camera exposure with a sub-millisecond laser pulse, allowing us to image cell cultures using five orders of magnitude less light energy than with confocal. This can be done with hardware costing ~ 1000. We use the instrument to image growth of MCF7 breast cancer cells and p. pastoras yeast. We acknowledge support from NSF GRFP.

  18. Biological applications of fluorescence lifetime imaging beyond microscopy

    NASA Astrophysics Data System (ADS)

    Akers, Walter J.; Berezin, Mikhail Y.; Lee, Hyeran; Guo, Kevin; Almutairi, Adah; Fréchet, Jean M. J.; Fischer, Georg M.; Daltrozzo, Ewald; Achilefu, Samuel

    2010-02-01

    Fluorescence lifetime is a relatively new contrast mechanism for optical imaging in living subjects that relies on intrinsic properties of fluorophores rather than concentration dependent intensity. Drawing upon the success of fluorescence lifetime imaging microscopy (FLIM) for investigation of protein-protein interactions and intracellular physiology, in vivo fluorescence lifetime imaging (FLI) promises to dramatically increase the utility of fluorescencebased imaging in preclinical and clinical applications. Intrinsic fluorescence lifetime measurements in living tissues can distinguish pathologies such as cancer from healthy tissue. Unfortunately, intrinsic FLT contrast is limited to superficial measurements. Conventional intensity-based agents have been reported for measuring these phenomena in vitro, but translation into living animals is difficult due to optical properties of tissues. For this reason, contrast agents that can be detected in the near infrared (NIR) wavelengths are being developed by our lab and others to enhance the capabilities of this modality. FLT is less affected by concentration and thus is better for detecting small changes in physiology, as long as sufficient fluorescence signal can be measured. FLT can also improve localization of signals for improved deep tissue imaging. Examples of the utility of exogenous contrast agents will be discussed, including applications in monitoring physiologic functions, controlled drug release and cancer biology. Instrumentation for FLI will also be discussed, including planar and diffuse optical imaging in time and frequency domains. Future applications will also be discussed that are being developed in this exciting field that complement other optical modalities.

  19. Nonlinear optical microscopy and ultrasound imaging of human cervical structure

    NASA Astrophysics Data System (ADS)

    Reusch, Lisa M.; Feltovich, Helen; Carlson, Lindsey C.; Hall, Gunnsteinn; Campagnola, Paul J.; Eliceiri, Kevin W.; Hall, Timothy J.

    2013-03-01

    The cervix softens and shortens as its collagen microstructure rearranges in preparation for birth, but premature change may lead to premature birth. The global preterm birth rate has not decreased despite decades of research, likely because cervical microstructure is poorly understood. Our group has developed a multilevel approach to evaluating the human cervix. We are developing quantitative ultrasound (QUS) techniques for noninvasive interrogation of cervical microstructure and corroborating those results with high-resolution images of microstructure from second harmonic generation imaging (SHG) microscopy. We obtain ultrasound measurements from hysterectomy specimens, prepare the tissue for SHG, and stitch together several hundred images to create a comprehensive view of large areas of cervix. The images are analyzed for collagen orientation and alignment with curvelet transform, and registered with QUS data, facilitating multiscale analysis in which the micron-scale SHG images and millimeter-scale ultrasound data interpretation inform each other. This novel combination of modalities allows comprehensive characterization of cervical microstructure in high resolution. Through a detailed comparative study, we demonstrate that SHG imaging both corroborates the quantitative ultrasound measurements and provides further insight. Ultimately, a comprehensive understanding of specific microstructural cervical change in pregnancy should lead to novel approaches to the prevention of preterm birth.

  20. Compressive fluorescence microscopy for biological and hyperspectral imaging.

    PubMed

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed Shams; Candes, Emmanuel; Dahan, Maxime

    2012-06-26

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy. PMID:22689950

  1. Template-driven segmentation of confocal microscopy images.

    PubMed

    Chen, Ying-Cheng; Chen, Yung-Chang; Chiang, Ann-Shyn

    2008-03-01

    High quality 3D visualization of anatomic structures is necessary for many applications. The anatomic structures first need to be segmented. A variety of segmentation algorithms have been developed for this purpose. For confocal microscopy images, the noise introduced during the specimen preparation process, such as the procedure of penetration or staining, may cause images to be of low contrast in some regions. This property will make segmentation difficult. Also, the segmented structures may have rugged surfaces in 3D visualization. In this paper, we present a hybrid method that is suitable for segmentation of confocal microscopy images. A rough segmentation result is obtained from the atlas-based segmentation via affine registration. The boundaries of the segmentation result are close to the object boundaries, and are regarded as the initial contours of the active contour models. After convergence of the snake algorithm, the resulting contours in regions of low contrast are locally refined by parametric bicubic surfaces to alleviate the problem of incorrect convergence. The proposed method increases the accuracy of the snake algorithm because of better initial contours. Besides, it can provide smoother segmented results in 3D visualization. PMID:18178286

  2. Nuclear uptake of ultrasmall gold-doxorubicin conjugates imaged by fluorescence lifetime imaging microscopy (FLIM) and electron microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Xuan; Shastry, Sathvik; Bradforth, Stephen E.; Nadeau, Jay L.

    2014-11-01

    Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in Dox-resistant cancers.Fluorescence lifetime imaging microscopy (FLIM) has been used to image free and encapsulated doxorubicin (Dox) uptake into cells, since interaction of Dox with DNA leads to a characteristic lifetime change. However, none of the reported Dox conjugates were able to enter cell nuclei. In this work, we use FLIM to show nuclear uptake of 2.7 nm mean diameter Au nanoparticles conjugated to Dox. The pattern of labelling differed substantially from what was seen with free Dox, with slower nuclear entry and stronger cytoplasmic labelling at all time points. As the cells died, the pattern of labelling changed further as intracellular structures disintegrated, consistent with association of Au-Dox to membranes. The patterns of Au distribution and intracellular structure changes were confirmed using electron microscopy, and indicate different mechanisms of cytotoxicity with stable Au-Dox conjugates compared to Dox alone. Such conjugates are promising tools for overcoming resistance in

  3. Orientation Imaging Microscopy: New possibilities for microstructural investigations using automated BKD analysis

    SciTech Connect

    Adams, B.L.; Kunze, K.; Dingley, D.J.; Wright, S.I.

    1993-10-01

    A new microscopy, called Orientation Imaging Microscopy, is described. Imaging results from precise measurements of local lattice orientation rapidly obtained by Backscattered Kikuchi Diffraction. The hardware configuration of the microscope is described and a description of image formation presented. Applications to several materials of differing lattice structure are described. Connections of the microscopy with various aspects of modern texture analysis are emphasized.

  4. Nonlinear optical imaging characteristics of colonic adenocarcinoma using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Nenrong; Chen, Rong; Li, Hongsheng; Chen, Jianxin

    2012-12-01

    Multiphoton microscopy (MPM), a noninvasive optical method with high resolution and high sensitivity, can obtain detailed microstructures of biotissues at submolecular level. In this study, MPM is used to image microstructure varieties of human colonic mucosa and submucosa with adenocarcinoma. Some parameters, such as gland configuration, SHG/TPEF intensity ratio, and collagen orientation and so on, should serve the indicators of early colorectal cancer. The exploratory results show that it's potential for the development of multiphoton mini-endoscopy in real-time early diagnosis of colorectal cancer.

  5. Scattering imaging method in transmission x-ray microscopy.

    PubMed

    Chen, Jian; Gao, Kun; Ge, Xin; Wang, Zhili; Zhang, Kai; Hong, Youli; Pan, Zhiyun; Wu, Zhao; Zhu, Peiping; Yun, Wenbing; Wu, Ziyu

    2013-06-15

    We present a x-ray microscopy technique based on structured illumination in a microscope that characterizes the size of the subresolution-limit features. The technique is effective for characterizing fine structures substantially beyond the Rayleigh resolution of the microscope. We carried out optical experiments to demonstrate the basic principle of this new technique. Experimental results show good agreement with theoretical predictions. This technique should find a wide range of important imaging applications with a feature size down to nanometer scale, such as oil and gas reservoir rocks, advanced composites, and functional nanodevices and materials. PMID:23938979

  6. Photonic near-field imaging in multiphoton photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Fitzgerald, J. P. S.; Word, R. C.; Saliba, S. D.; Könenkamp, R.

    2013-05-01

    We report the observation of optical near fields in a photonic waveguide of conductive indium tin oxide (ITO) using multiphoton photoemission electron microscopy (PEEM). Nonlinear two-photon photoelectron emission is enhanced at field maxima created by interference between incident 410-nm and coherently excited guided photonic waves, providing strong phase contrast. Guided modes are observed under both transverse magnetic field (TM) and transverse electric field (TE) polarized illuminations and are consistent with classical electromagnetic theory. Implications on the role of multiphoton PEEM in optical near-field imaging are discussed.

  7. Computational methods for microfluidic microscopy and phase-space imaging

    NASA Astrophysics Data System (ADS)

    Pegard, Nicolas Christian Richard

    Modern optical devices are made by assembling separate components such as lenses, objectives, and cameras. Traditionally, each part is optimized separately, even though the trade-offs typically limit the performance of the system overall. This component-based approach is particularly unfit to solve the new challenges brought by modern biology: 3D imaging, in vivo environments, and high sample throughput. In the first part of this thesis, we introduce a general method to design integrated optical systems. The laws of wave propagation, the performance of available technology, as well as other design parameters are combined as constraints into a single optimization problem. The solution provides qualitative design rules to improve optical systems as well as quantitative task-specific methods to minimize loss of information. Our results have applications in optical data storage, holography, and microscopy. The second part of this dissertation presents a direct application. We propose a more efficient design for wide-field microscopy with coherent light, based on double transmission through the sample. Historically, speckle noise and aberrations caused by undesired interferences have made coherent illumination unpopular for imaging. We were able to dramatically reduce speckle noise and unwanted interferences using optimized holographic wavefront reconstruction. The resulting microscope not only yields clear coherent images with low aberration---even in thick samples---but also increases contrast and enables optical filtering and in-depth sectioning. In the third part, we develop new imaging techniques that better respond to the needs of modern biology research through implementing optical design optimization. Using a 4D phase-space distribution, we first represent the state and propagation of incoherent light. We then introduce an additional degree of freedom by putting samples in motion in a microfluidic channel, increasing image diversity. From there, we develop a

  8. Stable blue phosphorescent organic light emitting devices

    SciTech Connect

    Forrest, Stephen R.; Thompson, Mark; Giebink, Noel

    2014-08-26

    Novel combination of materials and device architectures for organic light emitting devices is provided. An organic light emitting device, is provided, having an anode, a cathode, and an emissive layer disposed between the anode and the cathode. The emissive layer includes a host and a phosphorescent emissive dopant having a peak emissive wavelength less than 500 nm, and a radiative phosphorescent lifetime less than 1 microsecond. Preferably, the phosphorescent emissive dopant includes a ligand having a carbazole group.

  9. Robust supervised segmentation of neuropathology whole-slide microscopy images.

    PubMed

    Vandenberghe, Michel E; Balbastre, Yaël; Souedet, Nicolas; Hérard, Anne-Sophie; Dhenain, Marc; Frouin, Frédérique; Delzescaux, Thierry

    2015-08-01

    Alzheimer's disease is characterized by brain pathological aggregates such as Aβ plaques and neurofibrillary tangles which trigger neuroinflammation and participate to neuronal loss. Quantification of these pathological markers on histological sections is widely performed to study the disease and to evaluate new therapies. However, segmentation of neuropathology images presents difficulties inherent to histology (presence of debris, tissue folding, non-specific staining) as well as specific challenges (sparse staining, irregular shape of the lesions). Here, we present a supervised classification approach for the robust pixel-level classification of large neuropathology whole slide images. We propose a weighted form of Random Forest in order to fit nonlinear decision boundaries that take into account class imbalance. Both color and texture descriptors were used as predictors and model selection was performed via a leave-one-image-out cross-validation scheme. Our method showed superior results compared to the current state of the art method when applied to the segmentation of Aβ plaques and neurofibrillary tangles in a human brain sample. Furthermore, using parallel computing, our approach easily scales-up to large gigabyte-sized images. To show this, we segmented a whole brain histology dataset of a mouse model of Alzheimer's disease. This demonstrates our method relevance as a routine tool for whole slide microscopy images analysis in clinical and preclinical research settings. PMID:26737134

  10. Modeling of optical quadrature microscopy for imaging mouse embryos

    NASA Astrophysics Data System (ADS)

    Warger, William C., II; DiMarzio, Charles A.

    2008-02-01

    Optical quadrature microscopy (OQM) has been shown to provide the optical path difference through a mouse embryo, and has led to a novel method to count the total number of cells further into development than current non-toxic imaging techniques used in the clinic. The cell counting method has the potential to provide an additional quantitative viability marker for blastocyst transfer during in vitro fertilization. OQM uses a 633 nm laser within a modified Mach-Zehnder interferometer configuration to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras preceded by multiple beamsplitters record the four interferograms that are used within a reconstruction algorithm to produce an image of the complex electric field amplitude. Here we present a model for the electric field through the primary optical components in the imaging configuration and the reconstruction algorithm to calculate the signal to noise ratio when imaging mouse embryos. The model includes magnitude and phase errors in the individual reference and sample paths, fixed pattern noise, and noise within the laser and detectors. This analysis provides the foundation for determining the imaging limitations of OQM and the basis to optimize the cell counting method in order to introduce additional quantitative viability markers.

  11. Multispectral imaging fluorescence microscopy for lymphoid tissue analysis

    NASA Astrophysics Data System (ADS)

    Monici, Monica; Agati, Giovanni; Fusi, Franco; Mazzinghi, Piero; Romano, Salvatore; Pratesi, Riccardo; Alterini, Renato; Bernabei, Pietro A.; Rigacci, Luigi

    1999-01-01

    Multispectral imaging autofluorescence microscopy (MIAM) is used here for the analysis of lymphatic tissues. Lymph node biopsies, from patients with lympthoadenopathy of different origin have been examined. Natural fluorescence (NF) images of 3 micrometers sections were obtained using three filters peaked at 450, 550 and 680 nm with 50 nm bandpass. Monochrome images were combined together in a single RGB image. NF images of lymph node tissue sections show intense blue-green fluorescence of the connective stroma. Normal tissue shows follicles with faintly fluorescent lymphocytes, as expected fro the morphologic and functional characteristics of these cells. Other more fluorescent cells (e.g., plasma cells and macrophages) are evidenced. Intense green fluorescence if localized in the inner wall of the vessels. Tissues coming from patients affected by Hodgkin's lymphoma show spread fluorescence due to connective infiltration and no evidence of follicle organization. Brightly fluorescent large cells, presumably Hodgkin cells, are also observed. These results indicate that MIAM can discriminate between normal and pathological tissues on the basis of their natural fluorescence pattern, and, therefore, represent a potentially useful technique for diagnostic applications. Analysis of the fluorescence spectra of both normal and malignant lymphoid tissues resulted much less discriminatory than MIAM.

  12. Biofunctionalization of carbon nanotubes for atomic force microscopy imaging.

    PubMed

    Woolley, Adam T

    2004-01-01

    The study of biological processes relies increasingly on methods for probing structure and function of biochemical machinery (proteins, nucleic acids, and so on) with submolecular resolution. Atomic force microscopy (AFM) has recently emerged as a promising approach for imaging biological structures with resolution approaching the nanometer scale. Two important limitations of AFM in biological imaging are (1) resolution is constrained by probe tip dimensions, and (2) typical probe tips lack chemical specificity to differentiate between functional groups in biological structures. Single-walled carbon nanotubes (SWNTs) offer an intriguing possibility for providing both high resolution and chemical selectivity in AFM imaging, thus overcoming the enumerated limitations. Procedures for generating SWNT tips for AFM will be described. Carboxylic acid functional groups at the SWNT ends can be functionalized using covalent coupling chemistry to attach biological moieties via primary amine groups. Herein, the focus will be on describing methods for attaching biotin to SWNT tips and probing streptavidin on surfaces; importantly, this same coupling chemistry can also be applied to other biomolecules possessing primary amine groups. Underivatized SWNT tips can also provide high-resolution AFM images of DNA. Biofunctionalization of SWNT AFM tips offers great potential to enable high-resolution, chemically selective imaging of biological structures. PMID:15197321

  13. Multiphoton microscopy as a diagnostic imaging modality for lung cancer

    NASA Astrophysics Data System (ADS)

    Pavlova, Ina; Hume, Kelly R.; Yazinski, Stephanie A.; Peters, Rachel M.; Weiss, Robert S.; Webb, Watt W.

    2010-02-01

    Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

  14. Recent Progress in Molecular Recognition Imaging Using Atomic Force Microscopy.

    PubMed

    Senapati, Subhadip; Lindsay, Stuart

    2016-03-15

    Atomic force microscopy (AFM) is an extremely powerful tool in the field of bionanotechnology because of its ability to image single molecules and make measurements of molecular interaction forces with piconewton sensitivity. It works in aqueous media, enabling studies of molecular phenomenon taking place under physiological conditions. Samples can be imaged in their near-native state without any further modifications such as staining or tagging. The combination of AFM imaging with the force measurement added a new feature to the AFM technique, that is, molecular recognition imaging. Molecular recognition imaging enables mapping of specific interactions between two molecules (one attached to the AFM tip and the other to the imaging substrate) by generating simultaneous topography and recognition images (TREC). Since its discovery, the recognition imaging technique has been successfully applied to different systems such as antibody-protein, aptamer-protein, peptide-protein, chromatin, antigen-antibody, cells, and so forth. Because the technique is based on specific binding between the ligand and receptor, it has the ability to detect a particular protein in a mixture of proteins or monitor a biological phenomenon in the native physiological state. One key step for recognition imaging technique is the functionalization of the AFM tips (generally, silicon, silicon nitrides, gold, etc.). Several different functionalization methods have been reported in the literature depending on the molecules of interest and the material of the tip. Polyethylene glycol is routinely used to provide flexibility needed for proper binding as a part of the linker that carries the affinity molecule. Recently, a heterofunctional triarm linker has been synthesized and successfully attached with two different affinity molecules. This novel linker, when attached to AFM tip, helped to detect two different proteins simultaneously from a mixture of proteins using a so-called "two

  15. Lanthanide-based laser-induced phosphorescence for spray diagnostics

    NASA Astrophysics Data System (ADS)

    van der Voort, D. D.; Maes, N. C. J.; Lamberts, T.; Sweep, A. M.; van de Water, W.; Kunnen, R. P. J.; Clercx, H. J. H.; van Heijst, G. J. F.; Dam, N. J.

    2016-03-01

    Laser-induced phosphorescence (LIP) is a relatively recent and versatile development for studying flow dynamics. This work investigates certain lanthanide-based molecular complexes for their use in LIP for high-speed sprays. Lanthanide complexes in solutions have been shown to possess long phosphorescence lifetimes (˜1-2 ms) and to emit light in the visible wavelength range. In particular, europium and terbium complexes are investigated using fluorescence/phosphorescence spectrometry, showing that europium-thenoyltrifluoracetone-trioctylphosphineoxide (Eu-TTA-TOPO) can be easily and efficiently excited using a standard frequency-tripled Nd:YAG laser. The emitted spectrum, with maximum intensity at a wavelength of 614 nm, is shown not to vary strongly with temperature (293-383 K). The decay constant of the phosphorescence, while independent of ambient pressure, decreases by approximately 12 μs/K between 323 and 373 K, with the base level of the decay constant dependent on the used solvent. The complex does not luminesce in the gas or solid state, meaning only the liquid phase is visualized, even in an evaporating spray. By using an internally excited spray containing the phosphorescent complex, the effect of vaporization is shown through the decrease in measured intensity over the length of the spray, together with droplet size measurements using interferometric particle imaging. This study shows that LIP, using the Eu-TTA-TOPO complex, can be used with different solvents, including diesel surrogates. Furthermore, it can be easily handled and used in sprays to investigate spray breakup and evaporation.

  16. Lanthanide-based laser-induced phosphorescence for spray diagnostics.

    PubMed

    van der Voort, D D; Maes, N C J; Lamberts, T; Sweep, A M; van de Water, W; Kunnen, R P J; Clercx, H J H; van Heijst, G J F; Dam, N J

    2016-03-01

    Laser-induced phosphorescence (LIP) is a relatively recent and versatile development for studying flow dynamics. This work investigates certain lanthanide-based molecular complexes for their use in LIP for high-speed sprays. Lanthanide complexes in solutions have been shown to possess long phosphorescence lifetimes (∼1-2 ms) and to emit light in the visible wavelength range. In particular, europium and terbium complexes are investigated using fluorescence/phosphorescence spectrometry, showing that europium-thenoyltrifluoracetone-trioctylphosphineoxide (Eu-TTA-TOPO) can be easily and efficiently excited using a standard frequency-tripled Nd:YAG laser. The emitted spectrum, with maximum intensity at a wavelength of 614 nm, is shown not to vary strongly with temperature (293-383 K). The decay constant of the phosphorescence, while independent of ambient pressure, decreases by approximately 12 μs/K between 323 and 373 K, with the base level of the decay constant dependent on the used solvent. The complex does not luminesce in the gas or solid state, meaning only the liquid phase is visualized, even in an evaporating spray. By using an internally excited spray containing the phosphorescent complex, the effect of vaporization is shown through the decrease in measured intensity over the length of the spray, together with droplet size measurements using interferometric particle imaging. This study shows that LIP, using the Eu-TTA-TOPO complex, can be used with different solvents, including diesel surrogates. Furthermore, it can be easily handled and used in sprays to investigate spray breakup and evaporation. PMID:27036779

  17. Watershed Merge Tree Classification for Electron Microscopy Image Segmentation

    SciTech Connect

    Liu, TIng; Jurrus, Elizabeth R.; Seyedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2012-11-11

    Automated segmentation of electron microscopy (EM) images is a challenging problem. In this paper, we present a novel method that utilizes a hierarchical structure and boundary classification for 2D neuron segmentation. With a membrane detection probability map, a watershed merge tree is built for the representation of hierarchical region merging from the watershed algorithm. A boundary classifier is learned with non-local image features to predict each potential merge in the tree, upon which merge decisions are made with consistency constraints in the sense of optimization to acquire the final segmentation. Independent of classifiers and decision strategies, our approach proposes a general framework for efficient hierarchical segmentation with statistical learning. We demonstrate that our method leads to a substantial improvement in segmentation accuracy.

  18. Imaging nonlocal transport in graphene using scanning gate microscopy

    NASA Astrophysics Data System (ADS)

    Connolly, Malcolm; Dou, Ziwei; Morikawa, Sei; Wang, Shu-Wei; Smith, Charles; Watanabe, Kenji; Taniguchi, Takashi; Masubuchi, Satoru; Machida, Tomoki

    Nonlocal transport measurements are designed to detect when charge injected by a current probe induces voltages far from the classical current path. While a range of exotic forces can induce nonlocal transport of Dirac fermions in graphene such as bandstructure topology, Zeeman spin Hall, and many-body interactions, it is important to understand the role of density fluctuations around the Dirac point where nonlocality can be most pronounced. We use scanning gate microscopy to image current flow and nonlocal signals directly in high-mobility graphene encapsulated by hexagonal boron nitride. Despite being located several mean-free paths from the current injector, Hall voltage probes parallel with current path display an order of magnitude larger nonlocal signal than expected around the Dirac point. SGM images captured at different carrier density are consistent with current spreading due to percolation. Such long range charge transport should be considered when designing devices and calculating the relaxation length of nonlocal currents. Supported by EPSRC.

  19. Atomic force microscopy images of lyotropic lamellar phases.

    PubMed

    Garza, C; Thieghi, L T; Castillo, R

    2007-02-01

    For the very first time, atomic force microscope images of lamellar phases were observed combined with a freeze fracture technique that does not involve the use of replicas. Samples are rapidly frozen, fractured, and scanned directly with atomic force microscopy, at liquid nitrogen temperature and in high vacuum. This procedure can be used to investigate micro-structured liquids. The lamellar phases in Sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/water and in C12E5/water systems were used to asses this new technique. Our observations were compared with x-ray diffraction measurements and with other freeze fracture methods reported in the literature. Our results show that this technique is useful to image lyotropic lamellar phases and the estimated repeat distances for lamellar periodicity are consistent with those obtained by x-ray diffraction. PMID:17302467

  20. Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

    NASA Astrophysics Data System (ADS)

    Morikawa, Sei; Dou, Ziwei; Wang, Shu-Wei; Smith, Charles G.; Watanabe, Kenji; Taniguchi, Takashi; Masubuchi, Satoru; Machida, Tomoki; Connolly, Malcolm R.

    2015-12-01

    We use scanning gate microscopy to map out the trajectories of ballistic carriers in high-mobility graphene encapsulated by hexagonal boron nitride and subject to a weak magnetic field. We employ a magnetic focusing geometry to image carriers that emerge ballistically from an injector, follow a cyclotron path due to the Lorentz force from an applied magnetic field, and land on an adjacent collector probe. The local electric field generated by the scanning tip in the vicinity of the carriers deflects their trajectories, modifying the proportion of carriers focused into the collector. By measuring the voltage at the collector while scanning the tip, we are able to obtain images with arcs that are consistent with the expected cyclotron motion. We also demonstrate that the tip can be used to redirect misaligned carriers back to the collector.

  1. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    NASA Astrophysics Data System (ADS)

    Kuo, Yung; Hsu, Tsung-Yuan; Wu, Yi-Chun; Hsu, Jui-Hung; Chang, Huan-Cheng

    2013-03-01

    The negatively charged nitrogen-vacancy (NV-) center in bulk diamond is a photostable fluorophore with a radiative lifetime of 11.6 ns at room temperature. The lifetime substantially increases to ~20 ns for diamond nanoparticles (size ~ 100 nm) suspended in water due to the change in refractive index of the surrounding medium of the NV- centers. This fluorescence decay time is much longer than that (typically 1 - 4 ns) of endogenous and exogenous fluorophores commonly used in biological imaging, making it possible to detect NV--containing nanodiamonds in vivo at the single particle level by fluorescence lifetime imaging microscopy (FLIM). We demonstrate the feasibility of this approach using Caenorhabditis elegans (C. elegans) as a model organism.

  2. Atomic force microscopy images of lyotropic lamellar phases

    NASA Astrophysics Data System (ADS)

    Garza, C.; Thieghi, L. T.; Castillo, R.

    2007-02-01

    For the very first time, atomic force microscope images of lamellar phases were observed combined with a freeze fracture technique that does not involve the use of replicas. Samples are rapidly frozen, fractured, and scanned directly with atomic force microscopy, at liquid nitrogen temperature and in high vacuum. This procedure can be used to investigate micro-structured liquids. The lamellar phases in Sodium bis(2-ethylhexyl) sulfosuccinate (AOT)/water and in C12E5/water systems were used to asses this new technique. Our observations were compared with x-ray diffraction measurements and with other freeze fracture methods reported in the literature. Our results show that this technique is useful to image lyotropic lamellar phases and the estimated repeat distances for lamellar periodicity are consistent with those obtained by x-ray diffraction.

  3. Microscopy and chemical imaging of Behcet brain tissue

    NASA Astrophysics Data System (ADS)

    Aranyosiova, Monika; Michalka, Miroslav; Kopani, Martin; Rychly, Boris; Jakubovsky, Jan; Velic, Dusan

    2008-12-01

    Chemical composition and distribution of molecules and elements in a human brain tissue of Behcet diseased patient are of interest. Behcet disease is a multi-system disorder of which pathogenesis and chemical causality are still uncertain. Time-of-flight secondary ion mass spectrometry is used along with scanning electron microscopy and energy dispersive X-ray analysis providing complex composition in Behcet disease and control tissues. Determined organic compounds are represented by fragments of carbohydrates, phospholipids, amino acids, and peptides. The distributions of inorganic species are well represented by heavy trace elements and by oxides in positive and negative polarities of time-of-flight secondary ion mass spectrometry, respectively. Organic and inorganic compounds are qualitatively determined in both samples, Behcet and control, providing complementary chemical images. The complementary chemical images interestingly change with the quantitative regression of organic compounds distribution, characteristic for the healthy control, towards inorganic compounds distribution, characteristic for Behcet tissue.

  4. Imaging ballistic carrier trajectories in graphene using scanning gate microscopy

    SciTech Connect

    Morikawa, Sei; Masubuchi, Satoru; Dou, Ziwei; Wang, Shu-Wei; Smith, Charles G.; Connolly, Malcolm R.; Watanabe, Kenji; Taniguchi, Takashi; Machida, Tomoki

    2015-12-14

    We use scanning gate microscopy to map out the trajectories of ballistic carriers in high-mobility graphene encapsulated by hexagonal boron nitride and subject to a weak magnetic field. We employ a magnetic focusing geometry to image carriers that emerge ballistically from an injector, follow a cyclotron path due to the Lorentz force from an applied magnetic field, and land on an adjacent collector probe. The local electric field generated by the scanning tip in the vicinity of the carriers deflects their trajectories, modifying the proportion of carriers focused into the collector. By measuring the voltage at the collector while scanning the tip, we are able to obtain images with arcs that are consistent with the expected cyclotron motion. We also demonstrate that the tip can be used to redirect misaligned carriers back to the collector.

  5. FT-IR microscopy imaging on oral cavity tumours, II

    NASA Astrophysics Data System (ADS)

    Conti, C.; Giorgini, E.; Pieramici, T.; Rubini, C.; Tosi, G.

    2005-06-01

    Changes in the biochemistry of oral cavity tissues have been studied by FT-IR microscopy. Various aspects of squamous cell carcinomas of cheek mucosa, of tongue, of gingiva, and of the floor of the mouth have been analyzed through FT-IR imaging with the aim to relate spectral patterns with histopathological results. In particular, changes in frequency and intensity of proteins, connective and nucleic acids vibrational modes as well as the visualization of biochemical single wavenumber or band ratio images allowed a quali- and quantitative evaluation of the changes in the proliferating activity from displastic to neoplastic states. 'Supervised' and 'unsupervised' procedures of data handling afforded a satisfactory degree of accordance between spectroscopic and histological findings.

  6. Fast Neuronal Imaging using Objective Coupled Planar Illumination Microscopy

    NASA Astrophysics Data System (ADS)

    Tarantino, Walter

    Complex computations performed by the brain are produced by activities of neuronal populations. There is a large diversity in the functions of each individual neuron, and neuronal activities occur in the time scale of milliseconds. In order to gain a fundamental understanding of the neuronal populations, one has to measure activity of each neuron at high temporal resolution, while investigating enough neurons to encapsulate the neuronal diversity. Traditional neurotechniques such as electrophysiology and optical imaging are constrained by the number of neurons whose activities can be simultaneously measured or the speed of measuring such activities. We have developed a novel light-sheet based technique called Objective Coupled Planar Illumination (OCPI) microscopy which is capable of measuring simultaneous activities of thousands of neurons at high speeds. In this thesis I pursue the following two aims: · Improve OCPI microscopy by enhancing the spatial resolution deeper in tissue. Tissue inhomogeneity and refractive index mismatch at the surface of the tissue lead to optical aberrations. We have compensated for such aberrations by (1) miniaturizing the OCPI illumination optics, so as to enable more vertical imaging of the tissue, (2) correcting for the angular defocus caused by the refraction at the immersion fluid/tissue interface, and (3) applying adaptive optics to correct for higher order optical aberrations. The improvement in the depth at which one can image tissue will enable the measurement of activities of neuronal populations in cortical areas. · Measure the diversity in the expression pattern of VSNs responsive to sulfated steroids. Nodari et al. have identified sulfated steroids as a novel family of ligands which activate vomeronasal sensory neurons (VSNs). Due to the experimental constraints, it has not been possible to obtain a comprehensive understanding of the number, location and functional characteristics of the sulfated steroid responsive VSNs

  7. Cell imaging by transient fluorescence detected infrared microscopy

    NASA Astrophysics Data System (ADS)

    Ohmori, Tsutomu; Sakai, Makoto; Ishihara, Miya; Kikuchi, Makoto; Fujii, Masaaki

    2008-02-01

    Transient fluorescence detected infrared (TFD-IR) microscopy was developed to overcome the diffraction limit of infrared (IR) light without a near-field system. This microscopic technique is based on TFD-IR spectroscopy, which converts information on IR absorption to fluorescence intensity by further electronic excitation of vibrationally excited molecules by a probing UV/visible light. Roots of Arabidopsis thaliana and living A549 cells with fluorescent dyes were chosen as samples. In the measurements using the TFD-IR microscope, tunable IR picosecond laser pulses were used in the wavelength range from 2700 to 3700 nm, corresponding to CH, NH, and OH stretching modes. Fluorescence images of the root cells of A. thaliana by the TFD-IR scheme were obtained with super-resolution compared with the resolution of conventional IR microscopy. The resolution is estimated to be less than 2.6 μm by fitting of a gaussian function. However, the TFD-IR images were dominated mainly by the fluorescent dyes because they were almost the same as a conventional fluorescence image. To investigate other contributions hidden by that of fluorescent dyes, we plotted the fluorescence intensity in several 5 μm squares at various IR wavelengths, called a TFD-IR spectrum. For root cells of A. thaliana, the TFD-IR spectra show shapes similar to those of a conventional IR absorption spectrum of the fluorescent dye. Therefore, the TFD-IR images are not due to the cellular components. For an A549 cell, the TFD-IR spectra were different from a conventional IR absorption spectrum of fluorescent dyes in the wavelength region shorter than 3100 nm. We speculate that the spectral difference is due to the cellular components, possibly assigned to the combination band related to amino groups of cellular components bonded covalently to the fluorescent dyes.

  8. Photon budget analysis for fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Zhao, Qiaole; Young, Ian T.; de Jong, Jan Geert Sander

    2011-08-01

    We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon ``budget.'' These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

  9. Coupling EELS/EFTEM Imaging with Environmental Fluid Cell Microscopy

    SciTech Connect

    Unocic, Raymond R; Baggetto, Loic; Veith, Gabriel M; Dudney, Nancy J; More, Karren Leslie

    2012-01-01

    Insight into dynamically evolving electrochemical reactions and mechanisms encountered in electrical energy storage (EES) and conversion technologies (batteries, fuel cells, and supercapacitors), materials science (corrosion and oxidation), and materials synthesis (electrodeposition) remains limited due to the present lack of in situ high-resolution characterization methodologies. Electrochemical fluid cell microscopy is an emerging in-situ method that allows for the direct, real-time imaging of electrochemical processes within a fluid environment. This technique is facilitated by the use of MEMS-based biasing microchip platforms that serve the purpose of sealing the highly volatile electrolyte between two electron transparent SiNx membranes and interfacing electrodes to an external potentiostat for controlled nanoscale electrochemislly experiments [!]. In order to elucidate both stmctural and chemical changes during such in situ electrochemical experiments, it is impmtant to first improve upon the spatial resolution by utilizing energy-filtered transmission electron microscopy (EFTEM) (to minimize chromatic aben ation), then to detennine the chemical changes via electron energy loss spectroscopy (EELS). This presents a formidable challenge since the overall thickness through which electrons are scattered through the multiple layers of the cell can be on the order of hundreds of nanometers to microns, scattering through which has the deleterious effect of degrading image resolution and decreasing signal-to noise for spectroscopy [2].

  10. IMAGING RED BLOOD CELL DYNAMICS BY QUANTITATIVE PHASE MICROSCOPY

    PubMed Central

    Popescu, Gabriel; Park, YoungKeun; Choi, Wonshik; Dasari, Ramachandra R.; Feld, Michael S.; Badizadegan, Kamran

    2008-01-01

    Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease. PMID:18387320

  11. Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell

    PubMed Central

    Kikuchi, Hiroyuki; Prasad, Ankush; Matsuoka, Ryo; Aoyagi, Shigeo; Matsue, Tomokazu; Kasai, Shigenobu

    2016-01-01

    Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials. PMID:26903876

  12. Functional transcranial brain imaging by optical-resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Hu, Song; Maslov, Konstantin; Tsytsarev, Vassiliy; Wang, Lihong V.

    2009-07-01

    Optical-resolution photoacoustic microscopy (OR-PAM) is applied to functional brain imaging in living mice. A near-diffraction-limited bright-field optical illumination is employed to achieve micrometer lateral resolution, and a dual-wavelength measurement is utilized to extract the blood oxygenation information. The variation in hemoglobin oxygen saturation (sO2) along vascular branching has been imaged in a precapillary arteriolar tree and a postcapillary venular tree, respectively. To the best of our knowledge, this is the first report on in vivo volumetric imaging of brain microvascular morphology and oxygenation down to single capillaries through intact mouse skulls. It is anticipated that: (i) chronic imaging enabled by this minimally invasive procedure will advance the study of cortical plasticity and neurological diseases; (ii) revealing the neuroactivity-dependent changes in hemoglobin concentration and oxygenation will facilitate the understanding of neurovascular coupling at the capillary level; and (iii) combining functional OR-PAM and high-resolution blood flowmetry will have the potential to explore cellular pathways of brain energy metabolism.

  13. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  14. Interpretation of Scanning Tunneling Microscopy Images of Graphite.

    NASA Astrophysics Data System (ADS)

    Mizes, Howard Albert

    This dissertation analyzes scanning tunneling microscopy (STM) images of graphite. Graphite is an important substrate for molecular imaging and nanometer lithography. Because it is a layered structure with a simple unit cell, its electronic structure can be described simply using tight binding theory. More interestingly, the charge density at the Fermi level, which is the quantity that the STM probes, is well approximated by the six leading Fourier components. The effect of multiple atomic tips on the STM images of graphite can be predicted using the three sine wave description. Both the varying asymmetry between the two inequivalent atoms, and the loss of three fold symmetry observed in experimental images, can be attributed to differing tip configurations. The existence of multiple atomic tips is directly confirmed by the observation of moire patterns occurring near grain boundaries. Physisorbed and intercalated atoms and molecules will produce weak perturbations in the electronic structure of the graphite. These perturbations can be measured with the STM as localized changes in the tunneling current, and appear as bright areas in STM gray-scale images. The dependence of the brightness with scanning height is calculated and can be used as a measure to help identify the atom or molecule. Chemisorbed atoms and molecules will produce a stronger long-range perturbation in the electronic structure of the graphite. It is shown that any strong perturbation should give rise to oscillations in the Fermi level charge density with a wavelength surd{3} times that of the graphite lattice. The intensity of the oscillations and their symmetry about the defect is shown to be a probe of the geometry of the bonding to the surface. Predicted images that arise from multiple tips, along with those arising from physisorbed and chemisorbed atoms, are compared with available experiments.

  15. Post-processing strategies in image scanning microscopy.

    PubMed

    McGregor, J E; Mitchell, C A; Hartell, N A

    2015-10-15

    Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view. PMID:25962644

  16. Imaging chromophores with undetectable fluorescence by stimulated emission microscopy.

    PubMed

    Min, Wei; Lu, Sijia; Chong, Shasha; Roy, Rahul; Holtom, Gary R; Xie, X Sunney

    2009-10-22

    Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging. PMID:19847261

  17. Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

    PubMed Central

    Warren, Sean C.; Margineanu, Anca; Alibhai, Dominic; Kelly, Douglas J.; Talbot, Clifford; Alexandrov, Yuriy; Munro, Ian; Katan, Matilda

    2013-01-01

    Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell

  18. Quantitative biological imaging by ptychographic x-ray diffraction microscopy

    PubMed Central

    Giewekemeyer, Klaus; Thibault, Pierre; Kalbfleisch, Sebastian; Beerlink, André; Kewish, Cameron M.; Dierolf, Martin; Pfeiffer, Franz; Salditt, Tim

    2010-01-01

    Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·105 Gy, and were shown to scale with photon fluence. PMID:20018650

  19. Imaging metal oxide nanoparticles in biological structures with CARS microscopy.

    PubMed

    Moger, Julian; Johnston, Blair D; Tyler, Charles R

    2008-03-01

    Metal oxide nanomaterials are being used for an increasing number of commercial applications, such as fillers, opacifiers, catalysts, semiconductors, cosmetics, microelectronics, and as drug delivery vehicles. The effects of these nanoparticles on the physiology of animals and in the environment are largely unknown and their potential associated health risks are currently a topic of hot debate. Information regarding the entry route of nanoparticles into exposed organisms and their subsequent localization within tissues and cells in the body are essential for understanding their biological impact. However, there is currently no imaging modality available that can simultaneously image these nanoparticles and the surrounding tissues without disturbing the biological structure. Due to their large nonlinear optical susceptibilities, which are enhanced by two-photon electronic resonance, metal oxides are efficient sources of coherent anti-Stokes Raman Scattering (CARS). We show that CARS microscopy can provide localization of metal oxide nanoparticles within biological structures at the cellular level. Nanoparticles of 20 - 70 nm in size were imaged within the fish gill; a structure that is a primary site of pollutant uptake into fish from the aquatic environment. PMID:18542432

  20. Atomic-Scale Imaging and Spectroscopy Using Scanning Tunneling Microscopy.

    NASA Astrophysics Data System (ADS)

    Youngquist, Michael George

    Advances in scanning tunneling microscopy (STM) instrumentation and applications are presented. An ultrahigh vacuum (UHV) scanning tunneling microscope incorporating computer-controlled two-dimensional sample translation and in vacuo tip and sample transfer was developed. Its performance is documented through large-area and atomic -resolution imaging of highly stepped Si(111) 7 x 7 reconstructed surfaces and physisorbed clusters on graphite. An STM with automated approach and intra-Dewar spring suspension was developed for operation in cryogenic liquids. A high performance digital signal processor (DSP) based control system was constructed, and software with advanced spectroscopic imaging and data processing capabilities was developed. The feasibility of individual-molecule vibrational spectroscopy via STM-detected inelastic electron tunneling is assessed. In preliminary experiments, a low-temperature STM was used for energy gap and phonon spectroscopy of superconducting Pb films. The first STM observation of phonon density of states effects in a superconductor is reported. A systematic UHV STM imaging and spectroscopy study of 2H-MoS_2 was conducted. Atom -resolved images from three distinct imaging modes are presented. Occasional appearance of negative differential resistance (NDR) in I vs. V measurements is traced to changing tip electronic structure rather than localized surface states. Other potential NDR mechanisms are discussed including electron trap charging and resonant tunneling through a double-barrier quantum well structure arising from layer separation in the MoS_2 crystal. DNA was imaged at atomic resolution with a UHV STM. Images show double-helical structure, base pairs, and atomic-scale substructure. Experimental STM profiles have atom-for-atom correlation with the A-DNA van der Waals surface. This work demonstrates the potential of the STM for characterization of large biomolecular structures. Impurity-pinned steps on silicon and gold surfaces

  1. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    PubMed

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives. PMID:25265458

  2. Imaging doped silicon test structures using low energy electron microscopy.

    SciTech Connect

    Nakakura, Craig Yoshimi; Anderson, Meredith Lynn; Kellogg, Gary Lee

    2010-01-01

    This document is the final SAND Report for the LDRD Project 105877 - 'Novel Diagnostic for Advanced Measurements of Semiconductor Devices Exposed to Adverse Environments' - funded through the Nanoscience to Microsystems investment area. Along with the continuous decrease in the feature size of semiconductor device structures comes a growing need for inspection tools with high spatial resolution and high sample throughput. Ideally, such tools should be able to characterize both the surface morphology and local conductivity associated with the structures. The imaging capabilities and wide availability of scanning electron microscopes (SEMs) make them an obvious choice for imaging device structures. Dopant contrast from pn junctions using secondary electrons in the SEM was first reported in 1967 and more recently starting in the mid-1990s. However, the serial acquisition process associated with scanning techniques places limits on the sample throughput. Significantly improved throughput is possible with the use of a parallel imaging scheme such as that found in photoelectron emission microscopy (PEEM) and low energy electron microscopy (LEEM). The application of PEEM and LEEM to device structures relies on contrast mechanisms that distinguish differences in dopant type and concentration. Interestingly, one of the first applications of PEEM was a study of the doping of semiconductors, which showed that the PEEM contrast was very sensitive to the doping level and that dopant concentrations as low as 10{sup 16} cm{sup -3} could be detected. More recent PEEM investigations of Schottky contacts were reported in the late 1990s by Giesen et al., followed by a series of papers in the early 2000s addressing doping contrast in PEEM by Ballarotto and co-workers and Frank and co-workers. In contrast to PEEM, comparatively little has been done to identify contrast mechanisms and assess the capabilities of LEEM for imaging semiconductor device strictures. The one exception is the

  3. Magnetic resonance microscopy of prostate tissue: How basic science can inform clinical imaging development

    SciTech Connect

    Bourne, Roger

    2013-03-15

    This commentary outlines how magnetic resonance imaging (MRI) microscopy studies of prostate tissue samples and whole organs have shed light on a number of clinical imaging mysteries and may enable more effective development of new clinical imaging methods.

  4. Phosphorescent Nanocluster Light-Emitting Diodes.

    PubMed

    Kuttipillai, Padmanaban S; Zhao, Yimu; Traverse, Christopher J; Staples, Richard J; Levine, Benjamin G; Lunt, Richard R

    2016-01-13

    Devices utilizing an entirely new class of earth abundant, inexpensive phosphorescent emitters based on metal-halide nanoclusters are reported. Light-emitting diodes with tunable performance are demonstrated by varying cation substitution to these nanoclusters. Theoretical calculations provide insight about the nature of the phosphorescent emitting states, which involves a strong pseudo-Jahn-Teller distortion. PMID:26568044

  5. Dark-field microscopy in imaging of plasmon resonant nanoparticles.

    PubMed

    Liu, Mengmeng; Chao, Jie; Deng, Suhui; Wang, Kun; Li, Kun; Fan, Chunhai

    2014-12-01

    Dark-field microscopy (DFM) and spectroscopy base on localized surface plasmon resonance (LSPR) have been widely applied in biological sensing and single-molecule imaging. Using plasmonic nanoparticles with controlled geometrical, optical, and surface chemical properties as the probes, the scattering light depending on the surrounding environment can be detected by DF microscope. Signal-to-noise radio and time resolution of the conventional DFM is not sufficient to identify single molecular dynamics. To break these limitations, significant improvements have been made in recent years. This critical review is focused on the developments of the DFM and the utilization of DFM as a powerful technology in the application of LSPR detection. PMID:25009105

  6. Fluorescence Lifetime Imaging Microscopy of Intracellular Glucose Dynamics

    PubMed Central

    Veetil, Jithesh V.; Jin, Sha; Ye, Kaiming

    2012-01-01

    Background One of the major hurdles in studying diabetes pathophysiology is the lack of adequate methodology that allows for direct and real-time determination of glucose transport and metabolism in cells and tissues. In this article, we present a new methodology that adopts frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) to visualize and quantify the dynamics of intracellular glucose within living cells using a biosensor protein based on fluorescence resonance energy transfer (FRET). Method The biosensor protein was developed by fusing a FRET pair, an AcGFP1 donor and a mCherry acceptor to N- and C- termini of a mutant glucose-binding protein (GBP), respectively. The probe was expressed and biosynthesized inside the cells, offering continuous monitoring of glucose dynamics in real time through fluorescence lifetime imaging microscopy (FLIM) measurement. Results We transfected the deoxyribonucleic acid of the AcGFP1-GBP-mCherry sensor into murine myoblast cells, C2C12, and continuously monitored the changes in intracellular glucose concentrations in response to the variation in extracellular glucose, from which we determined glucose uptake and clearance rates. The distribution of intracellular glucose concentration was also characterized. We detected a high glucose concentration in a region close to the cell membrane and a low glucose concentration in a region close to the nucleus. The monoexponential decay of AcGFP1 was distinguished using FD-FLIM. Conclusions This work enables continuous glucose monitoring (CGM) within living cells using FD-FLIM and a biosensor protein. The sensor protein developed offers a new means for quantitatively analyzing glucose homeostasis at the cellular level. Data accumulated from these studies will help increase our understanding of the pathology of diabetes. PMID:23294772

  7. Second harmonic generation imaging microscopy of cellular structure and function

    NASA Astrophysics Data System (ADS)

    Millard, Andrew C.; Jin, Lei; Loew, Leslie M.

    2005-03-01

    Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, with many advantages over existing one- or two-photon fluorescence techniques. A non-linear phenomenon employing mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry. Many important structural proteins such as collagen and cellulose show intrinsic SHG, thus providing access to sub-resolution information on symmetry. However, we are particularly interested here in "resonance-enhanced" SHG from styryl dyes. In general SHG is a combination of a true second-order process and a third-order process dependent on a static electric field, such that SHG from membrane-bound dyes depends on a cell's trans-membrane potential. With simultaneous patch-clamping and non-linear imaging of cells, we have found that SHG is a sensitive probe of trans-membrane potential with sensitivities that are up to four times better than those obtained under optimal conditions using one-photon fluorescence imaging. With the sensitivity of SHG to local electric fields from other sources such as the membrane dipole potential as well as the quadratic dependence of SHG on concentration, we have found that SHG imaging of styryl dyes is also a powerful technique for the investigation of lipid phases and rafts and for the visualization of the dynamics of membrane-vesicle fusion following fertilization of an ovum.

  8. An advanced image analysis tool for the quantification and characterization of breast cancer in microscopy images.

    PubMed

    Goudas, Theodosios; Maglogiannis, Ilias

    2015-03-01

    The paper presents an advanced image analysis tool for the accurate and fast characterization and quantification of cancer and apoptotic cells in microscopy images. The proposed tool utilizes adaptive thresholding and a Support Vector Machines classifier. The segmentation results are enhanced through a Majority Voting and a Watershed technique, while an object labeling algorithm has been developed for the fast and accurate validation of the recognized cells. Expert pathologists evaluated the tool and the reported results are satisfying and reproducible. PMID:25681102

  9. Circularly polarised phosphorescent photoluminescence and electroluminescence of iridium complexes

    PubMed Central

    Li, Tian-Yi; Jing, Yi-Ming; Liu, Xuan; Zhao, Yue; Shi, Lin; Tang, Zhiyong; Zheng, You-Xuan; Zuo, Jing-Lin

    2015-01-01

    Nearly all the neutral iridium complexes widely used as dopants in PhOLEDs are racemic mixtures; however, this study observed that these complexes can be separated into stable optically active Λ and ∆ isomers and that their chirality is an intrinsic property. The circularly polarised phosphorescent photoluminescence (CPPPL) signals of Λ/Δ isomers are perfect mirror images with opposite polarisation and equal intensity exhibiting a “handedness” for the polarisation. For the first time, we applied the Λ/Δ iridium isomers as emitters in OLEDs, and the circularly polarised phosphorescent electroluminescence (CPPEL) spectra reveal completely positive or negative broad peaks consistent with the CPPPL spectra. The results demonstrate that the Λ/Δ isomers have potential application for 3D OLEDs because they can exhibit high efficiency and luminance, and 3D display technology based on circularly polarised light is the most comfortable for the eyes. PMID:26446521

  10. 3D image reconstruction algorithms for cryo-electron-microscopy images of virus particles

    NASA Astrophysics Data System (ADS)

    Doerschuk, Peter C.; Johnson, John E.

    2000-11-01

    A statistical model for the object and the complete image formation process in cryo electron microscopy of viruses is presented. Using this model, maximum likelihood reconstructions of the 3D structure of viruses are computed using the expectation maximization algorithm and an example based on Cowpea mosaic virus is provided.

  11. Imaging leukocytes in vivo with third harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Tsai, Cheng-Kun; Chen, Chien-Kuo; Chen, Yu-Shing; Wu, Pei-Chun; Hsieh, Tsung-Yuan; Liu, Han-Wen; Yeh, Chiou-Yueh; Lin, Win-Li; Chia, Jean-San; Liu, Tzu-Ming

    2013-02-01

    Without a labeling, we demonstrated that lipid granules in leukocytes have distinctive third harmonic generation (THG) contrast. Excited by a 1230nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order smaller. These characteristic THG features can also be observed in vivo to trace the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. Furthermore, using video-rate THG microscopy, we also captured images of blood cells in human capillaries. Quite different from red-blood-cells, every now and then, round and granule rich blood cells with strong THG contrast appeared in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts. These results suggested that labeling-free THG imaging may provide timely tracing of leukocyte movement and hematology inspection without disturbing the normal cellular or physiological status.

  12. Analysis of virus textures in transmission electron microscopy images.

    PubMed

    Nanni, Loris; Paci, Michelangelo; Caetano Dos Santos, Florentino Luciano; Brahnam, Sheryl; Hyttinen, Jari

    2014-01-01

    In this paper we propose an ensemble of texture descriptors for analyzing virus textures in transmission electron microscopy images. Specifically, we present several novel multi-quinary (MQ) codings of local binary pattern (LBP) variants: the MQ version of the dense LBP, the MQ version of the rotation invariant co-occurrence among adjacent LBPs, and the MQ version of the LBP histogram Fourier. To reduce computation time as well as to improve performance, a feature selection approach is utilized to select the thresholds used in the MQ approaches. In addition, we propose new variants of descriptors where two histograms, instead of the standard one histogram, are produced for each descriptor. The two histograms (one for edge pixels and the other for non-edge pixels) are calculated for training two different SVMs, whose results are then combined by sum rule. We show that a bag of features approach works well with this problem. Our experiments, using a publicly available dataset of 1500 images with 15 classes and same protocol as in previous works, demonstrate the superiority of our new proposed ensemble of texture descriptors. The MATLAB code of our approach is available at https://www.dei.unipd.it/node/2357. PMID:25488214

  13. Rapid microscopy measurement of very large spectral images.

    PubMed

    Lindner, Moshe; Shotan, Zav; Garini, Yuval

    2016-05-01

    The spectral content of a sample provides important information that cannot be detected by the human eye or by using an ordinary RGB camera. The spectrum is typically a fingerprint of the chemical compound, its environmental conditions, phase and geometry. Thus measuring the spectrum at each point of a sample is important for a large range of applications from art preservation through forensics to pathological analysis of a tissue section. To date, however, there is no system that can measure the spectral image of a large sample in a reasonable time. Here we present a novel method for scanning very large spectral images of microscopy samples even if they cannot be viewed in a single field of view of the camera. The system is based on capturing information while the sample is being scanned continuously 'on the fly'. Spectral separation implements Fourier spectroscopy by using an interferometer mounted along the optical axis. High spectral resolution of ~5 nm at 500 nm could be achieved with a diffraction-limited spatial resolution. The acquisition time is fairly high and takes 6-8 minutes for a sample size of 10mm x 10mm measured under a bright-field microscope using a 20X magnification. PMID:27137565

  14. Photoswitchable Nanoparticles Enable High-Resolution Cell Imaging: PULSAR Microscopy

    SciTech Connect

    Hu, Dehong; Tian, Z.; Wu, Wuwei; Wan, Wei; Li, Alexander D.

    2008-10-22

    Fluorescence imaging has transformed biological sciences and opened a window to reveal biological mechanisms in real time despite Abbe’s diffraction limit restricts current microscope resolution to 300 nm?.HDH2 Recently, two high-resolution fluorescence microscopic techniques emerged: one uses a special photoactivatable green fluorescent proteinHDH3 and the other employs a pair of cy3/cy5 dyes.HDH4 Both avoid Abbe’s diffraction limit by photoswitching nearby fluorophores off. Thus, photoswitching fluorescence between a bright and a dark state promises to deliver a wealth of information regarding biological phenomena at the nanoscale. The ideal probe is a key-enabling single molecule that can be photoswitched on and off. Such wonderful properties, albeit implausible to imagine at first, were realized in spiropyran derivatives. While being photoswitched, one molecule alternates red-fluorescence on-and-off. Using such photo-actuated unimolecular logical switching attained reconstruction (PULSAR) microscopy, we achieved high-resolution fluorescence imaging down to 80 nm? in nanostructures and cellular organelles.

  15. Oxygen tomography by Čerenkov-excited phosphorescence during external beam irradiation

    PubMed Central

    Zhang, Rongxiao; Davis, Scott C.; Demers, Jennifer-Lynn H.; Glaser, Adam K.; Gladstone, David J.; Esipova, Tatiana V.; Vinogradov, Sergei A.

    2013-01-01

    Abstract. The efficacy of radiation therapy depends strongly on tumor oxygenation during irradiation. However, current techniques to measure this parameter in vivo do not facilitate routine monitoring in patients. Herein, we demonstrate a noninvasive method for tomographic imaging of oxygen partial pressure (pO2) in deep tissue using the phosphorescence decay of an oxygen-sensitive probe excited by Čerenkov radiation induced by external beam radiotherapy. Tissue-simulating scattering phantoms (60 mm diameter with a 20 mm anomaly) containing platinum(II)-G4 (PtG4), a dendritic porphyrin-based phosphor, whose phosphorescence is quenched in the presence of oxygen, were irradiated with a clinical linear accelerator. The emitted phosphorescence was measured at various positions on the phantom boundary using a spectrograph coupled to an intensified charge-coupled device (ICCD). At each position, PtG4 phosphorescence decay curves were measured by synchronizing the ICCD to the linear accelerator pulses. Tomographic images of phosphorescence yield and lifetime were recovered for phantoms with homogenous PtG4 concentrations and heterogeneous pO2. Since PtG4 lifetime is strongly and predictably dependent on pO2 through the Stern-Volmer relationship, tomographic images of pO2 were also reported, and showed excellent agreement with independent oxygenation measurements. Translating this approach to the clinic could facilitate direct sensing of pO2 during radiotherapy. PMID:23644902

  16. Oxygen tomography by Čerenkov-excited phosphorescence during external beam irradiation

    NASA Astrophysics Data System (ADS)

    Zhang, Rongxiao; Davis, Scott C.; Demers, Jennifer-Lynn H.; Glaser, Adam K.; Gladstone, David J.; Esipova, Tatiana V.; Vinogradov, Sergei A.; Pogue, Brian W.

    2013-05-01

    The efficacy of radiation therapy depends strongly on tumor oxygenation during irradiation. However, current techniques to measure this parameter in vivo do not facilitate routine monitoring in patients. Herein, we demonstrate a noninvasive method for tomographic imaging of oxygen partial pressure (pO) in deep tissue using the phosphorescence decay of an oxygen-sensitive probe excited by Čerenkov radiation induced by external beam radiotherapy. Tissue-simulating scattering phantoms (60 mm diameter with a 20 mm anomaly) containing platinum(II)-G4 (PtG4), a dendritic porphyrin-based phosphor, whose phosphorescence is quenched in the presence of oxygen, were irradiated with a clinical linear accelerator. The emitted phosphorescence was measured at various positions on the phantom boundary using a spectrograph coupled to an intensified charge-coupled device (ICCD). At each position, PtG4 phosphorescence decay curves were measured by synchronizing the ICCD to the linear accelerator pulses. Tomographic images of phosphorescence yield and lifetime were recovered for phantoms with homogenous PtG4 concentrations and heterogeneous pO2. Since PtG4 lifetime is strongly and predictably dependent on pO through the Stern-Volmer relationship, tomographic images of pO were also reported, and showed excellent agreement with independent oxygenation measurements. Translating this approach to the clinic could facilitate direct sensing of pO during radiotherapy.

  17. Comparison of mouse mammary gland imaging techniques and applications: Reflectance confocal microscopy, GFP Imaging, and ultrasound

    PubMed Central

    Tilli, Maddalena T; Parrish, Angela R; Cotarla, Ion; Jones, Laundette P; Johnson, Michael D; Furth, Priscilla A

    2008-01-01

    Background Genetically engineered mouse models of mammary gland cancer enable the in vivo study of molecular mechanisms and signaling during development and cancer pathophysiology. However, traditional whole mount and histological imaging modalities are only applicable to non-viable tissue. Methods We evaluated three techniques that can be quickly applied to living tissue for imaging normal and cancerous mammary gland: reflectance confocal microscopy, green fluorescent protein imaging, and ultrasound imaging. Results In the current study, reflectance confocal imaging offered the highest resolution and was used to optically section mammary ductal structures in the whole mammary gland. Glands remained viable in mammary gland whole organ culture when 1% acetic acid was used as a contrast agent. Our application of using green fluorescent protein expressing transgenic mice in our study allowed for whole mammary gland ductal structures imaging and enabled straightforward serial imaging of mammary gland ducts in whole organ culture to visualize the growth and differentiation process. Ultrasound imaging showed the lowest resolution. However, ultrasound was able to detect mammary preneoplastic lesions 0.2 mm in size and was used to follow cancer growth with serial imaging in living mice. Conclusion In conclusion, each technique enabled serial imaging of living mammary tissue and visualization of growth and development, quickly and with minimal tissue preparation. The use of the higher resolution reflectance confocal and green fluorescent protein imaging techniques and lower resolution ultrasound were complementary. PMID:18215290

  18. Total variation based image deconvolution for extended depth-of-field microscopy images

    NASA Astrophysics Data System (ADS)

    Hausser, F.; Beckers, I.; Gierlak, M.; Kahraman, O.

    2015-03-01

    One approach for a detailed understanding of dynamical cellular processes during drug delivery is the use of functionalized biocompatible nanoparticles and fluorescent markers. An appropriate imaging system has to detect these moving particles so as whole cell volumes in real time with high lateral resolution in a range of a few 100 nm. In a previous study Extended depth-of-field microscopy (EDF-microscopy) has been applied to fluorescent beads and tradiscantia stamen hair cells and the concept of real-time imaging has been proved in different microscopic modes. In principle a phase retardation system like a programmable space light modulator or a static waveplate is incorporated in the light path and modulates the wavefront of light. Hence the focal ellipsoid is smeared out and images seem to be blurred in a first step. An image restoration by deconvolution using the known point-spread-function (PSF) of the optical system is necessary to achieve sharp microscopic images of an extended depth-of-field. This work is focused on the investigation and optimization of deconvolution algorithms to solve this restoration problem satisfactorily. This inverse problem is challenging due to presence of Poisson distributed noise and Gaussian noise, and since the PSF used for deconvolution exactly fits in just one plane within the object. We use non-linear Total Variation based image restoration techniques, where different types of noise can be treated properly. Various algorithms are evaluated for artificially generated 3D images as well as for fluorescence measurements of BPAE cells.

  19. Characterization of gold nanoparticle films: Rutherford backscattering spectroscopy, scanning electron microscopy with image analysis, and atomic force microscopy

    SciTech Connect

    Lansåker, Pia C. Niklasson, Gunnar A.; Granqvist, Claes G.; Hallén, Anders

    2014-10-15

    Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness d{sub g}—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM) combined with image analysis as well as by atomic force microscopy (AFM). The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for d{sub g} were obtained by SEM with image analysis and by AFM.

  20. Biological imaging by soft X-ray diffraction microscopy

    NASA Astrophysics Data System (ADS)

    Shapiro, David

    We have developed a microscope for soft x-ray diffraction imaging of dry or frozen hydrated biological specimens. This lensless imaging system does not suffer from the resolution or specimen thickness limitations that other short wavelength microscopes experience. The microscope, currently situated at beamline 9.0.1 of the Advanced Light Source, can collect diffraction data to 12 nm resolution with 750 eV photons and 17 nm resolution with 520 eV photons. The specimen can be rotated with a precision goniometer through an angle of 160 degrees allowing for the collection of nearly complete three-dimensional diffraction data. The microscope is fully computer controlled through a graphical user interface and a scripting language automates the collection of both two-dimensional and three-dimensional data. Diffraction data from a freeze-dried dwarf yeast cell, Saccharomyces cerevisiae carrying the CLN3-1 mutation, was collected to 12 run resolution from 8 specimen orientations spanning a total rotation of 8 degrees. The diffraction data was phased using the difference map algorithm and the reconstructions provide real space images of the cell to 30 nm resolution from each of the orientations. The agreement of the different reconstructions provides confidence in the recovered, and previously unknown, structure and indicates the three dimensionality of the cell. This work represents the first imaging of the natural complex refractive contrast from a whole unstained cell by the diffraction microscopy method and has achieved a resolution superior to lens based x-ray tomographic reconstructions of similar specimens. Studies of the effects of exposure to large radiation doses were also carried out. It was determined that the freeze-dried cell suffers from an initial collapse, which is followed by a uniform, but slow, shrinkage. This structural damage to the cell is not accompanied by a diminished ability to see small features in the specimen. Preliminary measurements on frozen

  1. Extracting twins from orientation imaging microscopy scan data.

    PubMed

    Wright, S I; Larsen, R J

    2002-03-01

    Automated electron backscatter diffraction or orientation imaging microscopy (OIM) provides spatially specific measurements of crystallographic orientation. These measurements are typically collected on regular grids. By inspecting the misorientation between neighbouring measurements on the grid, potential twin boundaries can be identified. If the misorientation is within some given tolerance of a specified twin misorientation, the boundary separating the two measurements may be identified as a potential twin boundary. In addition, for a coherent twin, the twinning planes must be coincident with the grain boundary plane. As OIM scans are inherently two-dimensional, the scan data provide only limited information on the boundary plane. Thus, it is not possible to ascertain definitively whether the twinning planes are coincident with the boundary plane. Nonetheless, the alignment of the surface traces of the twinning planes with the trace of the boundary provides a partial indication of coincidence. An automated approach has been developed that allows data concerning both twin criterion to be extracted from OIM scans. Application of the methodology to deformed zirconium suggests that the twinning planes remain coherent during deformation. The methodology was also used to improve grain size distributions measured by OIM. These results more closely match those obtained by conventional metallography. PMID:11996188

  2. Nanometric depth resolution from multi-focal images in microscopy

    PubMed Central

    Dalgarno, Heather I. C.; Dalgarno, Paul A.; Dada, Adetunmise C.; Towers, Catherine E.; Gibson, Gavin J.; Parton, Richard M.; Davis, Ilan; Warburton, Richard J.; Greenaway, Alan H.

    2011-01-01

    We describe a method for tracking the position of small features in three dimensions from images recorded on a standard microscope with an inexpensive attachment between the microscope and the camera. The depth-measurement accuracy of this method is tested experimentally on a wide-field, inverted microscope and is shown to give approximately 8 nm depth resolution, over a specimen depth of approximately 6 µm, when using a 12-bit charge-coupled device (CCD) camera and very bright but unresolved particles. To assess low-flux limitations a theoretical model is used to derive an analytical expression for the minimum variance bound. The approximations used in the analytical treatment are tested using numerical simulations. It is concluded that approximately 14 nm depth resolution is achievable with flux levels available when tracking fluorescent sources in three dimensions in live-cell biology and that the method is suitable for three-dimensional photo-activated localization microscopy resolution. Sub-nanometre resolution could be achieved with photon-counting techniques at high flux levels. PMID:21247948

  3. Imaging heart development using high-resolution episcopic microscopy.

    PubMed

    Mohun, Timothy J; Weninger, Wolfgang J

    2011-10-01

    Development of the heart in vertebrate embryos is a complex process in which the organ is continually remodelled as chambers are formed, valves sculpted and connections established to the developing vascular system. Investigating the genetic programmes driving these changes and the environmental factors that may influence them is critical for our understanding of congenital heart disease. A recurrent challenge in this work is how to integrate studies as diverse as those of cardiac gene function and regulation with an appreciation of the localised interactions between cardiac tissues not to mention the manner in which both may be affected by cardiac function itself. Meeting this challenge requires an accurate way to analyse the changes in 3D morphology of the developing heart, which can be swift or protracted and both dramatic or subtle in consequence. Here we review the use of high-resolution episcopic microscopy as a simple and effective means to examine organ structure and one that allows modern computing methods pioneered by clinical imaging to be applied to the embryonic heart. PMID:21893408

  4. Image processing pipeline for synchrotron-radiation-based tomographic microscopy.

    PubMed

    Hintermüller, C; Marone, F; Isenegger, A; Stampanoni, M

    2010-07-01

    With synchrotron-radiation-based tomographic microscopy, three-dimensional structures down to the micrometer level can be visualized. Tomographic data sets typically consist of 1000 to 1500 projections of 1024 x 1024 to 2048 x 2048 pixels and are acquired in 5-15 min. A processing pipeline has been developed to handle this large amount of data efficiently and to reconstruct the tomographic volume within a few minutes after the end of a scan. Just a few seconds after the raw data have been acquired, a selection of reconstructed slices is accessible through a web interface for preview and to fine tune the reconstruction parameters. The same interface allows initiation and control of the reconstruction process on the computer cluster. By integrating all programs and tools, required for tomographic reconstruction into the pipeline, the necessary user interaction is reduced to a minimum. The modularity of the pipeline allows functionality for new scan protocols to be added, such as an extended field of view, or new physical signals such as phase-contrast or dark-field imaging etc. PMID:20567088

  5. Two-photon microscopy of oxygen: polymersomes as probe carrier vehicles

    PubMed Central

    Sinks, Louise E.; Robbins, Gregory P.; Roussakis, Emmanuel; Troxler, Thomas; Hammer, Daniel A.; Vinogradov, Sergei A.

    2010-01-01

    Oxygen concentration distributions in biological systems can be imaged by the phosphorescence quenching method in combination with two-photon laser scanning microscopy. In this paper we identified the excitation regime in which the signal of a two-photon-enhanced phosphorescent probe1 is dependent quadratically on the excitation power (quadratic regime), and performed simulations that relate the photophysical properties of the probe to the imaging resolution. Further, we characterized polymersomes as a method of probe encapsulation and delivery. Photo-physical and oxygen sensing properties of the probe were found unchanged when the probe is encapsulated in polymersomes. Polymersomes were found capable of sustaining high probe concentrations, thereby serving to improve the signal-to-noise ratios for oxygen detection compared to the previously employed probe delivery methods. Imaging of polymersomes loaded with the probe was used as a test-bed for a new method. PMID:20462225

  6. Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging

    PubMed Central

    Husvogt, Lennart; Vardeh, Hilde; Faulkner-Jones, Beverly E.; Hornegger, Joachim; Connolly, James L.; Fujimoto, James G.

    2016-01-01

    We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems. PMID:27500636

  7. Host compounds for red phosphorescent OLEDs

    SciTech Connect

    Xia, Chuanjun; Cheon, Kwang -Ohk

    2015-08-25

    Novel compounds containing a triphenylene moiety linked to an .alpha..beta. connected binaphthyl ring system are provided. These compounds have surprisingly good solubility in organic solvents and are useful as host compounds in red phosphorescent OLEDs.

  8. Cell cycle imaging with quantitative differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Kostyk, Piotr; Phelan, Shelley; Xu, Min

    2013-02-01

    We report a microscopic approach for determining cell cycle stages by measuring the nuclear optical path length (OPL) with quantitative differential interference contrast (DIC) microscopy. The approach is validated by the excellent agreement between the proportion of proliferating-to-quiescent cancerous breast epithelial cells obtained from DIC microscopy, and that from a standard immunofluorescence assay.

  9. Image analysis in fluorescence microscopy: Bacterial dynamics as a case study

    PubMed Central

    van Teeffelen, Sven; Shaevitz, Joshua W.; Gitai, Zemer

    2012-01-01

    Fluorescence microscopy is the primary tool for studying complex processes inside individual living cells. Technical advances in both molecular biology and microscopy have made it possible to image cells from many genetic and environmental backgrounds. These images contain a vast amount of information, but this information is often hidden behind various sources of noise, convoluted with other information, and stochastic in nature. Accessing the desired biological information therefore requires new tools of computational image analysis and modeling. Here, we review some of the recent advances in computational analysis of images obtained from fluorescence microscopy, focusing on bacterial systems. We emphasize techniques that are readily available to molecular and cell biologists but also point out examples where problem-specific image analyses are necessary. Thus, image analysis is not only a toolkit to be applied to new images but is an integral part of the design and implementation of a microscopy experiment. PMID:22415868

  10. Prototype study on a miniaturized dual-modality imaging system for photoacoustic microscopy and confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Xie, Zhixing; Guo, L. Jay; Wang, Xueding

    2014-03-01

    It is beneficial to study tumor angiogenesis and microenvironments by imaging the microvasculature and cells at the same time. Photoacoustic microscopy (PAM) is capable of sensitive three-dimensional mapping of microvasculature, while fluorescence microscopy may be applied to assessment of tissue pathology. In this work, a fiber-optic based PAM and confocal fluorescence microscopy (CFM) dual-modality imaging system was designed and built, serving as a prototype of a miniaturized dual-modality imaging probe for endoscopic applications. As for the design, we employed miniature components, including a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector. The system resolutions were calibrated as 8.8 μm in the lateral directions for both PAM and CFM, and 19 μm and 53 μm in the axial direction for PAM and CFM, respectively. Images of the animal bladders ex vivo were demonstrated to show the ability of the system in imaging not only microvasculature but also cellular structure.