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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. 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.

  3. Photon counting phosphorescence lifetime imaging with TimepixCam

    NASA Astrophysics Data System (ADS)

    Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; Nomerotski, Andrei

    2017-01-01

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window and read out by a Timepix Application Specific Integrated Circuit. The 256 × 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting imaging. We have characterised the photon detection capabilities of this detector system and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200 μm diameter polystyrene beads.

  4. Photon counting phosphorescence lifetime imaging with TimepixCam

    DOE PAGES

    Hirvonen, Liisa M.; Fisher-Levine, Merlin; Suhling, Klaus; ...

    2017-01-12

    TimepixCam is a novel fast optical imager based on an optimized silicon pixel sensor with a thin entrance window, and read out by a Timepix ASIC. The 256 x 256 pixel sensor has a time resolution of 15 ns at a sustained frame rate of 10 Hz. We used this sensor in combination with an image intensifier for wide-field time-correlated single photon counting (TCSPC) imaging. We have characterised the photon detection capabilities of this detector system, and employed it on a wide-field epifluorescence microscope to map phosphorescence decays of various iridium complexes with lifetimes of about 1 μs in 200more » μm diameter polystyrene beads.« less

  5. Water-soluble triscyclometalated organoiridium complex: phosphorescent nanoparticle formation, nonlinear optics, and application for cell imaging.

    PubMed

    Fan, Yuanpeng; Zhao, Jingyi; Yan, Qifan; Chen, Peng R; Zhao, Dahui

    2014-03-12

    Two water-soluble triscyclometalated organoiridium complexes, 1 and 2, with polar side chains that form nanoparticles emitting bright-red phosphorescence in water were synthesized. The optimal emitting properties are related to both the triscyclometalated structure and nanoparticle-forming ability in aqueous solution. Nonlinear optical properties are also observed with the nanoparticles. Because of their proper cellular uptake in addition to high emission brightness and effective two-photon absorbing ability, cell imaging can be achieved with nanoparticles of 2 bearing quaternary ammonium side chains at ultra-low effective concentrations using NIR incident light via the multiphoton excitation phosphorescence process.

  6. 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

  7. Multifunctional Phosphorescent Conjugated Polymer Dots for Hypoxia Imaging and Photodynamic Therapy of Cancer Cells

    PubMed Central

    Zhou, Xiaobo; Liang, Hua; Jiang, Pengfei; Zhang, Kenneth Yin; Liu, Shujuan; Yang, Tianshe; Yang, Lijuan; Lv, Wen; Yu, Qi

    2015-01-01

    Molecular oxygen (O2) plays a key role in many physiological processes, and becomes a toxicant to kill cells when excited to 1O2. Intracellular O2 levels, or the degree of hypoxia, are always viewed as an indicator of cancers. Due to the highly efficient cancer therapy ability and low side effect, photodynamic therapy (PDT) becomes one of the most promising treatments for cancers. Herein, an early‐stage diagnosis and therapy system is reported based on the phosphorescent conjugated polymer dots (Pdots) containing Pt(II) porphyrin as an oxygen‐responsive phosphorescent group and 1O2 photosensitizer. Intracellular hypoxia detection has been investigated. Results show that cells treated with Pdots display longer lifetimes under hypoxic conditions, and time‐resolved luminescence images exhibit a higher signal‐to‐noise ratio after gating off the short‐lived background fluorescence. Quantification of O2 is realized by the ratiometric emission intensity of phosphorescence/fluorescence and the lifetime of phosphorescence. Additionally, the PDT efficiency of Pdots is estimated by flow cytometry, MTT cell viability assay, and in situ imaging of PDT induced cell death. Interestingly, Pdots exhibit a high PDT efficiency and would be promising in clinical applications. PMID:27722081

  8. 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.

  9. Photothermal imaging scanning microscopy

    DOEpatents

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

    2006-07-11

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

  10. 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.

  11. 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

    2015-04-09

    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.

  12. 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.

  13. Optical imaging. Expansion microscopy.

    PubMed

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

    2015-01-30

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

  14. Light microscopy digital imaging.

    PubMed

    Joubert, James; Sharma, Deepak

    2011-10-01

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

  15. Image Force Microscopy

    NASA Astrophysics Data System (ADS)

    Rajapaksa, Indrajith

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

  16. 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%)

  17. 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.''

  18. Cyclometalated iridium(III) complexes for phosphorescence sensing of biological metal ions.

    PubMed

    You, Youngmin; Cho, Somin; Nam, Wonwoo

    2014-02-17

    using confocal laser scanning microscopy and photoluminescence lifetime imaging microscopy techniques. We hope that the significant knowledge gained from our studies will be of great help in the design of new molecules as phosphorescence sensors.

  19. Phosphorescent iridium(III) complexes as multicolor probes for specific mitochondrial imaging and tracking.

    PubMed

    Chen, Yu; Qiao, Liping; Ji, Liangnian; Chao, Hui

    2014-01-01

    In the present study, four phosphorescent iridium(III) complexes [Ir(C-N)2(PhenSe)](+) (Ir1-Ir4, in which C-N = 2-(2,4-difluorophenyl)pyridine (dfppy), dibenzo[f,h]quinoxaline (dbq), 2-phenylquinoline (2-pq) and 2-phenylpyridine (ppy), PhenSe = 1,10-phenanthrolineselenazole) with tunable emission colors were developed to image mitochondria and track the dynamics of the mitochondrial morphology. In comparison with commercially available mitochondrial trackers, Ir1-Ir4 possess high specificity to mitochondria in live and fixed cells without requiring prior membrane permeabilization or the replacement of the culture medium. Due to the high resistance of Ir1-Ir4 to the loss of mitochondrial membrane potential as well as the appreciable tolerance to environmental changes, these complexes are applicable for the imaging and tracking of the mitochondrial morphological changes over long periods of time. In addition, Ir2-Ir4 exhibited superior photostability compared to the commercially available mitochondrial trackers. These colorful iridium(III) complexes may contribute to the future development of staining agents for organelle-selective imaging in living cells.

  20. 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

  1. Chip-scale microscopy imaging.

    PubMed

    Zheng, Guoan

    2012-08-01

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

  2. Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue.

    PubMed

    Son, Aoi; Kawasaki, Atsushi; Hara, Daiki; Ito, Takeo; Tanabe, Kazuhito

    2015-02-02

    Understanding oxygen fluctuation in a cancerous tumor is important for effective treatment, especially during radiotherapy. In this paper, ruthenium complexes bearing a nitroimidazole group are shown to report the oxygen status in tumor tissue directly. The nitroimidazole group was known to be accumulated in hypoxic tumor tissues. On the other hand, the ruthenium complex showed strong phosphorescence around 600 nm. The emission of ruthenium is quenched instantaneously by molecular oxygen due to energy transfer between triplet states of oxygen and ruthenium complex, but the emission is then recovered by the removal of oxygen. Thus, we could observe oxygen fluctuation in tumor tissue in a real-time manner by monitoring the phosphorescence of the ruthenium complex. The versatility of the probe is demonstrated by monitoring oxygen fluctuation in living cells and tumor tissue planted in mice. The ruthenium complex promptly penetrated plasma membrane and accumulated in cells to emit its oxygen-dependent phosphorescence. In vivo experiments revealed that the oxygen level in tumor tissue seems to fluctuate at the sub-minute timescale.

  3. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2016-07-12

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

  4. 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.

  5. Boron Polylactide Nanoparticles Exhibiting Fluorescence and Phosphorescence in Aqueous Medium

    PubMed Central

    Pfister, Anne; Zhang, Guoqing; Zareno, Jessica; Horwitz, Alan F.; Fraser, Cassandra L.

    2008-01-01

    Difluoroboron dibenzoylmethane-polylactide, BF2dbmPLA, a biocompatible polymerluminophore conjugate was fabricated as nanoparticles. Spherical particles <100 nm in size were generated via nanoprecipitation. Intense blue fluorescence, two-photon absorption, and long-lived room temperature phosphorescence (RTP) are retained in aqueous suspension. The nanoparticles were internalized by cells and visualized by fluorescence microscopy. Luminescent boron biomaterials show potential for imaging and sensing. PMID:19081748

  6. Image scanning microscopy with radially polarized light

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  7. 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.

  8. A Convenient Approach To Synthesize o-Carborane-Functionalized Phosphorescent Iridium(III) Complexes for Endocellular Hypoxia Imaging.

    PubMed

    Li, Xiang; Tong, Xiao; Yan, Hong; Lu, Changsheng; Zhao, Qiang; Huang, Wei

    2016-11-21

    The structure-property relationship of carborane-modified iridium(III) complexes was investigated. Firstly, an efficient approach for the synthesis of o-carborane-containing pyridine ligands a-f in high yields was developed by utilizing stable and cheap B10 H10 (Et4 N)2 as the starting material. By using these ligands, iridium(III) complexes I-VII were efficiently prepared. In combination with DFT calculations, the photophysical and electrochemical properties of these complexes were studied. The hydrophilic nido-o-carborane-based iridium(III) complex VII showed the highest phosphorescence efficiency (abs. ϕP =0.48) among known water-soluble homoleptic cyclometalated iridium(III) complexes and long emission lifetime (τ=1.24 μs) in aqueous solution. Both of them are sensitive to O2 , and thus endocellular hypoxia imaging of complex VII was realized by time-resolved luminescence imaging (TRLI). This is the first example of applying TRLI in endocellular oxygen detection with a water-soluble nido-carborane functionalized iridium(III) complex.

  9. Fidelity imaging for atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Ghosal, Sayan; Salapaka, Murti

    2015-01-01

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

  10. 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.

  11. 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.

  12. Microscopy image segmentation tool: robust image data analysis.

    PubMed

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

    2014-03-01

    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.

  13. 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.

  14. 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

  15. Phase imaging with intermodulation atomic force microscopy.

    PubMed

    Platz, Daniel; Tholén, Erik A; Hutter, Carsten; von Bieren, Arndt C; Haviland, David B

    2010-05-01

    Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.

  16. Microscopy imaging device with advanced imaging properties

    DOEpatents

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

    2016-10-25

    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.

  17. Microscopy imaging device with advanced imaging properties

    DOEpatents

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

    2016-11-22

    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.

  18. Microscopy imaging device with advanced imaging properties

    DOEpatents

    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.

  19. 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

  20. 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.

  1. Improvement of image quality in holographic microscopy.

    PubMed

    Budhiraja, C J; Som, S C

    1981-05-15

    A novel technique of noise reduction in holographic microscopy has been experimentally studied. It has been shown that significant improvement in the holomicroscopic images of actual low-contrast continuous tone biological objects can be achieved without trade off in image resolution. The technique makes use of holographically produced multidirectional phase gratings used as diffusers and the continuous addition of subchannel holograms. It has been shown that the self-imaging property of this type of diffuser makes the use of these diffusers ideal for microscopic objects. Experimental results have also been presented to demonstrate real-time image processing capability of this technique.

  2. Quantitative imaging of bilirubin by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

    2013-03-01

    Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

  3. 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

  4. 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.

  5. Deep Imaging: the next frontier in microscopy.

    PubMed

    Roukos, Vassilis; Misteli, Tom

    2014-08-01

    The microscope is the quintessential tool for discovery in cell biology. From its earliest incarnation as a tool to make the unseen visible, microscopes have been at the center of most revolutionizing developments in cell biology, histology and pathology. Major quantum leaps in imaging involved the dramatic improvements in resolution to see increasingly smaller structures, methods to visualize specific molecules inside of cells and tissues, and the ability to peer into living cells to study dynamics of molecules and cellular structures. The latest revolution in microscopy is Deep Imaging-the ability to look at very large numbers of samples by high-throughput microscopy at high spatial and temporal resolution. This approach is rooted in the development of fully automated high-resolution microscopes and the application of advanced computational image analysis and mining methods. Deep Imaging is enabling two novel, powerful approaches in cell biology: the ability to image thousands of samples with high optical precision allows every discernible morphological pattern to be used as a read-out in large-scale imaging-based screens, particularly in conjunction with RNAi-based screening technology; in addition, the capacity to capture large numbers of images, combined with advanced computational image analysis methods, has also opened the door to detect and analyze very rare cellular events. These two applications of Deep Imaging are revolutionizing cell biology.

  6. 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 ...

  7. Decreasing luminescence lifetime of evaporating phosphorescent droplets

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

    Laser-induced phosphorescence has been used extensively to study spray dynamics. It is important to understand the influence of droplet evaporation in the interpretation of such measurements, as it increases luminescence quenching. By suspending a single evaporating n-heptane droplet in an acoustic levitator, the properties of lanthanide-complex europium-thenoyltrifluoroacetone-trioctylphosphine oxide (Eu-TTA-TOPO) phosphorescence are determined through high-speed imaging. A decrease was found in the measured phosphorescence decay coefficient (780 → 200 μs) with decreasing droplet volumes (10-9 → 10-11 m3) corresponding to increasing concentrations (10-4 → 10-2 M). This decrease continues up to the point of shell-formation at supersaturated concentrations. The diminished luminescence is shown not to be attributable to triplet-triplet annihilation, quenching between excited triplet-state molecules. Instead, the pure exponential decays found in the measurements show that a non-phosphorescent quencher, such as free TTA/TOPO, can be attributable to this decay. The concentration dependence of the phosphorescence lifetime can therefore be used as a diagnostic of evaporation in sprays.

  8. Stimulated Raman scattering microscopy for biomedical imaging

    NASA Astrophysics Data System (ADS)

    Min, Wei; Freudiger, Christian W.; Lu, Sijia; He, Chengwei; Kang, Jing X.; Xie, X. Sunney

    2009-02-01

    Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is significantly greater than that of spontaneous Raman scattering, and is further enhanced by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and easily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast.

  9. 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

  10. Image Restoration in Cryo-electron Microscopy

    PubMed Central

    Penczek, Pawel A.

    2011-01-01

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

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

    PubMed

    Sims, Paul A; Hardin, Jeff D

    2007-01-01

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

  12. 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.

  13. 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

  14. Image segmentation for integrated multiphoton microscopy and reflectance confocal microscopy imaging of human skin in vivo

    PubMed Central

    Chen, Guannan; Lui, Harvey

    2015-01-01

    Background Non-invasive cellular imaging of the skin in vivo can be achieved in reflectance confocal microscopy (RCM) and multiphoton microscopy (MPM) modalities to yield complementary images of the skin based on different optical properties. One of the challenges of in vivo microscopy is the delineation (i.e., segmentation) of cellular and subcellular architectural features. Methods In this work we present a method for combining watershed and level-set models for segmentation of multimodality images obtained by an integrated MPM and RCM imaging system from human skin in vivo. Results Firstly, a segmentation model based on watershed is introduced for obtaining the accurate structure of cell borders from the RCM image. Secondly,, a global region based energy level-set model is constructed for extracting the nucleus of each cell from the MPM image. Thirdly, a local region-based Lagrange Continuous level-set approach is used for segmenting cytoplasm from the MPM image. Conclusions Experimental results demonstrated that cell borders from RCM image and boundaries of cytoplasm and nucleus from MPM image can be obtained by our segmentation method with better accuracy and effectiveness. We are planning to use this method to perform quantitative analysis of MPM and RCM images of in vivo human skin to study the variations of cellular parameters such as cell size, nucleus size and other mophormetric features with skin pathologies. PMID:25694949

  15. Phosphorescence bioimaging using cyclometalated Ir(III) complexes.

    PubMed

    You, Youngmin

    2013-08-01

    Recent advances in the development of the phosphorescent Ir(III) complexes have made it possible to implement the phosphorescence modality in bioimaging applications. A variety of phosphorescent Ir(III) complexes have been synthesized and assessed in the context of in vitro and in vivo imaging, especially in subcellular organelle staining and the sensing of biologically important analytes. The examples presented here demonstrate that Ir(III) complexes provide attractive alternatives to fluorescent organic compounds in the construction of biolabels and biosensors. The complexes are particularly advantageous with respect to fluorescent compounds in their compatibility with time-gated bioimaging techniques that completely eliminate background signals due to autofluorescence.

  16. Live cell imaging by multifocal multiphoton microscopy.

    PubMed

    Straub, M; Lodemann, P; Holroyd, P; Jahn, R; Hell, S W

    2000-10-01

    Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

  17. Difluoroboron β-Diketonate Materials with Long-Lived Phosphorescence Enable Lifetime Based Oxygen Imaging with a Portable Cost Effective Camera.

    PubMed

    Mathew, Alexander S; DeRosa, Christopher A; Demas, James N; Fraser, Cassandra L

    2016-04-21

    Lifetime-based oxygen imaging is useful in many biological applications but instrumentation can be stationary, expensive, and complex. Herein, we present a portable, cost effective, simple alternative with high spatiotemporal resolution that uses a complementary metal oxide silicon (CMOS) camera to measure oxygen sensitive lifetimes on the millisecond scale. We demonstrate its compatibility with difluoroboron β-diketonate poly(lactic acid) (BF2bdkPLA) polymers which are nontoxic and exhibit long-lived oxygen sensitive phosphorescence. Spatially resolved lifetimes of four BF2bdkPLA variants are measured using nonlinear least squares (NLS) and rapid lifetime determination (RLD) both of which are shown to be accurate and precise. Real-time imaging in a dynamic environment is demonstrated by determining lifetime pixel-wise. The setup costs less than $5000, easily fits into a backpack, and can operate on battery power alone. This versatility combined with the inherent utility of lifetime measurements make this system a useful tool for a wide variety of oxygen sensing applications. This study serves as an important foundation for the development of dual mode real time lifetime plus ratiometric imaging with bright, long lifetime difluoroboron β-diketonate probes.

  18. [Mobile phone based wireless microscopy imaging technology].

    PubMed

    Yuan, Yucheng; Liu, Jing

    2011-03-01

    This article proposes a new device named "Wireless Cellscope" that combining mobile phone and optical microscope together. The established wireless microscope platform consists of mobile phone, network monitor, miniaturized microscope or high resolution microscope etc. A series of conceptual experiments were performed on microscopic observation of ordinary objects and mice tumor tissue slices. It was demonstrated that, the new method could acquire microscopy images via a wireless way, which is spatially independent. With small size and low cost, the device thus developed has rather wide applicability in non-disturbing investigation of cell/tissue culture and long distance observation of dangerous biological sample etc.

  19. 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

  20. Image analysis applied to luminescence microscopy

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

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

  1. 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

  2. Nanoscale Imaging of RNA with Expansion Microscopy

    PubMed Central

    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.

    2016-01-01

    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 (ExM) 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, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) 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

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. Fully hydrated yeast cells imaged with electron microscopy.

    PubMed

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

    2011-05-18

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

  8. 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.

  9. A highly selective phosphorescence probe for histidine in living bodies.

    PubMed

    Gao, Quankun; Song, Bo; Ye, Zhiqiang; Yang, Liu; Liu, Ruoyang; Yuan, Jingli

    2015-11-14

    In this work, we designed and synthesized a heterobimetallic ruthenium(ii)-nickel(ii) complex, [Ru(bpy)2(phen-DPA)Ni](PF6)4 (Ru-Ni), as a highly selective phosphorescence probe for histidine. The probe exhibited weak emission at 603 nm because the phosphorescence of the Ru(ii) complex can be strongly quenched by the paramagnetic Ni(2+) ion. In the presence of histidine, reaction of Ru-Ni with histidine resulted in the release of nickel(ii) and an enhancement in the phosphorescence intensity at 603 nm. Ru-Ni showed high selectivity for histidine even in the presence of other amino acids and cellular abundant species. Cell imaging experimental results demonstrated that Ru-Ni is membrane permeable, and can be applied for visualizing histidine in live cells. More interestingly, Ru-Ni also can act as a novel reaction-based nuclear staining agent for visualizing exclusively the nuclei of living cells with a significant phosphorescence enhancement. In addition, the potential of the probe for biological applications was confirmed by employing it for phosphorescence imaging of histidine in larval zebrafish and Daphnia magna. These results demonstrated that Ru-Ni would be a useful tool for physiological and pathological studies involving histidine.

  10. Imaging DNA Structure by Atomic Force Microscopy.

    PubMed

    Pyne, Alice L B; Hoogenboom, Bart W

    2016-01-01

    Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine superstructure and secondary structure of surface-bound DNA. The method is also readily applicable to probe DNA-DNA interactions and DNA-protein complexes.

  11. 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

  12. 3D super-resolution imaging by localization microscopy.

    PubMed

    Magenau, Astrid; Gaus, Katharina

    2015-01-01

    Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

  13. 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.

  14. 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

  15. 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

  16. Multispectral imaging fluorescence microscopy for living cells.

    PubMed

    Hiraoka, Yasushi; Shimi, Takeshi; Haraguchi, Tokuko

    2002-10-01

    Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications. We introduce these systems with particular interest in live cell imaging. Finally we demonstrate examples of spectral imaging of living cells using commercially available systems with no need for user engineering.

  17. Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging.

    PubMed

    Rheinlaender, Johannes; Geisse, Nicholas A; Proksch, Roger; Schäffer, Tilman E

    2011-01-18

    We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

  18. Ferritin protein imaging and detection by magnetic force microscopy.

    PubMed

    Hsieh, Chiung-Wen; Zheng, Bin; Hsieh, Shuchen

    2010-03-14

    Magnetic force microscopy was used to image and detect ferritin proteins and the strength of the magnetic signal is discussed, revealing a large workable lift height between the magnetic tip and the ferritin sample.

  19. 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.

  20. 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

  1. Multiple fluorescence microscopy and optoelectronic imaging: possibilities and limits

    NASA Astrophysics Data System (ADS)

    Gundlach, Heinz

    1997-12-01

    The last 20 years have seen an unexpected great renaissance and a partial revolution in light microscopy. This recent progress is due to new design in optics and instrumentation as well as improvement of optical contrast enhancement techniques. Recent progress in fluorescence microscopy is achieved by multiparameter fluorescence techniques, by improvement of conventional photomicrography as well as by optoelectronic imaging, confocal laser scanning microscopy, image processing and analysis. Due to the increase in number of fluorescence dyes, double and triple bandpass filter sets permit a rapid changeover between different fluorochromes simultaneously.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. Imaging highly absorbing nanoparticles using photothermal microscopy

    NASA Astrophysics Data System (ADS)

    Lussier, Simon-Alexandre; Moradi, Hamid; Price, Alain; Murugkar, Sangeeta

    2015-03-01

    Gold nanoparticles (NPs) have tremendous potential in biomedicine. They can be used as absorbing labels inside living cells for the purpose of biomedical imaging, biosensing as well as for photothermal therapy. We demonstrate photothermal imaging of highly-absorbing particles using a pump-probe setup. The photothermal signal is recovered by heterodyne detection, where the excitation pump laser is at 532 nm and the probe laser is at 638 nm. The sample is moved by a scanning stage. Proof of concept images of red polystyrene microspheres and gold nanoparticles are obtained with this home-built multimodal microscope. The increase in temperature at the surface of the gold NPs, due to the pump laser beam, can be directly measured by means of this photothermal microscope and then compared with the results from theoretical predictions. This technique will be useful for characterization of nanoparticles of different shapes, sizes and materials that are used in cancer diagnosis and therapy.

  7. 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

  8. Scanning Tunneling Microscopy methods for spectroscopic imaging of subsurface interfaces

    NASA Technical Reports Server (NTRS)

    Bell, L. D.; Kaiser, W. J.

    1988-01-01

    A new method for spatially-resolved, spectroscopic investigation of subsurface interface structure has been developed. The method, Ballistic Electron Emission Microscopy (BEEM), is based on Scanning Tunneling Microscopy (STM) techniques. BEEM combines STM vacuum tunneling with unique ballistic electron spectroscopy capabilities. BEEM enables, for the first time, direct imaging of subsurface interface electronic properties with nanometer spatial resolution. STM topographic images of surface structure and BEEM images of subsurface properties are obtained simultaneously. BEEM capabilities are demonstrated by investigation of important metal-semiconductor interfaces.

  9. 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.

  10. Imaging mechanisms of force detected FMR microscopy

    SciTech Connect

    Midzor, M. M.; Wigen, P. E.; Pelekhov, D.; Chen, W.; Hammel, P. C.; Roukes, M. L.

    2000-05-01

    We demonstrate spatial resolution of ferromagnetic resonance in a microscopic sample of YIG using ferromagnetic resonance force microscopy (FMRFM). Measurements were performed on a small single crystal YIG film grown on a GGG substrate, roughly rectangular in shape 20 {mu}mx{approx}150 {mu}m and 3 {mu}m thick. The perpendicular and parallel force geometries of FMRFM, in conjunction with an external bias field both parallel and perpendicular to the film, were used to scan the sample. This enabled the detection of strong signals, even at atmospheric pressure and room temperature. The fundamental and higher-order magnetostatic modes were observed to have 26-29 Gauss separation. The intensity of these modes exhibited spatial variation as the magnetic tip was scanned over the sample, and this behavior is qualitatively explained by DE theory. An improved fabrication method for magnet on cantilever was employed, which yielded a spatial resolution of 15 {mu}m. These results demonstrate the potential of FMRFM for investigating the spatial dependence of ferromagnetic resonance, and for studying the anisotropy fields and exchange coupling effects within multilayer films and small magnetic systems. (c) 2000 American Institute of Physics.

  11. 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.

  12. 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.

  13. A water-soluble and highly phosphorescent cyclometallated iridium complex with versatile sensing capability.

    PubMed

    Yang, Zhen; Zhao, Yuan; Wang, Chan; Song, Qijun; Pang, Qingfeng

    2017-05-01

    A water-soluble and highly phosphorescent cyclometallated iridium complex [(pq)2Ir(bpy-COOK)](+)Cl(-) (where pq=2-phenylquinoline, bpy-COOK= potassium 2,2'-bipyridine-4,4'-dicarboxylate) (Ir) has been synthesized and characterized. Its phosphorescence can be sensitively and selectively quenched by tryptophan through a photoinduced electron-transfer (PET) process. Furthermore, the phosphorescence of Ir is drastically increased upon binding with bovine serum albumin (BSA), and the enhanced signal is effectively quenched in the presence of Cu(2+). Thus, Ir can be used as a multifunctional chemosensor for tryptophan, BSA, and Cu(2+) determination as well as for cell imaging.

  14. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    PubMed Central

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-01-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood. PMID:27845443

  15. Imaging Cytometry of Human Leukocytes with Third Harmonic Generation Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Cheng-Ham; Wang, Tzung-Dau; Hsieh, Chia-Hung; Huang, Shih-Hung; Lin, Jong-Wei; Hsu, Szu-Chun; Wu, Hau-Tieng; Wu, Yao-Ming; Liu, Tzu-Ming

    2016-11-01

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

  16. Imaging Local Electric Field Distribution by Plasmonic Impedance Microscopy.

    PubMed

    Wang, Yixian; Shan, Xiaonan; Wang, Shaopeng; Tao, Nongjian; Blanchard, Pierre-Yves; Hu, Keke; Mirkin, Michael V

    2016-02-02

    We report on imaging of local electric field on an electrode surface with plasmonic electrochemical impedance microscopy (P-EIM). The local electric field is created by putting an electrode inside a micropipet positioned over the electrode and applying a voltage between the two electrodes. We show that the distribution of the surface charge as well as the local electric field at the electrode surface can be imaged with P-EIM. The spatial distribution and the dependence of the local charge density and electric field on the distance between the micropipet and the surface are measured, and the results are compared with the finite element calculations. The work also demonstrates the possibility of integrating plasmonic imaging with scanning ion conductance microscopy (SICM) and other scanning probe microscopies.

  17. 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.

  18. 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.

  19. Single-shell carbon nanotubes imaged by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Höper, Ralf; Workman, Richard K.; Chen, Dong; Sarid, Dror; Yadav, Tapesh; Withers, James C.; Loutfy, Raouf O.

    1994-05-01

    Single-shell carbon nanotubes, approximately 1 nm in diameter, have been imaged for the first time by atomic force microscopy operating in both the contact and tapping modes. For the contact mode, the height of the imaged nanotubes has been calibrated using the atomic steps of the silicon substrate on which the nanotubes were deposited. For the tapping mode, the calibration was performed using an industry-standard grating. The paper discusses substrate and sample preparation methods for the characterization by scanning probe microscopy of nanotubes deposited on a substrate.

  20. 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.

  1. Localizing and extracting filament distributions from microscopy images.

    PubMed

    Basu, S; Liu, C; Rohde, G K

    2015-04-01

    Detailed quantitative measurements of biological filament networks represent a crucial step in understanding architecture and structure of cells and tissues, which in turn explain important biological events such as wound healing and cancer metastases. Microscopic images of biological specimens marked for different structural proteins constitute an important source for observing and measuring meaningful parameters of biological networks. Unfortunately, current efforts at quantitative estimation of architecture and orientation of biological filament networks from microscopy images are predominantly limited to visual estimation and indirect experimental inference. Here, we describe a new method for localizing and extracting filament distributions from 2D microscopy images of different modalities. The method combines a filter-based detection of pixels likely to contain a filament with a constrained reverse diffusion-based approach for localizing the filaments centrelines. We show with qualitative and quantitative experiments, using both simulated and real data, that the new method can provide more accurate centreline estimates of filament in comparison to other approaches currently available. In addition, we show the algorithm is more robust with respect to variations in the initial filter-based filament detection step often used. We demonstrate the application of the method in extracting quantitative parameters from confocal microscopy images of actin filaments and atomic force microscopy images of DNA fragments.

  2. Droplet Epitaxy Image Contrast in Mirror Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Kennedy, S. M.; Zheng, C. X.; Jesson, D. E.

    2017-01-01

    Image simulation methods are applied to interpret mirror electron microscopy (MEM) images obtained from a movie of GaAs droplet epitaxy. Cylindrical symmetry of structures grown by droplet epitaxy is assumed in the simulations which reproduce the main features of the experimental MEM image contrast, demonstrating that droplet epitaxy can be studied in real-time. It is therefore confirmed that an inner ring forms at the droplet contact line and an outer ring (or skirt) occurs outside the droplet periphery. We believe that MEM combined with image simulations will be increasingly used to study the formation and growth of quantum structures.

  3. Reconstruction of complementary images in second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Gao, Liang; Jin, Lei; Xue, Ping; Xu, Jun; Wang, Yi; Ma, Hui; Chen, Dieyan

    2006-05-01

    Second harmonic generation microscopy(SHGM) has become widely used to image biological samples. Due to the complexity of biological samples, more and more effort has been put on polarization imaging in SHGM technology to uncover their structures. In this work, we put forward a novel stitching method based on careful mathematical calculation, and accomplish it by rotating laser polarization. We first show its validity in imaging a perfectly synthesized bio-origin polymer poly (3-hyroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Then, we test its power by getting a true image of fibrillar collagen structure of rat-tail tendon.

  4. GPU-based image registration in aperture correlation microscopy, and reflection mode correlation microscopy

    NASA Astrophysics Data System (ADS)

    Fafchamps, Lionel J.; Neil, Mark A. A.; Juskaitis, Rimas

    2013-02-01

    Aperture Correlation Microscopy (ACM) is a fluorescence microscopy technique capable of depth resolved imaging and enhanced lateral resolution at real-time acquisition rates. It relies on the subtraction of 2 separate images from different cameras which must be registered to the sub-pixel level. In order to achieve real-time registration and subtraction, the graphics processing unit (GPU) is used to apply a transformation from one frame to the other, resulting in a system capable of processing over 200 frames per second on modest hardware (GeForce 330M). Currently, this rate is limited by camera acquision to 16fps. Additionally, a novel reflection mode correlation microscope is introduced which functions on similar principles as the fluorescent system but can be used to examine reflective samples. Images and z-stacks taken with this system are presented here.

  5. Imaging Nanotherapeutics in Inflamed Vasculature by Intravital Microscopy

    PubMed Central

    Wang, Zhenjia

    2016-01-01

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

  6. A Parallel Imaging Approach to Wide-field MR Microscopy

    PubMed Central

    McDougall, Mary Preston; Wright, Steven M.

    2011-01-01

    Magnetic resonance microscopy (MRM), suggested in the earliest papers on MRI, has always been limited by the low signal-to-noise ratio (SNR) resulting from the small voxel size. MRM has largely been enabled by the use of microcoils which provide the SNR improvement required to overcome this limitation. Concomitant with the small coils is a small field-of-view, which limits the use of MRM as a histological tool or for imaging large regions in general. This paper describes initial results in wide field-of-view MR microscopy using a large array of narrow, parallel coils, which provides an SNR enhancement as well as the ability to use parallel imaging techniques. Comparison images made between a volume coil and the proposed technique demonstrate reductions in imaging time of over 100 with no loss in SNR or resolution. PMID:22139858

  7. Biological imaging by soft x-ray diffraction microscopy

    DOE PAGES

    Shapiro, D.; Thibault, P.; Beetz, T.; ...

    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

  8. 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.

  9. High-speed synthetic aperture microscopy for live cell imaging

    PubMed Central

    Kim, Moonseok; Choi, Youngwoon; Fang-Yen, Christopher; Sung, Yongjin; Dasari, Ramachandra R.; Feld, Michael S.; Choi, Wonshik

    2011-01-01

    We present a high-speed synthetic aperture microscopy for quantitative phase imaging of live biological cells. We measure 361 complex amplitude images of an object with various directions of illumination covering an NA of 0.8 in less than one-thirteenth of a second and then combine the images with a phase-referencing method to create a synthesized phase image. Because of the increased depth selectivity, artifacts from diffraction that are typically present in coherent imaging are significantly suppressed, and lateral resolution of phase imaging is improved. We use the instrument to demonstrate high-quality phase imaging of live cells, both static and dynamic, and thickness measurements of a nanoscale cholesterol helical ribbon. PMID:21263482

  10. Super-resolution Microscopy Approaches for Live Cell Imaging

    PubMed Central

    Godin, Antoine G.; Lounis, Brahim; Cognet, Laurent

    2014-01-01

    By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells. PMID:25418158

  11. Cytology 3D structure formation based on optical microscopy images

    NASA Astrophysics Data System (ADS)

    Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Imaging of nucleic acids with atomic force microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.; Ando, Toshio

    2011-01-01

    Atomic force microscopy (AFM) is a key tool of nanotechnology with great importance in applications to DNA nanotechnology and to the recently emerging field of RNA nanotechnology. Advances in the methodology of AFM now enable reliable and reproducible imaging of DNA of various structures, topologies, and DNA and RNA nanostructures. These advances are reviewed here with emphasis on methods utilizing modification of mica to prepare the surfaces enabling reliable and reproducible imaging of DNA and RNA nanostructures. Since the AFM technology for DNA is more mature, AFM imaging of DNA is introduced in this review to provide experience and background for the improvement of AFM imaging of RNA. Examples of imaging different structures of RNA and DNA are discussed and illustrated. Special attention is given to the potential use of AFM to image the dynamics of nucleic acids at the nanometer scale. As such, we review recent advances with the use of time-lapse AFM. PMID:21310240

  17. 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.

  18. 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.

  19. 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.

  20. Divided-aperture differential confocal fast-imaging microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Yun; Qiu, Lirong; Zhao, Xiangye; Zhao, Weiqian

    2017-03-01

    A new method, laser divided-aperture differential confocal microscopy (DDCM), is proposed to achieve high-resolution 3D imaging of microstructures of large-scale sample surfaces. This method uses a divided-aperture confocal structure to significantly improve the axial resolution of confocal microscopy and keep a long working distance simultaneously; uses two radically offset point detectors to achieve differential detection to further improve the axial response sensitivity and realize fast imaging of a large-scale sample surface with a big axial scan-step interval. Theoretical analyses and experimental results show that the DDCM can reach an axial resolution of 5 nm with a 3.1 mm working distance with a 3 times imaging speed of a confocal system with the same resolution.

  1. 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.

  2. SEGMENTATION OF MITOCHONDRIA IN ELECTRON MICROSCOPY IMAGES USING ALGEBRAIC CURVES

    PubMed Central

    Seyedhosseini, Mojtaba; Ellisman, Mark H.; Tasdizen, Tolga

    2014-01-01

    High-resolution microscopy techniques have been used to generate large volumes of data with enough details for understanding the complex structure of the nervous system. However, automatic techniques are required to segment cells and intracellular structures in these multi-terabyte datasets and make anatomical analysis possible on a large scale. We propose a fully automated method that exploits both shape information and regional statistics to segment irregularly shaped intracellular structures such as mitochondria in electron microscopy (EM) images. The main idea is to use algebraic curves to extract shape features together with texture features from image patches. Then, these powerful features are used to learn a random forest classifier, which can predict mitochondria locations precisely. Finally, the algebraic curves together with regional information are used to segment the mitochondria at the predicted locations. We demonstrate that our method outperforms the state-of-the-art algorithms in segmentation of mitochondria in EM images. PMID:25132915

  3. 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.

  4. 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.

  5. High-speed atomic force microscopy: imaging and force spectroscopy.

    PubMed

    Eghiaian, Frédéric; Rico, Felix; Colom, Adai; Casuso, Ignacio; Scheuring, Simon

    2014-10-01

    Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.

  6. Imaging melanin by two-photon absorption microscopy

    NASA Astrophysics Data System (ADS)

    Ye, Tong; Yurtsever, Gunay; Fischer, Martin; Simon, John D.; Warren, Warren S.

    2006-02-01

    Multiphoton excitation fluorescence microscopy has proven to be a powerful method for non-invasive, in vivo, thick tissue imaging with molecular specificity. However, many important endogenous biomolecules do not fluoresce (NAD) or fluoresce with low efficiency (Melanin). In this report femtosecond pulse shaping methods are used to measure two-photon absorption (TPA) directly with very high sensitivity. Combining with the laser scanning microscope, this Two-photon Absorption Microscopy (TPAM) retains the penetration and localization advantages of two-photon fluorescence microscopy and permits direct observation of important endogenous molecular markers (melanin or hemoglobin) which are invisible in multiphoton fluorescence microscopy. We have demonstrated here for the first time that TPAM can successfully and more efficiently image melanoma cells and tissues and provide a good melanin contrast in optical sectioning of the melanoma lesions which are comparable to pathological histology. Combining with the two-photon fluorescence images acquired simultaneously, the distribution patterns of the melanocytes and their intratissue behavior could be studied without cutting the lesions from patients. TPAM will undoubtedly find the applications in the clinical diagnosis and biomedical research.

  7. A generalized Potts model for confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Máté, Gabriell; Heermann, Dieter W.

    2015-01-01

    Much as being among the least invasive mainstream imaging technologies in life sciences, the resolution of confocal microscopy is limited. Imaged structures, e.g., chromatin-fiber loops, have diameters around or beyond the diffraction limit, and microscopy images show seemingly random spatial density distributions only. While such images are important because the organization of the chromosomes influences different cell mechanisms, many interesting questions can also be related to the observed patterns. These concern their spatial aspects, the role of randomness, the possibility of modeling these images with a random generative process, the interaction between the densities of adjacent loci, the length-scales of these influences, etc. We answer these questions by implementing a generalization of the Potts model. We show how to estimate the model parameters, test the performance of the estimation process and numerically prove that the obtained values converge to the ground truth. Finally, we generate images with a trained model and show that they compare well to real cell images.

  8. Electromechanical imaging of biomaterials by scanning probe microscopy.

    PubMed

    Rodriguez, B J; Kalinin, S V; Shin, J; Jesse, S; Grichko, V; Thundat, T; Baddorf, A P; Gruverman, A

    2006-02-01

    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-10nm 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.

  9. In vivo volumetric imaging of subcutaneous microvasculature by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Hao F.; Maslov, Konstantin; Li, Meng-Lin; Stoica, George; Wang, Lihong V.

    2006-10-01

    Photoacoustic microscopy was developed to achieve volumetric imaging of the anatomy and functions of the subcutaneous microvasculature in both small animals and humans in vivo with high spatial resolution and high signal-to-background ratio. By following the skin contour in raster scanning, the ultrasonic transducer maintains focusing in the region of interest. Furthermore, off-focus lateral resolution is improved by using a synthetic-aperture focusing technique based on the virtual point detector concept. Structural images are acquired in both rats and humans, whereas functional images representing hemoglobin oxygen saturation are acquired in rats. After multiscale vesselness filtering, arterioles and venules in the image are separated based on the imaged oxygen saturation levels. Detailed structural information, such as vessel depth and spatial orientation, are revealed by volume rendering.

  10. 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.

  11. Quantitative single-molecule imaging by confocal laser scanning microscopy.

    PubMed

    Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

    2008-11-25

    A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

  12. 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.

  13. 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

  14. Volume scanning electron microscopy for imaging biological ultrastructure.

    PubMed

    Titze, Benjamin; Genoud, Christel

    2016-11-01

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

  15. 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.

  16. 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.

  17. Structured illumination microscopy for vibrational molecular imaging (Conference Presentation)

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    Raman microscopy is a powerful tool for analytical imaging. The wavelength shift of Raman scattering corresponds to molecular vibrational energy. Therefore, we can access rich chemical information, such as distribution, concentration, and chemical environment of sample molecules. Despite these strengths of Raman microscopy, the spatial resolution has been a limiting factor for many practical applications. In this study, we developed a large-area, high-resolution Raman microscope by utilizing structured illumination microscopy (SIM) to overcome the spatial resolution limit. A structured line-illumination (SLI) Raman microscope was constructed. The structured illumination is introduced along the line direction by the interference of two line-shaped beams. In SIM, the spatial frequency mixing between structured illumination and Raman scattering from the sample allows access to the high spatial frequency information beyond the conventional cut-off. As a result, the FWHM of 40-nm fluorescence particle images showed a clear resolution enhancement in the line direction: 366 nm in LI and 199 nm in SLI microscope. Using the developed microscope, we successfully demonstrated high-resolution Raman imaging of various kinds of specimens, such as few-layer graphene, graphite, mouse brain tissue, and polymer nanoparticles. The high resolution Raman images showed the capability to extract original spectral features from the mixed Raman spectra of a multi-component sample because of the enhanced spatial resolution, which is advantageous in observing complex spectral features. The Raman microscopy technique reported here enables us to see the detailed chemical structures of chemical, biological, and medical samples with a spatial resolution smaller than 200 nm.

  18. 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.

  19. Segmentation of virus particle candidates in transmission electron microscopy images.

    PubMed

    Kylberg, G; Uppström, M; Hedlund, K-O; Borgefors, G; Sintorn, I-M

    2012-02-01

    In this paper, we present an automatic segmentation method that detects virus particles of various shapes in transmission electron microscopy images. The method is based on a statistical analysis of local neighbourhoods of all the pixels in the image followed by an object width discrimination and finally, for elongated objects, a border refinement step. It requires only one input parameter, the approximate width of the virus particles searched for. The proposed method is evaluated on a large number of viruses. It successfully segments viruses regardless of shape, from polyhedral to highly pleomorphic.

  20. Microstructure imaging of human rectal mucosa using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Liu, N. R.; Chen, G.; Chen, J. X.; Yan, J.; Zhuo, S. M.; Zheng, L. Q.; Jiang, X. S.

    2011-01-01

    Multiphoton microscopy (MPM) has high resolution and sensitivity. In this study, MPM was used to image microstructure of human rectal mucosa. The morphology and distribution of the main components in mucosa layer, absorptive cells and goblet cells in the epithelium, abundant intestinal glands in the lamina propria and smooth muscle fibers in the muscularis mucosa were clearly monitored. The variations of these components were tightly relevant to the pathology in gastrointestine system, especially early rectal cancer. The obtained images will be helpful for the diagnosis of early colorectal cancer.

  1. Chromatin Imaging with Time-Lapse Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    Time-lapse atomic force microscopy (AFM) is widely used for direct visualization of the nanoscale dynamics of various biological systems. The advent of high-speed AFM instrumentation made it possible to image the dynamics of proteins and protein-DNA complexes within millisecond time range. This chapter describes protocols for studies of structure and dynamics of nucleosomes with time-lapse AFM including the high-speed AFM instrument. The necessary specifics for the preparation of chromatin samples for imaging with AFM including the protocols for the surface preparation are provided. PMID:25827873

  2. Localized charge imaging with scanning Kelvin probe microscopy

    NASA Astrophysics Data System (ADS)

    Orihuela, M. F.; Somoza, A. M.; Colchero, J.; Ortuño, M.; Palacios-Lidón, E.

    2017-01-01

    In this work, we propose an intuitive and easily implementable approach to model and interpret scanning Kelvin probe microscopy images of insulating samples with localized charges. The method, based on the image charges method, has been validated by a systematic comparison of its predictions with experimental measurements performed on charge domains of different sizes, injected in polymethyl methacrylate discontinuous films. The agreement between predictions and experimental lateral profiles, as well as with spectroscopy tip-sample distance curves, supports its consistency. The proposed procedure allows obtaining quantitative information such as total charge and the size of a charge domain and allows estimating the most adequate measurement parameters.

  3. Super-Resolution Real Imaging in Microsphere-Assisted Microscopy

    PubMed Central

    Wang, Feifei; Li, Yi; Jia, Boliang; Liu, Lianqing; Li, Wen Jung

    2016-01-01

    Microsphere-assisted microscopy has received a lot of attention recently due to its simplicity and its capability to surpass the diffraction limit. However, to date, sub-diffraction-limit features have only been observed in virtual images formed through the microspheres. We show that it is possible to form real, super-resolution images using high-refractive index microspheres. Also, we report on how changes to a microsphere’s refractive index and size affect image formation and planes. The relationship between the focus position and the additional magnification factor is also investigated using experimental and theoretical methods. We demonstrate that such a real imaging mode, combined with the use of larger microspheres, can enlarge sub-diffraction-limit features up to 10 times that of wide-field microscopy’s magnification with a field-of-view diameter of up to 9 μm. PMID:27768774

  4. Local dimensionality determines imaging speed in localization microscopy

    PubMed Central

    Fox-Roberts, Patrick; Marsh, Richard; Pfisterer, Karin; Jayo, Asier; Parsons, Maddy; Cox, Susan

    2017-01-01

    Localization microscopy allows biological samples to be imaged at a length scale of tens of nanometres. Live-cell super-resolution imaging is rare, as it is generally assumed to be too slow for dynamic samples. The speed of data acquisition can be optimized by tuning the density of activated fluorophores in each time frame. Here, we show that the maximum achievable imaging speed for a particular structure varies by orders of magnitude, depending on the sample dimensionality (that is, whether the sample is more like a point, a strand or an extended structure such as a focal adhesion). If too high an excitation density is used, we demonstrate that the analysis undergoes silent failure, resulting in reconstruction artefacts. We are releasing a tool to allow users to identify areas of the image in which the activation density was too high and correct for them, in both live- and fixed-cell experiments. PMID:28079054

  5. 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.

  6. Local dimensionality determines imaging speed in localization microscopy

    NASA Astrophysics Data System (ADS)

    Fox-Roberts, Patrick; Marsh, Richard; Pfisterer, Karin; Jayo, Asier; Parsons, Maddy; Cox, Susan

    2017-01-01

    Localization microscopy allows biological samples to be imaged at a length scale of tens of nanometres. Live-cell super-resolution imaging is rare, as it is generally assumed to be too slow for dynamic samples. The speed of data acquisition can be optimized by tuning the density of activated fluorophores in each time frame. Here, we show that the maximum achievable imaging speed for a particular structure varies by orders of magnitude, depending on the sample dimensionality (that is, whether the sample is more like a point, a strand or an extended structure such as a focal adhesion). If too high an excitation density is used, we demonstrate that the analysis undergoes silent failure, resulting in reconstruction artefacts. We are releasing a tool to allow users to identify areas of the image in which the activation density was too high and correct for them, in both live- and fixed-cell experiments.

  7. Superresolved multiphoton microscopy with spatial frequency-modulated imaging

    SciTech Connect

    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.

    2016-05-26

    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 2..eta.. below the diffraction limit, where ..eta.. 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.

  8. Superresolved multiphoton microscopy with spatial frequency-modulated imaging

    PubMed Central

    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.

    2016-01-01

    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

  9. 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.

  10. Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

    PubMed

    Shami, Gerald J; Cheng, Delfine; Braet, Filip

    2017-02-01

    Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc.

  11. Super-Resolution Microscopy Approaches to Nuclear Nanostructure Imaging.

    PubMed

    Cremer, Christoph; Szczurek, Aleksander; Schock, Florian; Gourram, Amine; Birk, Udo

    2017-04-05

    The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200 nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. Investigations in optoelectronic image processing in scanning laser microscopy

    NASA Astrophysics Data System (ADS)

    Chaliha, Hiranya Kumar

    A considerable amount of work has been done on scann-ing laser microscopy since its applications were first pointed out by Roberts and Young[1], Minsky [2] and Davidovits et al [3]. The advent of laser has made it possible to focus an intense beam of laser light in a scanning optical microscope (SOM) [4, 5] and hence explore regions of microscopy[6] uncovered by conven-tional microscopy. In the simple SOM [7, 8, 9], the upper spatial frequency in amplitude transmittance or reflectance of an object for which transfer function is nonzero is same as that in a conventional optical microscope. However, in Type II SOM [7] or confocal SOM that employs a coherent or a point detector, the spatial frequency bandwidth is twice that obtained in a conventional microscope. Besides this confocal set-up is found to be very useful in optical sectioning and consequently in 3-D image processing[10, 11, 12] specially of biological specimens. Such systems are also suitable for studies of semiconductor materials [13], super-resolution [14] and various imaginative ways of image processing[15, 16, 17] including phase imaging[18]. A brief survey of related advances in scanning optical microscopy has been covered in the chapter 1 of the thesis. The performance of SOM may be investigated by concent-rating also on signal derived by one dimensional scan of the object specimen. This simplified mode may also be adapted to give wealth of information for biological and semiconductor specimens. Hence we have investigated the design of a scanning laser system suited specifically for studies of line scan image signals of microscopic specimens when probed through a focused laser spot. An electro-mechanical method of scanning of the object specimen has been designed with this aim in mind. Chapter 2, Part A of the thesis deals with the design consider-ations of such a system. For analysis of scan signals at a later instant of time so as to facilitate further processing, an arrangement of microprocessor

  19. 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

  20. Deconvolved spatial light interference microscopy for live cell imaging.

    PubMed

    Haldar, Justin P; Wang, Zhuo; Popescu, Gabriel; Liang, Zhi-Pei

    2011-09-01

    Spatial light interference microscopy (SLIM) is a recently developed method for the label-free imaging of live cells, using the quantitative optical path length through the sample as an endogenous source of contrast. In conventional SLIM, spatial resolution is limited by diffraction and aberrations. This paper describes a novel constrained deconvolution method for improving resolution in SLIM. Constrained deconvolution is enabled by experimental measurement of the system point-spread function and the modeling of coherent image formation in SLIM. Results using simulated and experimental data demonstrate that the proposed method leads to significant improvements in the resolution and contrast of SLIM images. The proposed method should prove useful for high-resolution label-free studies of biological cells and subcellular processes.

  1. Automated microscopy and image analysis for androgen receptor function.

    PubMed

    Hartig, Sean M; Newberg, Justin Y; Bolt, Michael J; Szafran, Adam T; Marcelli, Marco; Mancini, Michael A

    2011-01-01

    Systems-level approaches have emerged that rely on analytical, microscopy-based technology for the discovery of novel drug targets and the mechanisms driving AR signaling, transcriptional activity, and ligand independence. Single cell behavior can be quantified by high-throughput microscopy methods through analysis of endogenous protein levels and localization or creation of biosensor cell lines that can simultaneously detect both acute and latent responses to known and unknown androgenic stimuli. The cell imaging and analytical protocols can be automated to discover agonist/antagonist response windows for nuclear translocation, reporter gene activity, nuclear export, and subnuclear transcription events, facilitating access to a multiplex model system that is inherently unavailable through classic biochemical approaches. In this chapter, we highlight the key steps needed for developing, conducting, and analyzing high-throughput screens to identify effectors of AR signaling.

  2. 3D Image Analysis of Geomaterials using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Mulukutla, G.; Proussevitch, A.; Sahagian, D.

    2009-05-01

    Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

  3. Directional bilateral filters for smoothing fluorescence microscopy images

    NASA Astrophysics Data System (ADS)

    Venkatesh, Manasij; Mohan, Kavya; Seelamantula, Chandra Sekhar

    2015-08-01

    Images obtained through fluorescence microscopy at low numerical aperture (NA) are noisy and have poor resolution. Images of specimens such as F-actin filaments obtained using confocal or widefield fluorescence microscopes contain directional information and it is important that an image smoothing or filtering technique preserve the directionality. F-actin filaments are widely studied in pathology because the abnormalities in actin dynamics play a key role in diagnosis of cancer, cardiac diseases, vascular diseases, myofibrillar myopathies, neurological disorders, etc. We develop the directional bilateral filter as a means of filtering out the noise in the image without significantly altering the directionality of the F-actin filaments. The bilateral filter is anisotropic to start with, but we add an additional degree of anisotropy by employing an oriented domain kernel for smoothing. The orientation is locally adapted using a structure tensor and the parameters of the bilateral filter are optimized for within the framework of statistical risk minimization. We show that the directional bilateral filter has better denoising performance than the traditional Gaussian bilateral filter and other denoising techniques such as SURE-LET, non-local means, and guided image filtering at various noise levels in terms of peak signal-to-noise ratio (PSNR). We also show quantitative improvements in low NA images of F-actin filaments.

  4. 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.

  5. Fluorescence lifetime imaging microscopy for the characterization of atherosclerotic plaques

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer; Sun, Yinghua; Saroufeem, Ramez; Hatami, Nisa; Marcu, Laura

    2009-02-01

    Atherosclerotic plaque composition has been associated with plaque instability and rupture. This study investigates the use of fluorescence lifetime imaging microscopy (FLIM) for mapping plaque composition and assessing features of vulnerability. Measurements were conducted in atherosclerotic human aortic samples using an endoscopic FLIM system (spatial resolution of 35 µm temporal resolution 200 ps) developed in our lab which allows mapping in one measurement the composition within a volume of 4 mm diameter x 250 µm depth. Each pixel in the image represents a corresponding fluorescence lifetime value; images are formed through a flexible 0.6 mm side-viewing imaging bundle which allows for further intravascular applications. Based on previously recorded spectra of human atherosclerotic plaque, fluorescence emission was collected through two filters: f1: 377/50 and f2: 460/60 (center wavelength/bandwidth), which together provides the greatest discrimination between intrinsic fluorophores related to plaque vulnerability. We have imaged nine aortas and lifetime images were retrieved using a Laguerre expansion deconvolution technique and correlated with histopathology. Early results demonstrate discrimination using fluorescence lifetime between early, lipid-rich, and collagen-rich lesions which are consistent with previously reported time-resolved atherosclerotic plaque measurements.

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

    PubMed Central

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

    2013-01-01

    Abstract. 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. PMID:23412434

  7. Imaging biological structures with fluorescence photoactivation localization microscopy.

    PubMed

    Gould, Travis J; Verkhusha, Vladislav V; Hess, Samuel T

    2009-01-01

    Fluorescence photoactivation localization microscopy (FPALM) images biological structures with subdiffraction-limited resolution. With repeated cycles of activation, readout and bleaching, large numbers of photoactivatable probes can be precisely localized to obtain a map (image) of labeled molecules with an effective resolution of tens of nanometers. FPALM has been applied to a variety of biological imaging applications, including membrane, cytoskeletal and cytosolic proteins in fixed and living cells. Molecular motions can be quantified. FPALM can also be applied to nonbiological samples, which can be labeled with photoactivatable probes. With emphasis on cellular imaging, we describe here the adaptation of a conventional widefield fluorescence microscope for FPALM and present step-by-step procedures to successfully obtain and analyze FPALM images. The fundamentals of this protocol may also be applicable to users of similar imaging techniques that apply localization of photoactivatable probes to achieve super-resolution. Once alignment of the setup has been completed, data acquisitions can be obtained in approximately 1-30 min and analyzed in approximately 0.5-4 h.

  8. Quantitative analysis of in vivo confocal microscopy images: a review.

    PubMed

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

  9. 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.

  10. Nanoscale cellular imaging with scanning angle interference microscopy.

    PubMed

    DuFort, Christopher; Paszek, Matthew

    2014-01-01

    Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate.

  11. Single particle maximum likelihood reconstruction from superresolution microscopy images.

    PubMed

    Verdier, Timothée; Gunzenhauser, Julia; Manley, Suliana; Castelnovo, Martin

    2017-01-01

    Point localization superresolution microscopy enables fluorescently tagged molecules to be imaged beyond the optical diffraction limit, reaching single molecule localization precisions down to a few nanometers. For small objects whose sizes are few times this precision, localization uncertainty prevents the straightforward extraction of a structural model from the reconstructed images. We demonstrate in the present work that this limitation can be overcome at the single particle level, requiring no particle averaging, by using a maximum likelihood reconstruction (MLR) method perfectly suited to the stochastic nature of such superresolution imaging. We validate this method by extracting structural information from both simulated and experimental PALM data of immature virus-like particles of the Human Immunodeficiency Virus (HIV-1). MLR allows us to measure the radii of individual viruses with precision of a few nanometers and confirms the incomplete closure of the viral protein lattice. The quantitative results of our analysis are consistent with previous cryoelectron microscopy characterizations. Our study establishes the framework for a method that can be broadly applied to PALM data to determine the structural parameters for an existing structural model, and is particularly well suited to heterogeneous features due to its single particle implementation.

  12. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  13. Scanning Transmission X-ray microscopy Imaging of Aerosol Particles

    NASA Astrophysics Data System (ADS)

    Gilles, M. K.; Kilcoyne, A.; Tyliszczak, T.; Shuh, D. K.; Fakra, S.; Robinson, M.; Chase, K.

    2003-12-01

    Scanning transmission x-ray microscopes (STXM) are used to image a diversity of carbon and metal containing items such as biofilms in soils, magnetic materials, polymers and meteorites. Studies on particles collected on SiO2 filters from biomass burns in Flagstaff, Arizona and individual aerosols collected in South Africa on TEM grids are underway at beamlines 5.3.2 and 11.0.2 at the Advanced Light Source of Lawrence Berkeley National Laboratory. Sub micron particles are imaged in the transmission mode over the energy range of 280 - 1900 eV. Spectromicroscopic studies on individual particles using near edge x-ray absorption fine structure (NEXAFS) probe multiple species within or on the same particle. In (STXM) an X-ray beam is focused with a zone plate onto a sample and the transmitted radiation is detected. Since the signal is obtained in the transmission mode, optically thin samples are required. Hence, atmospheric aerosols with submicron thickness and diameter are well suited for this method. Near edge spectra of various elements were scanned in step sizes from 0.1-0.5 eV around characteristic absorption edges, creating 2 dimensional images at each energy. While STXM images are taken with a lower spatial resolution (currently 40 nm) than microscopies such as scanning electron microscopy, transmission electron microscopy, and atomic force microscopy, detailed chemical information with spatial distributions, and oxidation states is obtained. A particular focus of this work is to obtain more detailed information on the type of carbons, multiply, or singly bonded and whether or not carbon is bonded to oxygen. The ultimate goal is discrimination between organic and black carbon within individual aerosol particles and determining if organic carbon, black carbon, and metal species are distributed homogeneously throughout aerosol particles. Initial scans of the samples from Flagstaff show spectral evidence of aromatic carbon, without distinct C=O signatures. NEXAFS

  14. Femtosecond digital lensless holographic microscopy to image biological samples.

    PubMed

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

    2013-09-01

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

  15. Mueller matrix signature in advanced fluorescence microscopy imaging

    NASA Astrophysics Data System (ADS)

    Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

    2017-02-01

    We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

  16. Image recombination transform algorithm for superresolution structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Xing; Lei, Ming; Dan, Dan; Yao, Baoli; Yang, Yanlong; Qian, Jia; Chen, Guangde; Bianco, Piero R.

    2016-09-01

    Structured illumination microscopy (SIM) is an attractive choice for fast superresolution imaging. The generation of structured illumination patterns made by interference of laser beams is broadly employed to obtain high modulation depth of patterns, while the polarizations of the laser beams must be elaborately controlled to guarantee the high contrast of interference intensity, which brings a more complex configuration for the polarization control. The emerging pattern projection strategy is much more compact, but the modulation depth of patterns is deteriorated by the optical transfer function of the optical system, especially in high spatial frequency near the diffraction limit. Therefore, the traditional superresolution reconstruction algorithm for interference-based SIM will suffer from many artifacts in the case of projection-based SIM that possesses a low modulation depth. Here, we propose an alternative reconstruction algorithm based on image recombination transform, which provides an alternative solution to address this problem even in a weak modulation depth. We demonstrated the effectiveness of this algorithm in the multicolor superresolution imaging of bovine pulmonary arterial endothelial cells in our developed projection-based SIM system, which applies a computer controlled digital micromirror device for fast fringe generation and multicolor light-emitting diodes for illumination. The merit of the system incorporated with the proposed algorithm allows for a low excitation intensity fluorescence imaging even less than 1 W/cm2, which is beneficial for the long-term, in vivo superresolved imaging of live cells and tissues.

  17. 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.

  18. 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.

  19. 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.

  20. Total variation versus wavelet-based methods for image denoising in fluorescence lifetime imaging microscopy.

    PubMed

    Chang, Ching-Wei; Mycek, Mary-Ann

    2012-05-01

    We report the first application of wavelet-based denoising (noise removal) methods to time-domain box-car fluorescence lifetime imaging microscopy (FLIM) images and compare the results to novel total variation (TV) denoising methods. Methods were tested first on artificial images and then applied to low-light live-cell images. Relative to undenoised images, TV methods could improve lifetime precision up to 10-fold in artificial images, while preserving the overall accuracy of lifetime and amplitude values of a single-exponential decay model and improving local lifetime fitting in live-cell images. Wavelet-based methods were at least 4-fold faster than TV methods, but could introduce significant inaccuracies in recovered lifetime values. The denoising methods discussed can potentially enhance a variety of FLIM applications, including live-cell, in vivo animal, or endoscopic imaging studies, especially under challenging imaging conditions such as low-light or fast video-rate imaging.

  1. 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

  2. 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.

  3. 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.

  4. Modeling atomic-resolution scanning transmission electron microscopy images.

    PubMed

    Findlay, Scott D; Oxley, Mark P; Allen, Leslie J

    2008-02-01

    A real-space description of inelastic scattering in scanning transmission electron microscopy is derived with particular attention given to the implementation of the projected potential approximation. A hierarchy of approximations to expressions for inelastic images is presented. Emphasis is placed on the conditions that must hold in each case. The expressions that justify the most direct, visual interpretation of experimental data are also the most approximate. Therefore, caution must be exercised in selecting experimental parameters that validate the approximations needed for the analysis technique used. To make the most direct, visual interpretation of electron-energy-loss spectroscopic images from core-shell excitations requires detector improvements commensurate with those that aberration correction provides for the probe-forming lens. Such conditions can be relaxed when detailed simulations are performed as part of the analysis of experimental data.

  5. Fluorescence microscopy imaging of electroperturbation in mammalian cells

    NASA Astrophysics Data System (ADS)

    Sun, Yinghua; Vernier, P. Thomas; Behrend, Matthew; Wang, Jingjing; Thu, Mya Mya; Gundersen, Martin A.; Marcu, Laura

    2006-03-01

    We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation-the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

  6. 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.

  7. 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

  8. Three dimensional reconstruction of neuron morphology from confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Fanti, Zian; Martinez-Perez, M. Elena

    2010-05-01

    In recent years it has been more common to see 3D visualization of objects applied in many different areas. In neuroscience research, 3D visualization of neurons acquired at different depth views (i.e. image stacks) by means of confocal microscopy are of increase use. However in the best case, these visualizations only help to have a qualitative description of the neuron shape. Since it is well know that neuronal function is intimately related to its morphology. Having a precise characterization of neuronal structures such as axons and dendrites is critical to perform a quantitative analysis and thus it allows to design neuronal functional models based on neuron morphology. Currently there exists different commercial software to reconstruct neuronal arbors, however these processes are labor intensive since in most of the cases they are manually made. In this paper we propose a new software capable to reconstruct 3D neurons from confocal microscopy views in a more efficient way, with minimal user intervention. The propose algorithm is based on finding the tubular structures present in the stack of images using a modify version of the minimal graph cut algorithm. The model is generated from the segmented stack with a modified version of the Marching Cubes algorithm to generate de 3D isosurface. Herein we describe the principles of our 3D segmentation technique and the preliminary results.

  9. Segmentation of fluorescence microscopy cell images using unsupervised mining.

    PubMed

    Du, Xian; Dua, Sumeet

    2010-05-28

    The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

  10. Residual Deconvolutional Networks for Brain Electron Microscopy Image Segmentation.

    PubMed

    Fakhry, Ahmed; Zeng, Tao; Ji, Shuiwang

    2017-02-01

    Accurate reconstruction of anatomical connections between neurons in the brain using electron microscopy (EM) images is considered to be the gold standard for circuit mapping. A key step in obtaining the reconstruction is the ability to automatically segment neurons with a precision close to human-level performance. Despite the recent technical advances in EM image segmentation, most of them rely on hand-crafted features to some extent that are specific to the data, limiting their ability to generalize. Here, we propose a simple yet powerful technique for EM image segmentation that is trained end-to-end and does not rely on prior knowledge of the data. Our proposed residual deconvolutional network consists of two information pathways that capture full-resolution features and contextual information, respectively. We showed that the proposed model is very effective in achieving the conflicting goals in dense output prediction; namely preserving full-resolution predictions and including sufficient contextual information. We applied our method to the ongoing open challenge of 3D neurite segmentation in EM images. Our method achieved one of the top results on this open challenge. We demonstrated the generality of our technique by evaluating it on the 2D neurite segmentation challenge dataset where consistently high performance was obtained. We thus expect our method to generalize well to other dense output prediction problems.

  11. Imaging red blood cell dynamics by quantitative phase microscopy.

    PubMed

    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.

  12. 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

  13. 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.

  14. Host compounds for red phosphorescent OLEDs

    DOEpatents

    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.

  15. Single nuclear pores visualized by confocal microscopy and image processing.

    PubMed Central

    Kubitscheck, U; Wedekind, P; Zeidler, O; Grote, M; Peters, R

    1996-01-01

    How nuclear pore complexes, mediating the transport of nucleic acids, proteins, and metabolites between cell nucleus and cytoplasm, are arranged in the nuclear envelope is essentially unknown. Here we describe a method combining high-resolution confocal imaging with image processing and pattern recognition to visualize single nuclear pore complexes (120 nm diameter), determine their relative positions with nanometer accuracy, and analyze their distribution in situ. The method was tested by means of a model system in which the very same sample areas could be imaged by confocal and electron microscopy. It was thus found that single fluorescent beads of 105 nm nominal diameter could be localized with a lateral accuracy of <20 nm and an axial accuracy of approximately 20 nm. The method was applied to digitonin-permeabilized 3T3 cells, whose nuclear pore complexes were fluorescently labeled with the anti-nucleoporin antibody mAb414. Stacks of optical sections were generated by confocal imaging at high resolution. Herein the nuclear pore complexes appeared as bright diffraction-limited spots whose centers were localized by fitting them by three-dimensional gaussians. The nearest-neighbor distribution function and the pair correlation function were calculated and found to agree well with those of randomly distributed hard cylinders of 138 +/- 17 nm diameter, but not with those of randomly distributed points or nonrandomly distributed cylinders. This was supported by a cluster analysis. Implications for the direct observation of the transport of single particles and molecules through individual nuclear pore complexes are discussed. Images FIGURE 1 FIGURE 2 FIGURE 4 PMID:9172731

  16. Brain tumor classification of microscopy images using deep residual learning

    NASA Astrophysics Data System (ADS)

    Ishikawa, Yota; Washiya, Kiyotada; Aoki, Kota; Nagahashi, Hiroshi

    2016-12-01

    The crisis rate of brain tumor is about one point four in ten thousands. In general, cytotechnologists take charge of cytologic diagnosis. However, the number of cytotechnologists who can diagnose brain tumors is not sufficient, because of the necessity of highly specialized skill. Computer-Aided Diagnosis by computational image analysis may dissolve the shortage of experts and support objective pathological examinations. Our purpose is to support a diagnosis from a microscopy image of brain cortex and to identify brain tumor by medical image processing. In this study, we analyze Astrocytes that is a type of glia cell of central nerve system. It is not easy for an expert to discriminate brain tumor correctly since the difference between astrocytes and low grade astrocytoma (tumors formed from Astrocyte) is very slight. In this study, we present a novel method to segment cell regions robustly using BING objectness estimation and to classify brain tumors using deep convolutional neural networks (CNNs) constructed by deep residual learning. BING is a fast object detection method and we use pretrained BING model to detect brain cells. After that, we apply a sequence of post-processing like Voronoi diagram, binarization, watershed transform to obtain fine segmentation. For classification using CNNs, a usual way of data argumentation is applied to brain cells database. Experimental results showed 98.5% accuracy of classification and 98.2% accuracy of segmentation.

  17. 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.

  18. Multiphoton fluorescence and second harmonic generation microscopy for imaging keratoconus

    NASA Astrophysics Data System (ADS)

    Sun, Yen; Lo, Wen; Lin, Sung-Jan; Lin, Wei-Chou; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Dong, Chen-Yuan

    2006-02-01

    The purpose of this study is to assess the possible application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging the structural features of keratoconus cornea and to evaluate its potential as being a clinical in vivo monitoring technique. Using the near-infrared excitation source from a titanium-sapphire laser pumped by a diode-pumped, solid state (DPSS) laser system, we can induce and simultaneously acquire multiphoton autofluorescence and SHG signals from the cornea specimens with keratoconus. A home-modified commercial microscope system with specified optical components is used for optimal signal detection. Keratoconus cornea button from patient with typical clinical presentation of keratoconus was obtained at the time of penetrating keratoplasty. The specimen was also sent for the histological examination as comparison. In all samples of keratoconus, destruction of lamellar structure with altered collagen fiber orientation was observed within whole layer of the diseased stromal area. In addition, the orientation of the altered collagen fibers within the cone area shows a trend directing toward the apex of the cone, which might implicate the biomechanical response of the keratoconus stroma to the intraocular pressure. Moreover, increased autofluorescent cells were also found in the cone area, with increased density as one approaches the apical area. In conclusion, multiphoton autofluorescence and SHG microscopy non-invasively demonstrated the morphological features of keratoconus cornea, especially the structural alternations of the stromal lamellae. We believe that in the future the multiphoton microscopy can be applied in vivo as an effective, non-invasive diagnostic and monitoring technique for keratoconus.

  19. 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.

  20. Microscopy image resolution improvement by deconvolution of complex fields.

    PubMed

    Cotte, Yann; Toy, M Fatih; Pavillon, Nicolas; Depeursinge, Christian

    2010-09-13

    Based on truncated inverse filtering, a theory for deconvolution of complex fields is studied. The validity of the theory is verified by comparing with experimental data from digital holographic microscopy (DHM) using a high-NA system (NA=0.95). Comparison with standard intensity deconvolution reveals that only complex deconvolution deals correctly with coherent cross-talk. With improved image resolution, complex deconvolution is demonstrated to exceed the Rayleigh limit. Gain in resolution arises by accessing the objects complex field - containing the information encoded in the phase - and deconvolving it with the reconstructed complex transfer function (CTF). Synthetic (based on Debye theory modeled with experimental parameters of MO) and experimental amplitude point spread functions (APSF) are used for the CTF reconstruction and compared. Thus, the optical system used for microscopy is characterized quantitatively by its APSF. The role of noise is discussed in the context of complex field deconvolution. As further results, we demonstrate that complex deconvolution does not require any additional optics in the DHM setup while extending the limit of resolution with coherent illumination by a factor of at least 1.64.

  1. Multiphoton, confocal, and lifetime microscopy for molecular imaging in cartilage

    NASA Astrophysics Data System (ADS)

    Wachsmann-Hogiu, Sebastian; Krakow, Deborah; Kirilova, Veneta T.; Cohn, Daniel H.; Bertolotto, Cristina; Acuna, Dora; Fang, Qiyin; Krivorov, Nikola; Farkas, Daniel L.

    2005-03-01

    It has recently been shown that mutations in Filamin A and B genes produce a large spectrum of skeletal disorders in developing fetuses. However, high-resolution optical microscopy in cartilage growth plate using fluorescent antibody assays, which should elucidate molecular aspects of these disorders, is extremely difficult due to the high level of autofluoresce in this tissue. We apply multiphoton, confocal, lifetime and spectral microscopy to (i) image and characterize autofluorophores in chondrocytes and subtract their contributions to obtain a corrected antibody-marker fluorescence signal, and (ii) measure the interaction between Filamin A and B proteins by detecting the fluorescence resonance energy transfer (FRET) between markers of the two proteins. Taking advantage of the different fluorescence spectra of the endogenous and exogenous markers, we can significantly reduce the autofluorescence background. Preliminary results of the FRET experiments suggest no interaction between Filamin A and B proteins. However, developing of new antibodies targeting the carboxy-terminal immunoglobulin-like domain may be necessary to confirm this result.

  2. Band Excitation in Scanning Probe Microscopy: Recognition and Functional Imaging

    NASA Astrophysics Data System (ADS)

    Jesse, S.; Vasudevan, R. K.; Collins, L.; Strelcov, E.; Okatan, M. B.; Belianinov, A.; Baddorf, A. P.; Proksch, R.; Kalinin, S. V.

    2014-04-01

    Field confinement at the junction between a biased scanning probe microscope's tip and solid surface enables local probing of various bias-induced transformations, such as polarization switching, ionic motion, and electrochemical reactions. The nanoscale size of the biased region, smaller or comparable to that of features such as grain boundaries and dislocations, potentially allows for the study of kinetics and thermodynamics at the level of a single defect. In contrast to classical statistically averaged approaches, this approach allows one to link structure to functionality and deterministically decipher associated mesoscopic and atomistic mechanisms. Furthermore, responses measured as a function of frequency and bias can serve as a fingerprint of local material functionality, allowing for local recognition imaging of inorganic and biological systems. This article reviews current progress in multidimensional scanning probe microscopy techniques based on band excitation time and voltage spectroscopies, including discussions on data acquisition, dimensionality reduction, and visualization, along with future challenges and opportunities for the field.

  3. 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.

  4. 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.

  5. 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.

  6. Rapid microscopy measurement of very large spectral images.

    PubMed

    Lindner, Moshe; Shotan, Zav; Garini, Yuval

    2016-05-02

    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.

  7. Ultrasound Backscatter Microscopy for Imaging of Oral Carcinoma

    PubMed Central

    Lam, Matthew; Chaudhari, Abhijit J.; Sun, Yang; Zhou, Feifei; Dobbie, Allison; Gandour-Edwards, Regina F.; Tinling, Steve P.; Farwell, D. Gregory; Monsky, Wayne L.; Shung, K. Kirk; Marcu, Laura

    2013-01-01

    Objectives Ultrasound backscatter microscopy (UBM), or ultrasound biomicroscopy, is a noninvasive, label-free, and ionizing radiation–free technique allowing high-resolution 3-dimensional structural imaging. The goal of this study was to evaluate UBM for resolving anatomic features associated with squamous cell carcinoma of the oral cavity. Methods The study was conducted in a hamster buccal pouch model. A carcinogen was topically applied to cheeks of 14 golden Syrian hamsters. Six additional hamsters served as healthy controls. A high-frequency (41 MHz, 6-mm focal depth, lateral and axial resolutions of 65 and 37 μm, respectively) UBM system was used for scanning the oral cavity after 14 weeks of carcinogen application. Histologic analyses were conducted on scanned regions. Results The histologic structure of buccal tissue and microvasculature networks could be visualized from the UBM images. Epithelial and mucosal hypertrophy and neoplastic changes were identified in animals subjected to the carcinogen. In animals with invasive squamous cell carcinoma, lesion development and destruction of the structural integrity of tissue layers were noted. Conclusions In this pilot study, UBM generated sufficient contrast for morphologic features associated with oral carcinoma compared to healthy tissue. This modality may present a practical technique for detection of oral neoplasms that is potentially translatable to humans. PMID:24065260

  8. Quantitative determination of maximal imaging depth in all-NIR multiphoton microscopy images of thick tissues

    NASA Astrophysics Data System (ADS)

    Sarder, Pinaki; Akers, Walter J.; Sudlow, Gail P.; Yazdanfar, Siavash; Achilefu, Samuel

    2014-02-01

    We report two methods for quantitatively determining maximal imaging depth from thick tissue images captured using all-near-infrared (NIR) multiphoton microscopy (MPM). All-NIR MPM is performed using 1550 nm laser excitation with NIR detection. This method enables imaging more than five-fold deep in thick tissues in comparison with other NIR excitation microscopy methods. In this study, we show a correlation between the multiphoton signal along the depth of tissue samples and the shape of the corresponding empirical probability density function (pdf) of the photon counts. Histograms from this analysis become increasingly symmetric with the imaging depth. This distribution transitions toward the background distribution at higher imaging depths. Inspired by these observations, we propose two independent methods based on which one can automatically determine maximal imaging depth in the all-NIR MPM images of thick tissues. At this point, the signal strength is expected to be weak and similar to the background. The first method suggests the maximal imaging depth corresponds to the deepest image plane where the ratio between the mean and median of the empirical photon-count pdf is outside the vicinity of 1. The second method suggests the maximal imaging depth corresponds to the deepest image plane where the squared distance between the empirical photon-count mean obtained from the object and the mean obtained from the background is greater than a threshold. We demonstrate the application of these methods in all-NIR MPM images of mouse kidney tissues to study maximal depth penetration in such tissues.

  9. All-optically integrated multimodality imaging system: combined photoacoustic microscopy, optical coherence tomography, and fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Chen, Zhongjiang; Yang, Sihua; Xing, Da

    2016-10-01

    We have developed a multimodality imaging system by optically integrating all-optical photoacoustic microscopy (AOPAM), optical coherence tomography (OCT) and fluorescence microscopy (FLM) to provide complementary information including optical absorption, optical back-scattering and fluorescence contrast of biological tissue. By sharing the same low-coherence Michelson interferometer, AOPAM and OCT could be organically optically combined to obtain the absorption and scattering information of the biological tissues. Also, owing to using the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence signals are obtained to present the radiative and nonradiative transition process of absorption. Simultaneously photoacoustic angiography, tissue structure and fluorescence molecular in vivo images of mouse ear were acquired to demonstrate the capabilities of the optically integrated trimodality imaging system, which can present more information to study tumor angiogenesis, vasculature, anatomical structure and microenvironments in vivo.

  10. 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.

  11. 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.

  12. Fluorescence lifetime imaging and reflectance confocal microscopy for multiscale imaging of oral precancer.

    PubMed

    Jabbour, Joey M; Cheng, Shuna; Malik, Bilal H; Cuenca, Rodrigo; Jo, Javier A; Wright, John; Cheng, Yi-Shing Lisa; Maitland, Kristen C

    2013-04-01

    Optical imaging techniques using a variety of contrast mechanisms are under evaluation for early detection of epithelial precancer; however, tradeoffs in field of view (FOV) and resolution may limit their application. Therefore, we present a multiscale multimodal optical imaging system combining macroscopic biochemical imaging of fluorescence lifetime imaging (FLIM) with subcellular morphologic imaging of reflectance confocal microscopy (RCM). The FLIM module images a 16×16 mm² tissue area with 62.5 μm lateral and 320 ps temporal resolution to guide cellular imaging of suspicious regions. Subsequently, coregistered RCM images are acquired at 7 Hz with 400 μm diameter FOV, <1  μm lateral and 3.5 μm axial resolution. FLIM-RCM imaging was performed on a tissue phantom, normal porcine buccal mucosa, and a hamster cheek pouch model of oral carcinogenesis. While FLIM is sensitive to biochemical and macroscopic architectural changes in tissue, RCM provides images of cell nuclear morphology, all key indicators of precancer progression.

  13. 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.

  14. 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

  15. Robust image alignment for cryogenic transmission electron microscopy.

    PubMed

    McLeod, Robert A; Kowal, Julia; Ringler, Philippe; Stahlberg, Henning

    2016-12-27

    Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd-frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro.

  16. Systematic and general method for quantifying localization in microscopy images

    PubMed Central

    Sheng, Huanjie; Stauffer, Weston

    2016-01-01

    ABSTRACT Quantifying the localization of molecules with respect to other molecules, cell structures and intracellular regions is essential to understanding their regulation and actions. However, measuring localization from microscopy images is often difficult with existing metrics. Here, we evaluate a metric for quantifying localization termed the threshold overlap score (TOS), and show it is simple to calculate, easy to interpret, able to be used to systematically characterize localization patterns, and generally applicable. TOS is calculated by: (i) measuring the overlap of pixels that are above the intensity thresholds for two signals; (ii) determining whether the overlap is more, less, or the same as expected by chance, i.e. colocalization, anti-colocalization, or non-colocalization; and (iii) rescaling to allow comparison at different thresholds. The above is repeated at multiple threshold combinations to generate a TOS matrix to systematically characterize the relationship between localization and signal intensities. TOS matrices were used to identify and distinguish localization patterns of different proteins in various simulations, cell types and organisms with greater specificity and sensitivity than common metrics. For all the above reasons, TOS is an excellent first line metric, particularly for cells with mixed localization patterns. PMID:27979831

  17. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    PubMed

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

  18. Confocal imaging at the nanoscale with two-color STED microscopy

    NASA Astrophysics Data System (ADS)

    Gugel, Hilmar; Giske, Arnold; Dyba, Marcus; Sieber, Jochen

    2011-03-01

    STED microscopy enables confocal imaging of biological samples with a resolution that is not limited by diffraction. It provides new insights in various fields of biology, such as membrane biology, neurobiology and physiology. Its three dimensional sectioning ability allows the acquisition of high resolution image stacks. Furthermore, STED microscopy enables the recording of dynamic processes and live cell images. We present two-color imaging in confocal STED microscopy with a single STED wavelength. Pulsed and continuous wave lasers in the visible and near infra-red wavelengths range are used for stimulated emission. The resolution enhancement is demonstrated in comparison to confocal imaging with biological specimens.

  19. Principles of phosphorescent organic light emitting devices.

    PubMed

    Minaev, Boris; Baryshnikov, Gleb; Agren, Hans

    2014-02-07

    Organic light-emitting device (OLED) technology has found numerous applications in the development of solid state lighting, flat panel displays and flexible screens. These applications are already commercialized in mobile phones and TV sets. White OLEDs are of especial importance for lighting; they now use multilayer combinations of organic and elementoorganic dyes which emit various colors in the red, green and blue parts of the visible spectrum. At the same time the stability of phosphorescent blue emitters is still a major challenge for OLED applications. In this review we highlight the basic principles and the main mechanisms behind phosphorescent light emission of various classes of photofunctional OLED materials, like organic polymers and oligomers, electron and hole transport molecules, elementoorganic complexes with heavy metal central ions, and clarify connections between the main features of electronic structure and the photo-physical properties of the phosphorescent OLED materials.

  20. Towards Building Computerized Image Analysis Framework for Nucleus Discrimination in Microscopy Images of Diffuse Glioma

    PubMed Central

    Kong, Jun; Cooper, Lee; Kurc, Tahsin; Brat, Daniel; Saltz, Joel

    2012-01-01

    As an effort to build an automated and objective system for pathologic image analysis, we present, in this paper, a computerized image processing method for identifying nuclei, a basic biological unit of diagnostic utility, in microscopy images of glioma tissue samples. The complete analysis includes multiple processing steps, involving mode detection with color and spatial information for pixel clustering, background normalization leveraging morphological operations, boundary refinement with deformable models, and clumped nuclei separation using watershed. In aggregate, our validation dataset includes 220 nuclei from 11 distinct tissue regions selected at random by an experienced neuropathologist. Computerized nuclei detection results are in good concordance with human markups by both visual appraisement and quantitative measures. We compare the performance of the proposed analysis algorithm with that of CellProfiler, a classical analysis software for cell image process, and present the superiority of our method to CellProfiler. PMID:22255853

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

    PubMed

    Giacomelli, Michael G; 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.

  2. 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

  3. Magnetic Force Microscopy Images of Magnetic Garnet With Thin-Film Magnetic Tip

    NASA Technical Reports Server (NTRS)

    Wadas, A.; Moreland, J.; Rice, P.; Katti, R.

    1993-01-01

    We present magnetic force microscopy images of YGdTmGa/YSmTmGa magnetic garnet, usinga thin Fe film deposited on Si_3N_5 tips. We have found correlations between the topography andthe magnetic domain structure. We have observed the domain wall contrast with a iron thin-film tip. We report on domain wall imaging of garnet with magnetic force microscopy.

  4. Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.

    PubMed

    Bottomley, Amy L; Turnbull, Lynne; Whitchurch, Cynthia B; Harry, Elizabeth J

    2017-01-01

    Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

  5. Coherent total internal reflection dark-field microscopy: label-free imaging beyond the diffraction limit.

    PubMed

    von Olshausen, Philipp; Rohrbach, Alexander

    2013-10-15

    Coherent imaging is barely applicable in life-science microscopy due to multiple interference artifacts. Here, we show how these interferences can be used to improve image resolution and contrast. We present a dark-field microscopy technique with evanescent illumination via total internal reflection that delivers high-contrast images of coherently scattering samples. By incoherent averaging of multiple coherent images illuminated from different directions we can resolve image structures that remain unresolved by conventional (incoherent) fluorescence microscopy. We provide images of 190 nm beads revealing resolution beyond the diffraction limit and slightly increased object distances. An analytical model is introduced that accounts for the observed effects and which is confirmed by numerical simulations. Our approach may be a route to fast, label-free, super-resolution imaging in live-cell microscopy.

  6. 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.

  7. Super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging

    NASA Astrophysics Data System (ADS)

    Wei, Lu; Zhu, Xinxin; Chen, Zhixing; Min, Wei

    2014-02-01

    Two-photon excited fluorescence microscopy (TPFM) offers the highest penetration depth with subcellular resolution in light microscopy, due to its unique advantage of nonlinear excitation. However, a fundamental imaging-depth limit, accompanied by a vanishing signal-to-background contrast, still exists for TPFM when imaging deep into scattering samples. Formally, the focusing depth, at which the in-focus signal and the out-of-focus background are equal to each other, is defined as the fundamental imaging-depth limit. To go beyond this imaging-depth limit of TPFM, we report a new class of super-nonlinear fluorescence microscopy for high-contrast deep tissue imaging, including multiphoton activation and imaging (MPAI) harnessing novel photo-activatable fluorophores, stimulated emission reduced fluorescence (SERF) microscopy by adding a weak laser beam for stimulated emission, and two-photon induced focal saturation imaging with preferential depletion of ground-state fluorophores at focus. The resulting image contrasts all exhibit a higher-order (third- or fourth- order) nonlinear signal dependence on laser intensity than that in the standard TPFM. Both the physical principles and the imaging demonstrations will be provided for each super-nonlinear microscopy. In all these techniques, the created super-nonlinearity significantly enhances the imaging contrast and concurrently extends the imaging depth-limit of TPFM. Conceptually different from conventional multiphoton processes mediated by virtual states, our strategy constitutes a new class of fluorescence microscopy where high-order nonlinearity is mediated by real population transfer.

  8. Imaging photonic crystals using Fourier plane imaging and Fourier ptychographic microscopy techniques implemented with a computer controlled hemispherical digital condenser

    NASA Astrophysics Data System (ADS)

    Sen, Sanchari; Desai, Darshan B.; Alsubaie, Meznh H.; Zhelyeznyakov, Maksym V.; Molina, L.; Sarraf, Hamed Sari; Bernussi, Ayrton A.; Peralta, Luis Grave de

    2017-01-01

    Fourier plane imaging (FPIM) and Fourier ptychographic (FPM) microscopy techniques were used to image photonic crystals. A computer-controlled hemispherical digital condenser provided required sample illumination with variable inclination. Notable improvement in image resolution was obtained with both methods. However, it was determined that the FPM technique cannot surpass the Rayleigh resolution limit when imaging photonic crystals.

  9. Combined atomic force microscopy and scanning tunneling microscopy imaging of cross-sectioned GaN light-emitting diodes.

    PubMed

    Bender, J W; Salmon, M E; Russell, P E

    2003-01-01

    Cross-sectional scanning tunneling microscopy (STM) was combined with atomic force microscopy (AFM) over the same area to characterize a cross-sectioned GaN light emitting diode. Because GaN is typically grown on a non-native substrate and also forms a wurtzite crystal structure, a cryogenic cleaving technique was developed to generate smooth surfaces. The depletion region surrounding the p-n junction was clearly identified using STM. Furthermore, by imaging under multiple sample biases, distinctions between the n-doped and p-doped GaN could be made.

  10. Integral imaging microscopy with enhanced depth-of-field using a spatial multiplexing.

    PubMed

    Kwon, Ki-Chul; Erdenebat, Munkh-Uchral; Alam, Md Ashraful; Lim, Young-Tae; Kim, Kwang Gi; Kim, Nam

    2016-02-08

    A depth-of-field enhancement method for integral imaging microscopy system using a spatial multiplexing structure consisting of a beamsplitter with dual video channels and micro lens arrays is proposed. A computational integral imaging reconstruction algorithm generates two sets of depth-sliced images for the acquired depth information of the captured elemental image arrays and the well-focused depth-slices of both image sets are combined where each is focused on a different depth plane of the specimen. A prototype is implemented, and the experimental results demonstrate that the depth-of-field of the reconstructed images in the proposed integral imaging microscopy is significantly increased compared with conventional integral imaging microscopy systems.

  11. Endoscopic fluorescence lifetime imaging microscopy (FLIM) images of aortic plaque: an automated classification method

    NASA Astrophysics Data System (ADS)

    Phipps, Jennifer; Sun, Yinghua; Hatami, Nisa; Fishbein, Michael C.; Rajaram, Amit; Saroufeem, Ramez; Marcu, Laura

    2010-02-01

    The objective of this study was to develop an automated algorithm which uses fluorescence lifetime imaging microscopy (FLIM) images of human aortic atherosclerotic plaque to provide quantitative and spatial information regarding compositional features related to plaque vulnerability such as collagen degradation, lipid accumulation, and macrophage infiltration. Images were acquired through a flexible fiber imaging bundle with intravascular potential at two wavelength bands optimal to recognizing markers of vulnerability: F377: 377/55 nm and F460: 460/50 nm (center wavelength/bandwidth). A classification method implementing principal components analysis and linear discriminant analysis to correlate FLIM data sets with histopathology was validated on a training set and then used to classify a validation set of FLIM images. The output of this algorithm was a false-color image with each pixel color coded to represent the chemical composition of the sample. Surface areas occupied by elastin, collagen, and lipid components were then calculated and used to define the vulnerability of each imaged location. Four groups were defined: early lesion, stable, mildly vulnerable and extremely vulnerable. Each imaged location was categorized in one of the groups based on histopathology and classification results; sensitivities (SE) and specificities (SP) were calculated (SE %/SP %): early lesion: 95/96, stable: 71/97, mildly vulnerable: 75/94, and extremely vulnerable: 100/93. The capability of this algorithm to use FLIM images to quickly determine the chemical composition of atherosclerotic plaque, particularly related to vulnerability, further enhances the potential of this system for implementation as an intravascular diagnostic modality.

  12. A specific Tween-80-Rhodamine S-MWNTs phosphorescent reagent for the detection of trace calcitonin.

    PubMed

    Liu, Jia-Ming; Huang, Xiao-Mei; Zhang, Li-Hong; Zheng, Zhi-Yong; Lin, Xuan; Zhang, Xiao-Yang; Jiao, Li; Cui, Ma-Lin; Jiang, Shu-Lian; Lin, Shao-Qin

    2012-09-26

    The present study proposed a simple sensitive and specific immunoassay for the quantification of calcitonin (CT) in human serum with water-soluble multi-walled carbon nanotubes (MWNTs). The COOH group of MWNTs could react with the NH group of rhodamine S (Rhod.S) molecules to form Rhod.S-MWNTs, which could emit room temperature phosphorescence (RTP) on acetate cellulose membrane (ACM) and react with Tween-80 to form micellar compound. Tween-80-Rhod.S-MWNTs (TRM), as a phosphorescent labelling reagent, could dramatically enhance the RTP signal of the system. The developed TRM phosphorescent reagent was used to label anti-calcitonin antibody (Ab(CT)) to form the TRM-Ab(CT) labelling product, which could take high specific immunoreaction with CT, and the ΔI(p) (= I(p2)-I(p1), I(p2) and I(p1) were the phosphorescence intensity of the test solution and the blank sample, respectively) of the system was linear to the content of CT. Hence, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) was established for the determination of CT in human serum. This sensitive (limit of quantification (LOQ) was 8.0×10(-14) g mL(-1)), accurate, selective and precise method has been applied to determine CT in human serum and predict primary osteoporosis and fractures, with the results in good agreement with those obtained by chemiluminescence immunoassay (CLIA). Simultaneously, the structure of MWNTs was characterized with scanning electron microscopy (SEM) and infrared spectroscopy (IR), and the reaction mechanisms of both labelling Ab(CT) with TRM and SSRTPIA for the determination of trace CT were discussed.

  13. New developments in electron microscopy for serial image acquisition of neuronal profiles.

    PubMed

    Kubota, Yoshiyuki

    2015-02-01

    Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed.

  14. Dental caries imaging using hyperspectral stimulated Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Zi; Zheng, Wei; Jian, Lin; Huang, Zhiwei

    2016-03-01

    We report the development of a polarization-resolved hyperspectral stimulated Raman scattering (SRS) imaging technique based on a picosecond (ps) laser-pumped optical parametric oscillator system for label-free imaging of dental caries. In our imaging system, hyperspectral SRS images (512×512 pixels) in both fingerprint region (800-1800 cm-1) and high-wavenumber region (2800-3600 cm-1) are acquired in minutes by scanning the wavelength of OPO output, which is a thousand times faster than conventional confocal micro Raman imaging. SRS spectra variations from normal enamel to caries obtained from the hyperspectral SRS images show the loss of phosphate and carbonate in the carious region. While polarization-resolved SRS images at 959 cm-1 demonstrate that the caries has higher depolarization ratio. Our results demonstrate that the polarization resolved-hyperspectral SRS imaging technique developed allows for rapid identification of the biochemical and structural changes of dental caries.

  15. Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA**

    PubMed Central

    Baggaley, Elizabeth; Gill, Martin R; Green, Nicola H; Turton, David; Sazanovich, Igor V; Botchway, Stanley W; Smythe, Carl; Haycock, John W; Weinstein, Julia A; Thomas, Jim A

    2014-01-01

    The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging. PMID:24458590

  16. Biological imaging with nonlinear photothermal microscopy using a compact supercontinuum fiber laser source.

    PubMed

    He, Jinping; Miyazaki, Jun; Wang, Nan; Tsurui, Hiromichi; Kobayashi, Takayoshi

    2015-04-20

    Nonlinear photothermal microscopy is applied in the imaging of biological tissues stained with chlorophyll and hematoxylin. Experimental results show that this type of organic molecules, which absorb light but transform dominant part of the absorbed energy into heat, may be ideal probes for photothermal imaging without photochemical toxicity. Picosecond pump and probe pulses, with central wavelengths of 488 and 632 nm, respectively, are spectrally filtered from a compact supercontinuum fiber laser source. Based on the light source, a compact and sensitive super-resolution imaging system is constructed. Further more, the imaging system is much less affected by thermal blurring than photothermal microscopes with continuous-wave light sources. The spatial resolution of nonlinear photothermal microscopy is ~ 188 nm. It is ~ 23% higher than commonly utilized linear photothermal microscopy experimentally and ~43% than conventional optical microscopy theoretically. The nonlinear photothermal imaging technology can be used in the evaluation of biological tissues with high-resolution and contrast.

  17. Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques

    PubMed Central

    D’Arco, Annalisa; Brancati, Nadia; Ferrara, Maria Antonietta; Indolfi, Maurizio; Frucci, Maria; Sirleto, Luigi

    2016-01-01

    The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed. PMID:27231626

  18. Speckle-based volume holographic microscopy for optically sectioned multi-plane fluorescent imaging.

    PubMed

    Chen, Hsi-Hsun; Singh, Vijay Raj; Luo, Yuan

    2015-03-23

    Structured illumination microscopy has been widely used to reconstruct optically sectioned fluorescence images in wide-field fashion; however, it still requires axial scanning to obtain multiple depth information of a volumetric sample. In this paper, a new imaging scheme, called speckle-based volume holographic microscopy system, is presented. The proposed system incorporates volumetric speckle illumination and multiplexed volume holographic gratings to acquire multi-plane images with optical sectioning capability, without any axial scanning. We present the design, implementation, and experimental image data demonstrating the proposed system's ability to simultaneously obtain wide-field, optically sectioned, and multi-depth resolved images of fluorescently labeled microspheres and tissue structures.

  19. X-ray Phase Imaging Microscopy using a Fresnel Zone Plate and a Transmission Grating

    SciTech Connect

    Yashiro, Wataru; Momose, Atsushi; Takeuchi, Akihisa; Suzuki, Yoshio

    2010-06-23

    We report on a hard X-ray phase imaging microscopy (a phase-difference microscopy) that consists of an objective and a transmission grating. The simple optical system provides a quantitative phase image, and does not need a wave field mostly coherent on the objective. Our method has a spatial resolution almost same as that of the absorption contrast microscope image obtained by removing the grating. We demonstrate how our approach provides a phase image from experimentally obtained images. Our approach is attractive for easily appending a quantitative phase-sensitive mode to normal X-ray microscopes, and has potentially broad applications in biology and material sciences.

  20. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

    PubMed Central

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-01-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm. PMID:27375935

  1. Frames-Based Denoising in 3D Confocal Microscopy Imaging.

    PubMed

    Konstantinidis, Ioannis; Santamaria-Pang, Alberto; Kakadiaris, Ioannis

    2005-01-01

    In this paper, we propose a novel denoising method for 3D confocal microscopy data based on robust edge detection. Our approach relies on the construction of a non-separable frame system in 3D that incorporates the Sobel operator in dual spatial directions. This multidirectional set of digital filters is capable of robustly detecting edge information by ensemble thresholding of the filtered data. We demonstrate the application of our method to both synthetic and real confocal microscopy data by comparing it to denoising methods based on separable 3D wavelets and 3D median filtering, and report very encouraging results.

  2. Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs

    NASA Astrophysics Data System (ADS)

    Rowlands, Christopher J.; Bruns, Oliver T.; Bawendi, Moungi G.; So, Peter T. C.

    2015-06-01

    Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue.

  3. Objective, comparative assessment of the penetration depth of temporal-focusing microscopy for imaging various organs

    PubMed Central

    Rowlands, Christopher J.; Bruns, Oliver T.; Bawendi, Moungi G.; So, Peter T. C.

    2015-01-01

    Abstract. Temporal focusing is a technique for performing axially resolved widefield multiphoton microscopy with a large field of view. Despite significant advantages over conventional point-scanning multiphoton microscopy in terms of imaging speed, the need to collect the whole image simultaneously means that it is expected to achieve a lower penetration depth in common biological samples compared to point-scanning. We assess the penetration depth using a rigorous objective criterion based on the modulation transfer function, comparing it to point-scanning multiphoton microscopy. Measurements are performed in a variety of mouse organs in order to provide practical guidance as to the achievable penetration depth for both imaging techniques. It is found that two-photon scanning microscopy has approximately twice the penetration depth of temporal-focusing microscopy, and that penetration depth is organ-specific; the heart has the lowest penetration depth, followed by the liver, lungs, and kidneys, then the spleen, and finally white adipose tissue. PMID:25844509

  4. Imaging calcium carbonate distribution in human sweat pore in vivo using nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Xueqin; Gasecka, Alicja; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-03-01

    Nonlinear microscopies, including two-photon excited autofluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), were used to study individual human sweat pore morphology and topically applied antiperspirant salt penetration inside sweat pore, in vivo on human palms. Sweat pore inner morphology in vivo was imaged up to the depth of 100 μm by TPEF microscopy. The 3D penetration and distribution of "in situ calcium carbonate" (isCC), an antiperspirant salt model, was investigated using CARS microscopy.

  5. Combining multiphoton and CARS microscopy for skin imaging

    NASA Astrophysics Data System (ADS)

    Breunig, H. G.; Weinigel, M.; Kellner-Höfer, M.; Bückle, R.; Darvin, M. E.; Lademann, J.; König, K.

    2013-02-01

    Microscopic imaging based on multiphoton fluorescence, second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been realized in one common platform which is appropriate for use in hospitals. The different optical modalities non-invasively provide in vivo images from human skin with subcellular resolution, at different depths based on endogenous fluorescent, SHG-active molecules as well as non-fluorescent molecules with vibrational resonances at 2845 cm-1, in particular lipids. An overview of the system employing a Ti:sapphire laser and photonic crystal fiber to generate the excitation light as well as several imaging examples are presented.

  6. Rigidification or interaction-induced phosphorescence of organic molecules.

    PubMed

    Baroncini, Massimo; Bergamini, Giacomo; Ceroni, Paola

    2017-02-09

    Phosphorescent materials are mostly based on metal complexes. Metal-free organic molecules usually display phosphorescence only in a rigid matrix at 77 K. In the last few years, there has been increasing interest in the design of organic molecules displaying long-lived and highly intense room-temperature phosphorescence, an extremely difficult task since these two properties are generally conflicting. This review reports the most recent and tutorial examples of molecules that are weakly or non-phosphorescent in deaerated fluid solution and whose room temperature phosphorescence is switched on upon aggregation. The examples are divided into two classes according to the mechanism responsible for switching on phosphorescence: (i) rigidification by crystallization or by encapsulation in a polymeric matrix and (ii) interaction with other molecules of the same type (self-aggregation) or a different type by taking advantage of heavy-atom effects.

  7. Limited-view light sheet fluorescence microscopy for three dimensional volume imaging

    NASA Astrophysics Data System (ADS)

    Rasmi, C. K.; Mohan, Kavya; Madhangi, M.; Rajan, K.; Nongthomba, U.; Mondal, Partha P.

    2015-12-01

    We propose and demonstrate a limited-view light sheet microscopy (LV-LSM) for three dimensional (3D) volume imaging. Realizing that longer and frequent image acquisition results in significant photobleaching, we have taken limited angular views (18 views) of the macroscopic specimen and integrated with maximum likelihood (ML) technique for reconstructing high quality 3D volume images. Existing variants of light-sheet microscopy require both rotation and translation with a total of approximately 10-fold more views to render a 3D volume image. Comparatively, LV-LSM technique reduces data acquisition time and consequently minimizes light-exposure by many-folds. Since ML is a post-processing technique and highly parallelizable, this does not cost precious imaging time. Results show noise-free and high contrast volume images when compared to the state-of-the-art selective plane illumination microscopy.

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

    PubMed Central

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

    2017-01-01

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

  9. Fluorescence lifetime imaging microscopy for brain tumor image-guided surgery

    NASA Astrophysics Data System (ADS)

    Sun, Yinghua; Hatami, Nisa; Yee, Matthew; Phipps, Jennifer; Elson, Daniel S.; Gorin, Fredric; Schrot, Rudolph J.; Marcu, Laura

    2010-09-01

    We demonstrate for the first time the application of an endoscopic fluorescence lifetime imaging microscopy (FLIM) system to the intraoperative diagnosis of glioblastoma multiforme (GBM). The clinically compatible FLIM prototype integrates a gated (down to 0.2 ns) intensifier imaging system with a fiber-bundle (fiber image guide of 0.5 mm diameter, 10,000 fibers with a gradient index lens objective 0.5 NA, and 4 mm field of view) to provide intraoperative access to the surgical field. Experiments conducted in three patients undergoing craniotomy for tumor resection demonstrate that FLIM-derived parameters allow for delineation of tumor from normal cortex. For example, at 460+/-25-nm wavelength band emission corresponding to NADH/NADPH fluorescence, GBM exhibited a weaker florescence intensity (35% less, p-value <0.05) and a longer lifetime τGBM-Amean=1.59+/-0.24 ns than normal cortex τNC-Amean=1.28+/-0.04 ns (p-value <0.005). Current results demonstrate the potential use of FLIM as a tool for image-guided surgery of brain tumors.

  10. Making Microscopy Motivating, Memorable, & Manageable for Undergraduate Students with Digital Imaging Laboratories

    ERIC Educational Resources Information Center

    Weeks, Andrea; Bachman. Beverly; Josway, Sarah; North, Brittany; Tsuchiya, Mirian T.N.

    2013-01-01

    Microscopy and precise observation are essential skills that are challenging to teach effectively to large numbers of undergraduate biology students. We implemented student-driven digital imaging assignments for microscopy in a large enrollment laboratory for organismal biology. We detail how we promoted student engagement with the material and…

  11. Label-free biomedical imaging of lipids by stimulated Raman scattering microscopy.

    PubMed

    Ramachandran, Prasanna V; Mutlu, Ayse Sena; Wang, Meng C

    2015-01-05

    Advances in modern optical microscopy have provided unparalleled tools to study intracellular structure and function, yet visualizing lipid molecules within a cell remains challenging. Stimulated Raman Scattering (SRS) microscopy is a recently developed imaging modality that addresses this challenge. By selectively imaging the vibration of chemical moieties enriched in lipids, this technique allows for rapid imaging of lipid molecules in vivo without the need for perturbative extrinsic labels. SRS microscopy has been effectively employed in the study of fat metabolism, helping uncover novel regulators of lipid storage. This unit provides a brief introduction to the principle of SRS microscopy, and describes methods for its use in imaging lipids in cells, tissues, and whole organisms.

  12. Resolution of oblique-plane images in sectioning microscopy.

    PubMed

    Smith, C W; Botcherby, E J; Wilson, T

    2011-01-31

    Live biological specimens exhibit time-varying behavior on the microscale in all three dimensions. Although scanning confocal and two-photon microscopes are able to record three-dimensional image stacks through these specimens, they do so at relatively low speeds which limits the time resolution of the biological processes that can be observed. One way to improve the data acquisition rate is to image only the regions of a specimen that are of interest and so researchers have recently begun to acquire two-dimensional images of inclined planes or surfaces extending significantly into the z-direction. As the resolution is not uniform in x, y and z, the images possess non-isotropic resolution. We explore this theoretically and show that images of an oblique plane may contain spectral content that could not have been generated by specimen features lying wholly within the plane but must instead arise from a spatial variation in another direction. In some cases we find that the image contains frequencies three times higher than the resolution limit for in-plane features. We confirm this finding through numerical simulations and experiments on a novel, oblique-plane imaging system and suggest that care be taken in the interpretation of such images.

  13. Imaging proteins with atomic force microscopy: an overview.

    PubMed

    Silva, Luciano Paulino

    2005-08-01

    Atomic force microscopy (AFM) has become a common tool for biophysical studies of proteins; mainly due its property to perform characterizations near physiological conditions. The tertiary and quaternary structures, forces driving folding-unfolding processes, and secondary structure elements can be studied in their native environments allowing high resolution level associated with small distortions. This review outlines the operational principles and applications of AFM for protein biophysics.

  14. Three-dimensional super-resolution structured illumination microscopy with maximum a posteriori probability image estimation.

    PubMed

    Lukeš, Tomáš; Křížek, Pavel; Švindrych, Zdeněk; Benda, Jakub; Ovesný, Martin; Fliegel, Karel; Klíma, Miloš; Hagen, Guy M

    2014-12-01

    We introduce and demonstrate a new high performance image reconstruction method for super-resolution structured illumination microscopy based on maximum a posteriori probability estimation (MAP-SIM). Imaging performance is demonstrated on a variety of fluorescent samples of different thickness, labeling density and noise levels. The method provides good suppression of out of focus light, improves spatial resolution, and allows reconstruction of both 2D and 3D images of cells even in the case of weak signals. The method can be used to process both optical sectioning and super-resolution structured illumination microscopy data to create high quality super-resolution images.

  15. Three-dimensional differential interference contrast microscopy using synthetic aperture imaging

    PubMed Central

    Kim, Moonseok; Choi, Youngwoon; Fang-Yen, Christopher; Sung, Yongjin; Kim, Kwanhyung; Dasari, Ramachandra R.; Feld, Michael S.

    2012-01-01

    Abstract. We implement differential interference contrast (DIC) microscopy using high-speed synthetic aperture imaging that expands the passband of coherent imaging by a factor of 2.2. For an aperture synthesized coherent image, we apply for the numerical post-processing and obtain a high-contrast DIC image for arbitrary shearing direction and bias retardation. In addition, we obtain images at different depths without a scanning objective lens by numerically propagating the acquired coherent images. Our method achieves high-resolution and high-contrast 3-D DIC imaging of live biological cells. The proposed method will be useful for monitoring 3-D dynamics of intracellular particles. PMID:22463035

  16. Electron Microscopy and Image Analysis for Selected Materials

    NASA Technical Reports Server (NTRS)

    Williams, George

    1999-01-01

    This particular project was completed in collaboration with the metallurgical diagnostics facility. The objective of this research had four major components. First, we required training in the operation of the environmental scanning electron microscope (ESEM) for imaging of selected materials including biological specimens. The types of materials range from cyanobacteria and diatoms to cloth, metals, sand, composites and other materials. Second, to obtain training in surface elemental analysis technology using energy dispersive x-ray (EDX) analysis, and in the preparation of x-ray maps of these same materials. Third, to provide training for the staff of the metallurgical diagnostics and failure analysis team in the area of image processing and image analysis technology using NIH Image software. Finally, we were to assist in the sample preparation, observing, imaging, and elemental analysis for Mr. Richard Hoover, one of NASA MSFC's solar physicists and Marshall's principal scientist for the agency-wide virtual Astrobiology Institute. These materials have been collected from various places around the world including the Fox Tunnel in Alaska, Siberia, Antarctica, ice core samples from near Lake Vostoc, thermal vents in the ocean floor, hot springs and many others. We were successful in our efforts to obtain high quality, high resolution images of various materials including selected biological ones. Surface analyses (EDX) and x-ray maps were easily prepared with this technology. We also discovered and used some applications for NIH Image software in the metallurgical diagnostics facility.

  17. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

    PubMed

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  18. Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C.; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E.

    2015-05-01

    In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1±2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6±8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

  19. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms

    PubMed Central

    Bouchard, Matthew B.; Voleti, Venkatakaushik; Mendes, César S.; Lacefield, Clay; Grueber, Wesley B.; Mann, Richard S.; Bruno, Randy M.; Hillman, Elizabeth M. C.

    2014-01-01

    We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae. PMID:25663846

  20. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.

    PubMed

    Bouchard, Matthew B; Voleti, Venkatakaushik; Mendes, César S; Lacefield, Clay; Grueber, Wesley B; Mann, Richard S; Bruno, Randy M; Hillman, Elizabeth M C

    2015-02-01

    We report a new 3D microscopy technique that allows volumetric imaging of living samples at ultra-high speeds: Swept, confocally-aligned planar excitation (SCAPE) microscopy. While confocal and two-photon microscopy have revolutionized biomedical research, current implementations are costly, complex and limited in their ability to image 3D volumes at high speeds. Light-sheet microscopy techniques using two-objective, orthogonal illumination and detection require a highly constrained sample geometry, and either physical sample translation or complex synchronization of illumination and detection planes. In contrast, SCAPE microscopy acquires images using an angled, swept light-sheet in a single-objective, en-face geometry. Unique confocal descanning and image rotation optics map this moving plane onto a stationary high-speed camera, permitting completely translationless 3D imaging of intact samples at rates exceeding 20 volumes per second. We demonstrate SCAPE microscopy by imaging spontaneous neuronal firing in the intact brain of awake behaving mice, as well as freely moving transgenic Drosophila larvae.

  1. Red long-lasting phosphorescence based on color conversion process

    NASA Astrophysics Data System (ADS)

    Li, Zhanjun; Zhang, Hongwu; Fu, Haixia

    2013-01-01

    The principle of color conversion process was used to generate red long-lasting phosphorescence (LLP) using SrAl2O4:Eu, Dy (SAO) as primary light source and rhodamine B encapsulated mesoporous silica nanoparticles (MCM-R) as effective color conversion agent. The phosphorescence spectra of MCM-R/SAO hybrid samples show green peaks from 425 nm to 550 nm and red peaks from 550 nm to 700 nm, which can be attributed to the phosphorescence of SAO and the fluorescence of MCM-R, respectively. The phosphorescence color can be adjusted from green to red by changing the mass ratio of MCM-R/SAO. When the mass ratio of MCM-R/SAO increases from 0.05 to 1.5, a blue shift for the green peak and a red shift for the red peak of the phosphorescence spectra can be observed and the intensity of the red emission peak increase relatively towards the green one. The phosphorescence decay curves show that MCM-R and SAO have similar decay dynamics and the MCM-R can inherit the LLP properties of SAO. The phosphorescence decay spectra indicate that the MCM-R/SAO hybrid can retain constant and steady visual phosphorescence color. The red phosphorescence can be seen in the dark with naked eyes for more than 5 h. So, the red LLP can be successfully achieved based on the principle of color conversion process.

  2. Laser phosphoroscope and applications to room-temperature phosphorescence.

    PubMed

    Payne, Sarah J; Zhang, Guoqing; Demas, James N; Fraser, Cassandra L; Degraff, Ben A

    2011-11-01

    A simple phosphoroscope with no moving parts is described. In one scan the total luminescence, the long-lived phosphorescence, and the short-lived fluorescence can be determined. A 50% duty cycle excitation from a diode laser is used to excite the sample, and from the digitized waveform the phosphorescence is extracted from the off period, the total emission from the full cycle, and the fluorescence from the on period corrected for the phosphorescence contribution. The performance of the system is demonstrated using room-temperature phosphorescence of organic dyes in boric acid glasses, a multi-emissive boron-polymer dye, and a europium chelate.

  3. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

    PubMed

    Xu, Jianquan; Tehrani, Kayvan F; Kner, Peter

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.

  4. Bacterial Immobilization for Imaging by Atomic Force Microscopy

    SciTech Connect

    Allison, David P; Sullivan, Claretta; Mortensen, Ninell P; Retterer, Scott T; Doktycz, Mitchel John

    2011-01-01

    AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

  5. Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

    PubMed

    Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

    2014-03-01

    One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

  6. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking.

    PubMed

    Dragoi, Ioan-Catalin; Stanciu, Stefan G; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E; Popescu, Marius; Coltuc, Dinu

    2016-04-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail.

  7. Embedding complementary imaging data in laser scanning microscopy micrographs by reversible watermarking

    PubMed Central

    Dragoi, Ioan-Catalin; Stanciu, Stefan G.; Hristu, Radu; Coanda, Henri-George; Tranca, Denis E.; Popescu, Marius; Coltuc, Dinu

    2016-01-01

    Complementary laser scanning microscopy micrographs are considered as pairs consisting in a master image (MI) and a slave image (SI), the latter with potential for facilitating the interpretation of the MI. We propose a strategy based on reversible watermarking for embedding a lossy compressed version of the SI into the MI. The use of reversible watermarking ensures the exact recovery of the host image. By storing and/or transmitting the watermarked MI in a single file, the information contained in both images that constitute the pair is made available to a potential end-user, which simplifies data association and transfer. Examples are presented using support images collected by two complementary techniques, confocal scanning laser microscopy and transmission laser scanning microscopy, on Hematoxylin and Eosin stained tissue fragments. A strategy for minimizing the watermarking distortions of the MI, while preserving the content of the SI, is discussed in detail. PMID:27446641

  8. Quantitative characterization of articular cartilage using Mueller matrix imaging and multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Ellingsen, Pa˚L. Gunnar; Lilledahl, Magnus Borstad; Aas, Lars Martin Sandvik; Davies, Catharina De Lange; Kildemo, Morten

    2011-11-01

    The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.

  9. High-speed quantitative interferometric microscopy based phase imaging cytometer

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Sun, Nan; Yan, Keding; Liu, Fei; Wang, Shouyu

    2014-11-01

    The paper proposed a simple large scale bio-sample phase detecting equipment called gravity driven phase detecting cytometer, which is based on quantitative interferometric microscopy to realize flowing red blood cells phase distribution detection. The method has advantages on high throughput phase detecting and statistical analysis with high detecting speed and in real-time. The statistical characteristics of red blood cells are useful for biological analysis and disease detection. We believe this method is shedding more light on quantitatively measurement of the phase distribution of bio-samples.

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

    NASA Astrophysics Data System (ADS)

    Han, Wenfeng; Xiao, Zexin; Wang, Xiaofen

    2009-11-01

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

  11. Whole slide imaging of unstained tissue using lensfree microscopy

    NASA Astrophysics Data System (ADS)

    Morel, Sophie Nhu An; Hervé, Lionel; Bordy, Thomas; Cioni, Olivier; Delon, Antoine; Fromentin, Catherine; Dinten, Jean-Marc; Allier, Cédric

    2016-04-01

    Pathologist examination of tissue slides provides insightful information about a patient's disease. Traditional analysis of tissue slides is performed under a binocular microscope, which requires staining of the sample and delays the examination. We present a simple cost-effective lensfree imaging method to record 2-4μm resolution wide-field (10 mm2 to 6 cm2) images of unstained tissue slides. The sample processing time is reduced as there is no need for staining. A wide field of view (10 mm2) lensfree hologram is recorded in a single shot and the image is reconstructed in 2s providing a very fast acquisition chain. The acquisition is multispectral, i.e. multiple holograms are recorded simultaneously at three different wavelengths, and a dedicated holographic reconstruction algorithm is used to retrieve both amplitude and phase. Whole tissue slides imaging is obtained by recording 130 holograms with X-Y translation stages and by computing the mosaic of a 25 x 25 mm2 reconstructed image. The reconstructed phase provides a phase-contrast-like image of the unstained specimen, revealing structures of healthy and diseased tissue. Slides from various organs can be reconstructed, e.g. lung, colon, ganglion, etc. To our knowledge, our method is the first technique that enables fast wide-field lensfree imaging of such unlabeled dense samples. This technique is much cheaper and compact than a conventional phase contrast microscope and could be made portable. In sum, we present a new methodology that could quickly provide useful information when a rapid diagnosis is needed, such as tumor margin identification on frozen section biopsies during surgery.

  12. Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

    PubMed Central

    Sakadžić, Sava; Roussakis, Emmanuel; Yaseen, Mohammad A.; Mandeville, Emiri T.; Srinivasan, Vivek J.; Arai, Ken; Ruvinskaya, Svetlana; Wu, Weicheng; Devor, Anna; Lo, Eng H.; Vinogradov, Sergei A.; Boas, David A.

    2011-01-01

    Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury. Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a

  13. Towards simultaneous single emission microscopy and magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Cai, Liang

    In recent years, the combined nuclear imaging and magnetic resonance imaging (MRI) has drawn extensive research effort. They can provide simultaneously acquired anatomical and functional information inside the human/small animal body in vivo. In this dissertation, the development of an ultrahigh resolution MR-compatible SPECT (Single Photon Emission Computed Tomography) system that can be operated inside a pre-existing clinical MR scanner for simultaneous dual-modality imaging of small animals will be discussed. This system is constructed with 40 small pixel CdTe detector modules assembled in a fully stationary ring SPECT geometry. Series of experiments have demonstrated that this system is capable of providing an imaging resolution of <500?m, when operated inside MR scanners. The ultrahigh resolution MR-compatible SPECT system is built around a small pixel CdTe detector module that we recently developed. Each module consists of CdTe detectors having an overall size of 2.2 cm x 1.1 cm, divided into 64 x 32 pixels of 350 mum in size. A novel hybrid pixel-waveform (HPWF) readout system is also designed to alleviate several challenges for using small-pixel CdTe detectors in ultrahigh-resolution SPECT imaging applications. The HPWF system utilizes a modified version of a 2048-channel 2-D CMOS ASIC to readout the anode pixel, and a digitizing circuitry to sample the signal waveform induced on the cathode. The cathode waveform acquired with the HPWF circuitry offers excellent spatial resolution, energy resolution and depth of interaction (DOI) information, even with the presence of excessive charge-sharing/charge-loss between the small anode pixels. The HPWF CdTe detector is designed and constructed with a minimum amount of ferromagnetic materials, to ensure the MR-compatibility. To achieve sub-500?m imaging resolution, two special designed SPECT apertures have been constructed with different pinhole sizes of 300?m and 500?m respectively. It has 40 pinhole inserts that

  14. Imaging of plant cell walls by confocal Raman microscopy.

    PubMed

    Gierlinger, Notburga; Keplinger, Tobias; Harrington, Michael

    2012-09-01

    Raman imaging of plant cell walls represents a nondestructive technique that can provide insights into chemical composition in context with structure at the micrometer level (<0.5 μm). The major steps of the experimental procedure are described: sample preparation (embedding and microcutting), setting the mapping parameters, and finally the calculation of chemical images on the basis of the acquired Raman spectra. Every Raman image is based on thousands of spectra, each being a spatially resolved molecular 'fingerprint' of the cell wall. Multiple components are analyzed within the native cell walls, and insights into polymer composition as well as the orientation of the cellulose microfibrils can be gained. The most labor-intensive step of this process is often the sample preparation, as the imaging approach requires a flat surface of the plant tissue with intact cell walls. After finishing the map (acquisition time is ∼10 min to 10 h, depending on the size of the region of interest and scanning parameters), many possibilities exist for the analysis of spectral data and image generation.

  15. Imaging of Protein Crystals with Two-Photon Microscopy

    SciTech Connect

    Padayatti, Pius; Palczewska, Grazyna; Sun, Wenyu; Palczewski, Krzysztof; Salom, David

    2012-05-02

    Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as 10 {mu}m in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials.

  16. Invited Review Article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy

    PubMed Central

    Carriles, Ramón; Schafer, Dawn N.; Sheetz, Kraig E.; Field, Jeffrey J.; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W.; Squier, Jeffrey A.

    2009-01-01

    We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences. PMID:19725639

  17. Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy.

    PubMed

    Carriles, Ramón; Schafer, Dawn N; Sheetz, Kraig E; Field, Jeffrey J; Cisek, Richard; Barzda, Virginijus; Sylvester, Anne W; Squier, Jeffrey A

    2009-08-01

    We review the current state of multiphoton microscopy. In particular, the requirements and limitations associated with high-speed multiphoton imaging are considered. A description of the different scanning technologies such as line scan, multifoci approaches, multidepth microscopy, and novel detection techniques is given. The main nonlinear optical contrast mechanisms employed in microscopy are reviewed, namely, multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation. Techniques for optimizing these nonlinear mechanisms through a careful measurement of the spatial and temporal characteristics of the focal volume are discussed, and a brief summary of photobleaching effects is provided. Finally, we consider three new applications of multiphoton microscopy: nonlinear imaging in microfluidics as applied to chemical analysis and the use of two-photon absorption and self-phase modulation as contrast mechanisms applied to imaging problems in the medical sciences.

  18. Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann; Kuo, Chun-Hung; Holland, Daniel B.; Chen, Yenyu; Ouyang, Mingxing; Blake, Geoffrey A.; Zadoyan, Ruben; Guo, Chin-Lin

    2011-11-01

    Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

  19. Observation of Localized Corrosion of Ni-Based Alloys Using Coupled Orientation Imaging Microscopy and Atomic Force Microscopy

    SciTech Connect

    Bedrossian, P.J.

    1999-11-24

    We present a method for assessing the relative vulnerabilities of distinct classes of grain boundaries to localized corrosion. Orientation imaging microscopy provides a spatial map which identifies and classifies grain boundaries at a metal surface. Once the microstructure of a region of a sample surface has been characterized, a sample can be exposed to repeated cycles of exposure to a corrosive environment alternating with topographic measurement by an atomic force microscope in the same region in which the microstructure had been mapped. When this procedure is applied to Ni and Ni-based alloys, we observe enhanced attack at random grain boundaries relative to special boundaries and twins in a variety of environments.

  20. Automatic segmentation and classification of human intestinal parasites from microscopy images.

    PubMed

    Suzuki, Celso T N; Gomes, Jancarlo F; Falcão, Alexandre X; Papa, João P; Hoshino-Shimizu, Sumie

    2013-03-01

    Human intestinal parasites constitute a problem in most tropical countries, causing death or physical and mental disorders. Their diagnosis usually relies on the visual analysis of microscopy images, with error rates that may range from moderate to high. The problem has been addressed via computational image analysis, but only for a few species and images free of fecal impurities. In routine, fecal impurities are a real challenge for automatic image analysis. We have circumvented this problem by a method that can segment and classify, from bright field microscopy images with fecal impurities, the 15 most common species of protozoan cysts, helminth eggs, and larvae in Brazil. Our approach exploits ellipse matching and image foresting transform for image segmentation, multiple object descriptors and their optimum combination by genetic programming for object representation, and the optimum-path forest classifier for object recognition. The results indicate that our method is a promising approach toward the fully automation of the enteroparasitosis diagnosis.

  1. Imaging of Microwave Circuits Using Near-Field Microwave Microscopy

    NASA Astrophysics Data System (ADS)

    Anlage, Steven M.; Dutta, Sudeep; Vlahacos, C. P.; Steinhauer, David E.; Wellstood, F. C.

    1997-03-01

    Detailed models and simulations have been a major tool in the development and evaluation of microwave devices (e.g. circulators, superconducting filters, antennas). However actual quantitative measurements of performance are generally limited to global characteristics (such as reflection/transmission coefficients) leaving the models unverified in detail. With the near-field scanning microwave microscope(C. P. Vlahacos, et al.) Appl. Phys. Lett. 69 (21), 3272 (1996) (which uses an open-ended coaxial cable scanned in proximity to the surface to be imaged), we present a method of probing the internal fields of devices. By imaging simple device configurations (e.g. microstrip), where field patterns are easily calculated, we show how quantitative values for the electric field and potential can be calculated directly from the data. Resolution is dependent on the size of the coaxial cable, and has a limit of approximately 20 μm. Images of both data and models will be shown for comparison.

  2. Neural imaging in songbirds using fiber optic fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Nooshabadi, Fatemeh; Hearn, Gentry; Lints, Thierry; Maitland, Kristen C.

    2012-02-01

    The song control system of juvenile songbirds is an important model for studying the developmental acquisition and generation of complex learned vocal motor sequences, two processes that are fundamental to human speech and language. To understand the neural mechanisms underlying song production, it is critical to characterize the activity of identified neurons in the song control system when the bird is singing. Neural imaging in unrestrained singing birds, although technically challenging, will advance our understanding of neural ensemble coding mechanisms in this system. We are exploring the use of a fiber optic microscope for functional imaging in the brain of behaving and singing birds in order to better understand the contribution of a key brain nucleus (high vocal center nucleus; HVC) to temporal aspects of song motor control. We have constructed a fluorescence microscope with LED illumination, a fiber bundle for transmission of fluorescence excitation and emission light, a ~2x GRIN lens, and a CCD for image acquisition. The system has 2 μm resolution, 375 μm field of view, 200 μm working distance, and 1 mm outer diameter. As an initial characterization of this setup, neurons in HVC were imaged using the fiber optic microscope after injection of quantum dots or fluorescent retrograde tracers into different song nuclei. A Lucid Vivascope confocal microscope was used to confirm the imaging results. Long-term imaging of the activity of these neurons in juvenile birds during singing may lead us to a better understanding of the central motor codes for song and the central mechanism by which auditory experience modifies song motor commands to enable vocal learning and imitation.

  3. Transillumination spatially modulated illumination microscopy for human chromosome imaging

    NASA Astrophysics Data System (ADS)

    Pitris, Costas; Heracleous, Peter; Patsalis, Philippos

    2005-03-01

    Human chromosome analysis is an essential task in cytogenetics, especially in prenatal screening, genetic syndrome diagnosis, cancer pathology research and mutagen dosimetry. Chromosomal analysis begins with the creation of a karyotype, which is a layout of chromosome images organized by decreasing size in pairs. Both manual and automatic classification of chromosomes are limited by the resolution of the microscope and imaging system used. One way to improve the results of classification and even detect subtleties now remaining undetected, is to enhance the resolution of the images. It is possible to achieve lateral resolution beyond the classical limit, by using spatially modulated illumination (SMI) in a wide-field, non-confocal microscope. In this case, the sample is illuminated with spatially modulated light, which makes normally inaccessible high-resolution information visible in the observed image by shifting higher frequencies within the OTF limits of the microscope. Although, SMI microscopes have been reported in the past, this manuscript reports the development of a transillumination microscope for opaque, non-fluorescent samples. The illumination path consisted of a light source illuminating a ruled grating which was subsequently imaged on the sample. The grating was mounted on a rotating and translating stage so that the magnification and rotation of the pattern could be adjusted. The imaging lens was a 1.25 NA oil immersion objective. Test samples showed resolution improvement, as judged from a comparison of the experimentally obtained FWHM. Further studies using smaller fringe distance or laser interference pattern illumination will be evaluated to further optimize the SMI results.

  4. Simulation of bright-field microscopy images depicting pap-smear specimen

    PubMed Central

    Malm, Patrik; Brun, Anders; Bengtsson, Ewert

    2015-01-01

    As digital imaging is becoming a fundamental part of medical and biomedical research, the demand for computer-based evaluation using advanced image analysis is becoming an integral part of many research projects. A common problem when developing new image analysis algorithms is the need of large datasets with ground truth on which the algorithms can be tested and optimized. Generating such datasets is often tedious and introduces subjectivity and interindividual and intraindividual variations. An alternative to manually created ground-truth data is to generate synthetic images where the ground truth is known. The challenge then is to make the images sufficiently similar to the real ones to be useful in algorithm development. One of the first and most widely studied medical image analysis tasks is to automate screening for cervical cancer through Pap-smear analysis. As part of an effort to develop a new generation cervical cancer screening system, we have developed a framework for the creation of realistic synthetic bright-field microscopy images that can be used for algorithm development and benchmarking. The resulting framework has been assessed through a visual evaluation by experts with extensive experience of Pap-smear images. The results show that images produced using our described methods are realistic enough to be mistaken for real microscopy images. The developed simulation framework is very flexible and can be modified to mimic many other types of bright-field microscopy images. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of ISAC PMID:25573002

  5. Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution.

    PubMed

    Azubel, Maia; Koivisto, Jaakko; Malola, Sami; Bushnell, David; Hura, Greg L; Koh, Ai Leen; Tsunoyama, Hironori; Tsukuda, Tatsuya; Pettersson, Mika; Häkkinen, Hannu; Kornberg, Roger D

    2014-08-22

    Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102-gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory.

  6. Fluorescence Cell Imaging and Manipulation Using Conventional Halogen Lamp Microscopy

    PubMed Central

    Yamagata, Kazuo; Iwamoto, Daisaku; Terashita, Yukari; Li, Chong; Wakayama, Sayaka; Hayashi-Takanaka, Yoko; Kimura, Hiroshi; Saeki, Kazuhiro; Wakayama, Teruhiko

    2012-01-01

    Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries. PMID:22347500

  7. Nanoscale imaging of Bacillus thuringiensis flagella using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Gillis, Annika; Dupres, Vincent; Delestrait, Guillaume; Mahillon, Jacques; Dufrêne, Yves F.

    2012-02-01

    Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in cell surface appendages.Because bacterial flagella play essential roles in various processes (motility, adhesion, host interactions, secretion), studying their expression in relation to function is an important challenge. Here, we use atomic force microscopy (AFM) to gain insight into the nanoscale surface properties of two wild-type and four mutant strains of Bacillus thuringiensis exhibiting various levels of flagellation. We show that, unlike AFM in liquid, AFM in air is a simple and reliable approach to observe the morphological details of the bacteria, and to quantify the density and dimensions of their flagella. We found that the amount of flagella expressed by the six strains, as observed at the nanoscale, correlates with their microscopic swarming motility. These observations provide novel information on flagella expression in Gram-positive bacteria and demonstrate the power of AFM in genetic studies for the fast assessment of the phenotypic characteristics of bacterial strains altered in

  8. Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

    NASA Astrophysics Data System (ADS)

    Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

    2011-01-01

    We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

  9. Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

    PubMed Central

    Korobchevskaya, Kseniya; Peres, Chiara; Li, Zhibin; Antipov, Alexei; Sheppard, Colin J. R.; Diaspro, Alberto; Bianchini, Paolo

    2016-01-01

    We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used. PMID:27174367

  10. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene

    PubMed Central

    Stefaniuk, Marzena; Gualda, Emilio J.; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  11. Subsurface defect of amorphous carbon film imaged by near field acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Zeng, J. T.; Zhao, K. Y.; Zeng, H. R.; Song, H. Z.; Zheng, L. Y.; Li, G. R.; Yin, Q. R.

    2008-05-01

    Amorphous carbon films were examined by low frequency scanning-probe acoustic microscopy (LF-SPAM). Local elastic properties as well as topography were imaged in the acoustic mode. Two kinds of subsurface defects were revealed by the LF-SPAM method. The influence of the subsurface defects on the elastic properties was also discussed. The ability to image subsurface defects was dependent on the scan area and the scan speed. Our results showed that the low frequency scanning-probe acoustic microscopy is a useful method for imaging subsurface defects with high resolution.

  12. Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development

    PubMed Central

    Bassi, Andrea; Schmid, Benjamin; Huisken, Jan

    2015-01-01

    Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs. PMID:25655702

  13. Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

    NASA Astrophysics Data System (ADS)

    Korobchevskaya, Kseniya; Peres, Chiara; Li, Zhibin; Antipov, Alexei; Sheppard, Colin J. R.; Diaspro, Alberto; Bianchini, Paolo

    2016-05-01

    We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used.

  14. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    SciTech Connect

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  15. A 3D imaging and visualization workflow, using confocal microscopy and advanced image processing for brachyuran crab larvae.

    PubMed

    Kamanli, S A; Kihara, T C; Ball, A D; Morritt, D; Clark, P F

    2017-03-07

    Confocal laser scanning microscopy is an excellent tool for nondestructive imaging of arthropods and can provide detailed information on morphology including fine surface detail. A methodology is presented here for the visualization by confocal microscopy of arthropods, using brachyuran crab zoeal stages as examples and postprocessing techniques derived from micro-CT protocols to improve the final images. This protocol is divided into description of the preprocessing steps (cleaning, staining, digesting and mounting), confocal laser scanning microscopy and data visualization using open-source, freeware programs ImageJ and Drishti. The advantages of using ImageJ to standardize stack data and Drishti for surface rendering are discussed. The methodology has been comprehensively tested using data acquired from all four brands of confocal microscope (Leica, Nikon, Olympus and Zeiss).

  16. Denoising time-resolved microscopy image sequences with singular value thresholding.

    PubMed

    Furnival, Tom; Leary, Rowan K; Midgley, Paul A

    2016-05-10

    Time-resolved imaging in microscopy is important for the direct observation of a range of dynamic processes in both the physical and life sciences. However, the image sequences are often corrupted by noise, either as a result of high frame rates or a need to limit the radiation dose received by the sample. Here we exploit both spatial and temporal correlations using low-rank matrix recovery methods to denoise microscopy image sequences. We also make use of an unbiased risk estimator to address the issue of how much thresholding to apply in a robust and automated manner. The performance of the technique is demonstrated using simulated image sequences, as well as experimental scanning transmission electron microscopy data, where surface adatom motion and nanoparticle structural dynamics are recovered at rates of up to 32 frames per second.

  17. Point scanning confocal microscopy facilitates 3D human hair follicle imaging in tissue sections.

    PubMed

    Kloepper, Jennifer E; Bíró, Tamás; Paus, Ralf; Cseresnyés, Zoltán

    2010-07-01

    Efficiency is a key factor in determining whether a scientific method becomes widely accepted in practical applications. In dermatology, morphological characterisation of intact hair follicles by traditional methods can be rather inefficient. Samples are embedded, sliced, imaged and digitally reconstructed, which can be time-consuming. Confocal microscopy, on the other hand, is more efficient and readily applicable to study intact hair follicles. Modern confocal microscopes deliver and collect light very efficiently and thus allow high spatial resolution imaging of relatively thick samples. In this letter, we report that we successfully imaged entire intact human hair follicles using point scanning confocal microscopy. Light delivery and light-collection were further improved by preparing the samples in 2,2'-Thiodiethanol (TDE), thus reducing refractive index gradients. The relatively short total scan times and the high quality of the acquired 3D images make confocal microscopy a desirable method for studying intact hair follicles under normal and pathological conditions.

  18. Comparing Fourier optics and contrast transfer function modeling of image formation in low energy electron microscopy.

    PubMed

    Yu, K M; Locatelli, A; Altman, M S

    2017-03-24

    A theoretical understanding of image formation in cathode lens microscopy can facilitate image interpretation. We compare Fourier Optics (FO) and Contrast Transfer Function (CTF) approaches that were recently adapted from other realms of microscopy to model image formation in low energy electron microscopy (LEEM). Although these two approaches incorporate imaging errors from several sources similarly, they differ in the way that the image intensity is calculated. The simplification that is used in the CTF calculation advantageously leads to its computational efficiency. However, we find that lens aberrations, and spatial and temporal coherence may affect the validity of the CTF approach to model LEEM image formation under certain conditions. In particular, these effects depend strongly on the nature of the object being imaged and also become more pronounced with increasing defocus. While the use of the CTF approach appears to be justified for objects that are routinely imaged with LEEM, comparison of theory to experimental observations of a focal image series for rippled, suspended graphene reveals one example where FO works, but CTF does not. This work alerts us to potential pitfalls and guides the effective use of FO and CTF approaches. It also lays the foundation for quantitative image evaluation using these methods.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  20. Quantitative 3D imaging of whole, unstained cells by using X-ray diffraction microscopy.

    PubMed

    Jiang, Huaidong; Song, Changyong; Chen, Chien-Chun; Xu, Rui; Raines, Kevin S; Fahimian, Benjamin P; Lu, Chien-Hung; Lee, Ting-Kuo; Nakashima, Akio; Urano, Jun; Ishikawa, Tetsuya; Tamanoi, Fuyuhiko; Miao, Jianwei

    2010-06-22

    Microscopy has greatly advanced our understanding of biology. Although significant progress has recently been made in optical microscopy to break the diffraction-limit barrier, reliance of such techniques on fluorescent labeling technologies prohibits quantitative 3D imaging of the entire contents of cells. Cryoelectron microscopy can image pleomorphic structures at a resolution of 3-5 nm, but is only applicable to thin or sectioned specimens. Here, we report quantitative 3D imaging of a whole, unstained cell at a resolution of 50-60 nm by X-ray diffraction microscopy. We identified the 3D morphology and structure of cellular organelles including cell wall, vacuole, endoplasmic reticulum, mitochondria, granules, nucleus, and nucleolus inside a yeast spore cell. Furthermore, we observed a 3D structure protruding from the reconstructed yeast spore, suggesting the spore germination process. Using cryogenic technologies, a 3D resolution of 5-10 nm should be achievable by X-ray diffraction microscopy. This work hence paves a way for quantitative 3D imaging of a wide range of biological specimens at nanometer-scale resolutions that are too thick for electron microscopy.

  1. Assessing the imaging performance of light sheet microscopies in highly scattering tissues

    PubMed Central

    Glaser, A. K.; Wang, Y.; Liu, J. T.C.

    2016-01-01

    Light sheet microscopy (LSM) has emerged as an optical-imaging method for high spatiotemporal volumetric imaging of relatively transparent samples. While this capability has allowed the technique to be highly impactful in fields such as developmental biology, applications involving highly scattering thick tissues have been largely unexplored. Herein, we employ Monte Carlo simulations to explore the use of LSM for imaging turbid media. In particular, due to its similarity to dual-axis confocal (DAC) microscopy, we compare LSM performance to point-scanned (PS-DAC) and line-scanned (LS-DAC) dual-axis confocal microscopy techniques that have been previously shown to produce high-quality images at round-trip optical lengths of ~9 – 10 and ~3 – 4 respectively. The results of this study indicate that LSM using widefield collection (WF-LSM) provides comparable performance to LS-DAC in thick tissues, due to the fact that they both utilize an illumination beam focused in one dimension (i.e. a line or sheet). On the other hand, LSM using confocal line detection (CL-LSM) is more analogous to PS-DAC microscopy, in which the illumination beam is focused in two dimensions to a point. The imaging depth of LSM is only slightly inferior to DAC (~2 – 3 and ~6 – 7 optical lengths for WF-LSM and CL-LSM respectively) due to the use of a lower numerical aperture (NA) illumination beam for extended imaging along the illumination axis. Therefore, we conclude that the ability to image deeply is dictated most by the confocality of the microscope technique. In addition, we find that imaging resolution is mostly dependent on the collection NA, and is relatively invariant to imaging depth in a homogeneous scattering medium. Our results indicate that superficial imaging of highly scattering tissues using light sheet microscopy is possible. PMID:26977355

  2. Dynamic structured illumination microscopy: Focused imaging and optical sectioning for moving objects

    NASA Astrophysics Data System (ADS)

    Krzewina, Leo G.; Kim, Myung K.

    2006-02-01

    Structured illumination microscopy (SIM) is a valuable tool for three-dimensional microscopy and has numerous applications in bioscience. Its success has been limited to static objects, though, as three sequential image acquisitions are required per final processed, focused image. To overcome this problem we have developed a multicolored grid which when used in tandem with a color camera is capable of performing SIM with just a single exposure. Images and movies demonstrating optical sectioning of three-dimensional objects are presented, and results of applying color SIM for wide-field focused imaging are compared to those of SIM. From computer modeling and analytical calculations a theoretical estimate of the maximum observable object velocity in both the lateral and axial directions is available, implying that the new method will be capable of imaging a variety of live objects. Sample images of the technique applied to lens paper and a pigeon feather are included to show both advantages and disadvantages of CSIM.

  3. Three-dimensional tooth imaging using multiphoton and second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Min-Huey; Chen, Wei-Liang; Sun, Yen; Fwu, Peter Tramyeon; Lin, Ming-Gu; Dong, Chen-Yuan

    2007-02-01

    Detailed morphological and cellular information relating to the human tooth have traditionally been obtained through histological studies that required decalcification, staining, and fixation. With the recent invention of multiphoton microscopy, it has become possible to acquire high resolution images without histological procedures. Using an epiilluminated multiphoton microscope, we obtained two-photon excited autofluorescence and second harmonic generation (SHG) images of ex vivo human tooth. By combining these two imaging modalities we obtained submicron resolution images of the enamel, dentin, and the periodontal ligaments. The enamel emits endogenous two-photon autofluorescence. The structure of the dentin is visible from both the autofluorescence and second harmonic generation signals. The periodontal ligament composed mostly of collagen can be visualized by SHG imaging. We also constructed three dimensional images of the enamel, dentin, and periodontal ligament. The effectiveness of using multiphoton and second harmonic generation microscopy to obtain structural information of teeth suggest its potential use in dental diagnostics.

  4. Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

    NASA Astrophysics Data System (ADS)

    Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

    2016-09-01

    We present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional data cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. We demonstrate this ‘big data’ approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.

  5. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

    PubMed

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

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  6. Correlated Atomic Force Microscopy and Flourescence Lifetime Imaging of Live Bacterial Cells

    SciTech Connect

    Micic, Miodrag; Hu, Dehong; Suh, Yung D.; Newton, Greg J.; Romine, Margaret F.; Lu, H PETER.

    2004-04-01

    We report on the imaging of living bacterial cells by using a new correlated tapping-mode atomic force microscopy (AFM) and confocal al fluorescence lifetime imaging microscopy (FLIM). Different methods of preparing the bacterial sample were explored for optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells on poly-1-lysine coated surfaces and agarose gel coated surfaces. We have found that the agarose gel containing 99% buffer can provide a local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and single-to-noise ration of the AFM images. Near-field AFM-tip enhanced fluorescence lifetime imaging (AFM-FLIM) holds great promise for obtaining fluorescence images beyond the optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond the diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging living bacterial cells, we demonstrate a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging to characterize cell polarity.

  7. High-Resolution Microscopy-Coil MR Imaging of Skin Tumors: Techniques and Novel Clinical Applications.

    PubMed

    Budak, Matthew J; Weir-McCall, Jonathan R; Yeap, Phey M; White, Richard D; Waugh, Shelley A; Sudarshan, Thiru A P; Zealley, Ian A

    2015-01-01

    High-resolution magnetic resonance (MR) imaging performed with a microscopy coil is a robust radiologic tool for the evaluation of skin lesions. Microscopy-coil MR imaging uses a small surface coil and a 1.5-T or higher MR imaging system. Simple T1- and T2-weighted imaging protocols can be implemented to yield high-quality, high-spatial-resolution images that provide an excellent depiction of dermal anatomy. The primary application of microscopy-coil MR imaging is to delineate the deep margins of skin tumors, thereby providing a preoperative road map for dermatologic surgeons. This information is particularly useful for surgeons who perform Mohs micrographic surgery and in cases of nasofacial neoplasms, where the underlying anatomy is complex. Basal cell carcinoma is the most common nonmelanocytic skin tumor and has a predilection to manifest on the face, where it can be challenging to achieve complete surgical excision while preserving the cosmetic dignity of the patient. Microscopy-coil MR imaging provides dermatologic surgeons with valuable preoperative anatomic information that is not available at conventional clinical examination.

  8. Deep Learning Segmentation of Optical Microscopy Images Improves 3D Neuron Reconstruction.

    PubMed

    Li, Rongjian; Zeng, Tao; Peng, Hanchuan; Ji, Shuiwang

    2017-03-08

    Digital reconstruction, or tracing, of 3-dimensional (3D) neuron structure from microscopy images is a critical step toward reversing engineering the wiring and anatomy of a brain. Despite a number of prior attempts, this task remains very challenging, especially when images are contaminated by noises or have discontinued segments of neurite patterns. An approach for addressing such problems is to identify the locations of neuronal voxels using image segmentation methods prior to applying tracing or reconstruction techniques. This preprocessing step is expected to remove noises in the data, thereby leading to improved reconstruction results. In this work, we proposed to use 3D Convolutional neural networks (CNNs) for segmenting the neuronal microscopy images. Specifically, we designed a novel CNN architecture that takes volumetric images as the inputs and their voxel-wise segmentation maps as the outputs. The developed architecture allows us to train and predict using large microscopy images in an end-to-end manner. We evaluated the performance of our model on a variety of challenging 3D microscopy images from different organisms. Results showed that the proposed methods improved the tracing performance significantly when combined with different reconstruction algorithms.

  9. Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

    NASA Astrophysics Data System (ADS)

    Sanchez-Armass, Sergio; Sennoune, Souad; Martinez, Gloria M.; Ortega, Filiberta; Martinez-Zaguilan, Raul

    2002-06-01

    Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

  10. Iplt--image processing library and toolkit for the electron microscopy community.

    PubMed

    Philippsen, Ansgar; Schenk, Andreas D; Stahlberg, Henning; Engel, Andreas

    2003-01-01

    We present the foundation for establishing a modular, collaborative, integrated, open-source architecture for image processing of electron microscopy images, named iplt. It is designed around object oriented paradigms and implemented using the programming languages C++ and Python. In many aspects it deviates from classical image processing approaches. This paper intends to motivate developers within the community to participate in this on-going project. The iplt homepage can be found at http://www.iplt.org.

  11. Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells.

    PubMed

    Peckys, Diana B; de Jonge, Niels

    2014-04-01

    Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

  12. Chemoselective imaging of mouse brain tissue via multiplex CARS microscopy.

    PubMed

    Pohling, Christoph; Buckup, Tiago; Pagenstecher, Axel; Motzkus, Marcus

    2011-08-01

    The fast and reliable characterization of pathological tissue is a debated topic in the application of vibrational spectroscopy in medicine. In the present work we apply multiplex coherent anti-Stokes Raman scattering (MCARS) to the investigation of fresh mouse brain tissue. The combination of imaginary part extraction followed by principal component analysis led to color contrast between grey and white matter as well as layers of granule and Purkinje cells. Additional quantitative information was obtained by using a decomposition algorithm. The results perfectly agree with HE stained references slides prepared separately making multiplex CARS an ideal approach for chemoselective imaging.

  13. An open data mining framework for the analysis of medical images: application on obstructive nephropathy microscopy images.

    PubMed

    Doukas, Charalampos; Goudas, Theodosis; Fischer, Simon; Mierswa, Ingo; Chatziioannou, Aristotle; Maglogiannis, Ilias

    2010-01-01

    This paper presents an open image-mining framework that provides access to tools and methods for the characterization of medical images. Several image processing and feature extraction operators have been implemented and exposed through Web Services. Rapid-Miner, an open source data mining system has been utilized for applying classification operators and creating the essential processing workflows. The proposed framework has been applied for the detection of salient objects in Obstructive Nephropathy microscopy images. Initial classification results are quite promising demonstrating the feasibility of automated characterization of kidney biopsy images.

  14. Imaging graphite in air by scanning tunneling microscopy - Role of the tip

    NASA Technical Reports Server (NTRS)

    Colton, R. J.; Baker, S. M.; Driscoll, R. J.; Youngquist, M. G.; Baldeschwieler, J. D.; Kaiser, W. J.

    1988-01-01

    Atomically resolved images of highly oriented pyrolytic graphite (HOPG) in air at point contact have been obtained. Direct contact between tip and sample or contact through a contamination layer provides a conduction mechanism in addition to the exponential tunneling mechanism responsible for scanning tunneling microscopy (STM) imaging. Current-voltage (I-V) spectra were obtained while scanning in the current imaging mode with the feedback circuit interrupted in order to study the graphite imaging mechanism. Multiple tunneling tips are probably responsible for images without the expected hexagonal or trigonal symmetry. The observations indicate that the use of HOPG for testing and calibration of STM instrumentation may be misleading.

  15. Atomic Force Microscopy Imaging of Filamentous Aggregates from an N-Terminal Peptide Fragment of Barnase

    NASA Astrophysics Data System (ADS)

    Shibata-Seki, Teiko; Masai, Junji; Yoshida, Kenji; Sato, Kazuki; Yanagawa, Hiroshi

    1993-06-01

    This paper reports the atomic force microscopy (AFM) imaging of filamentous aggregates derived from an N-terminal peptide fragment of barnase, a ribonuclease from Bacillus amyloliquefaciens. The sample was deposited on a freshly cleaved mica surface and observed in ambient conditions. The overall shapes of the filamentous structures imaged with two different kinds of AFMs were similar to those obtained with a transmission electron microscope (TEM), except that the filaments in AFM images were broader than those in TEM images. This broadening phenomenon characteristic of AFM images was explained in terms of the convolution-type distortion of the specimen diameter by the scanning-tip apex.

  16. Note: Fast imaging of DNA in atomic force microscopy enabled by a local raster scan algorithm

    SciTech Connect

    Huang, Peng; Andersson, Sean B.

    2014-06-15

    Approaches to high-speed atomic force microscopy typically involve some combination of novel mechanical design to increase the physical bandwidth and advanced controllers to take maximum advantage of the physical capabilities. For certain classes of samples, however, imaging time can be reduced on standard instruments by reducing the amount of measurement that is performed to image the sample. One such technique is the local raster scan algorithm, developed for imaging of string-like samples. Here we provide experimental results on the use of this technique to image DNA samples, demonstrating the efficacy of the scheme and illustrating the order-of-magnitude improvement in imaging time that it provides.

  17. Covalent and reversible short-range electrostatic imaging in noncontact atomic force microscopy.

    PubMed

    Dieska, Peter; Stich, Ivan; Pérez, Rubén

    2003-11-21

    We present a computational study of atomic-scale image formation in noncontact atomic force microscopy on metallic surfaces. We find two imaging scenarios: (1). atomic resolution arising due to very strong covalent tip-sample interaction exhibiting striking similarity with the imaging mechanism found on semiconductor surfaces, and (2). a completely new mechanism, reversible short-range electrostatic imaging, arising due to subtle charge-transfer interactions. Contrary to the strong covalent-bond imaging, the newly identified mechanism causes only negligible surface perturbation and can account for results recently observed experimentally.

  18. Automated podosome identification and characterization in fluorescence microscopy images.

    PubMed

    Meddens, Marjolein B M; Rieger, Bernd; Figdor, Carl G; Cambi, Alessandra; van den Dries, Koen

    2013-02-01

    Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

  19. Stochastic optical reconstruction microscopy (STORM) in comparison with stimulated emission depletion (STED) and other imaging methods.

    PubMed

    Tam, Johnny; Merino, David

    2015-11-01

    Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) are two super-resolution microscopy approaches that have rapidly gained popularity in recent years. STORM is based on the precise localization of a large number of individual molecules that together form a super-resolved image (bottom), whereas STED is based on the scanning of two super-imposed light sources which together allow for a super-resolved spot on the sample to be imaged (top). We discuss the specific advantages and disadvantages of each technique and explain the various parameters that affect image quality, which should be taken into consideration when planning experiments.

  20. Towards real-time image deconvolution: application to confocal and STED microscopy

    PubMed Central

    Zanella, R.; Zanghirati, G.; Cavicchioli, R.; Zanni, L.; Boccacci, P.; Bertero, M.; Vicidomini, G.

    2013-01-01

    Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings. PMID:23982127

  1. Fluorescent Nanodiamond-Gold Hybrid Particles for Multimodal Optical and Electron Microscopy Cellular Imaging.

    PubMed

    Liu, Weina; Naydenov, Boris; Chakrabortty, Sabyasachi; Wuensch, Bettina; Hübner, Kristina; Ritz, Sandra; Cölfen, Helmut; Barth, Holger; Koynov, Kaloian; Qi, Haoyuan; Leiter, Robert; Reuter, Rolf; Wrachtrup, Jörg; Boldt, Felix; Scheuer, Jonas; Kaiser, Ute; Sison, Miguel; Lasser, Theo; Tinnefeld, Philip; Jelezko, Fedor; Walther, Paul; Wu, Yuzhou; Weil, Tanja

    2016-10-12

    There is a continuous demand for imaging probes offering excellent performance in various microscopy techniques for comprehensive investigations of cellular processes by more than one technique. Fluorescent nanodiamond-gold nanoparticles (FND-Au) constitute a new class of "all-in-one" hybrid particles providing unique features for multimodal cellular imaging including optical imaging, electron microscopy, and, and potentially even quantum sensing. Confocal and optical coherence microscopy of the FND-Au allow fast investigations inside living cells via emission, scattering, and photothermal imaging techniques because the FND emission is not quenched by AuNPs. In electron microscopy, transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) analysis of FND-Au reveals greatly enhanced contrast due to the gold particles as well as an extraordinary flickering behavior in three-dimensional cellular environments originating from the nanodiamonds. The unique multimodal imaging characteristics of FND-Au enable detailed studies inside cells ranging from statistical distributions at the entire cellular level (micrometers) down to the tracking of individual particles in subcellular organelles (nanometers). Herein, the processes of endosomal membrane uptake and release of FNDs were elucidated for the first time by the imaging of individual FND-Au hybrid nanoparticles with single-particle resolution. Their convenient preparation, the availability of various surface groups, their flexible detection modalities, and their single-particle contrast in combination with the capability for endosomal penetration and low cytotoxicity make FND-Au unique candidates for multimodal optical-electronic imaging applications with great potential for emerging techniques, such as quantum sensing inside living cells.

  2. Imaging and manipulation of nanometer-size liquid droplets by scanning polarization force microscopy

    SciTech Connect

    Hu, J.; Carpick, R.W.; Salmeron, M.; Xiao, X.

    1996-03-01

    Using atomic force microscopy in noncontact mode, we have imaged nanometer-size liquid droplets of KOH water solutions on the surfaces of highly oriented pyrolitic graphite and mica. On graphite the droplets prefer to be adsorbed on atomic step edges. Droplets on the same step tend to be evenly spaced and of similar size. The droplets can be manipulated by the atomic force microscopy tip allowing the controllable formation of droplet patterns on the surface. {copyright} {ital 1996 American Vacuum Society}

  3. Digital image processing of crystalline specimens examined by electron microscopy.

    PubMed

    Kanaya, K

    1988-12-01

    Crystalline specimens imaged in the electron microscope are analysed using digital processing. Some principles of structural analysis using the method of Fourier decomposition are discussed. Complementary techniques, such as enhancement by gradient and Laplacian operators, have been found useful in analysing electron micrographs. The application of these techniques to some problems in Materials Science and Biology are reviewed. By selecting and phase-correcting spots in the computed diffraction pattern, it was possible to localize atoms, molecules, and their defective arrangement in evaporated gold, sputter-deposited tungsten films, and single crystals of cadmium selenide. Digital processing based on the theory of helical diffraction was used to explore the three-dimensional arrangement of molecules in cellular components of alveolar soft part sarcoma, Hirano bodies, and neurofibrillar tangles in the human brain.

  4. Band Excitation in Scanning Probe Microscopy: Recognition and Functional Imaging

    SciTech Connect

    Jesse, Stephen; Vasudevan, Dr. Rama; Collins, Liam; Strelcov, Evgheni; Okatan, Mahmut B; Belianinov, Alex; Baddorf, Arthur P; Proksch, Roger; Kalinin, Sergei V

    2014-01-01

    Field confinement at the junction between a biased scanning probe microscope s (SPM) tip and solid surface enables local probing of various bias-induced transformations such as polarization switching, ionic motion, or electrochemical reactions to name a few. The nanoscale size of the biased region is smaller or comparable to features like grain boundaries and dislocations, potentially allows for the study of kinetics and thermodynamics at the level of a single defect. In contrast to classical statistically averaged approaches, this allows one to link structure to functionality and deterministically decipher associated mesoscopic and atomistic mechanisms. Furthermore, this type of information can serve as a fingerprint of local material functionality, allowing for local recognition imaging. Here, current progress in multidimensional SPM techniques based on band-excitation time and voltage spectroscopies is illustrated, including discussions on data acquisition, dimensionality reduction, and visualization along with future challenges and opportunities for the field.

  5. Live cardiomyocyte imaging via hybrid TPEF-SHG microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Honghai; Qin, Wan; Shao, Yonghong; Liu, Qiuying; Ma, Zhen; Borg, Thomas K.; Gao, Bruce Z.

    2012-03-01

    Utilizing a custom-built, on-stage incubator-combined, two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) imaging system, we observed new-sarcomere addition in rat neonatal cardiomyocytes during 10 hours of on-stage incubation. This addition occurred at one end of an existing myofibril, the sides of existing myofibrils, and at the interstice of several separated myofibrils; in the cases of the latter two, we observed mature myofibrils acting as templates. We found that during sarcomeric addition, myosin filaments are assembled onto the premyofibril laterally. This lateral addition, which proceeds stepwise along the axial direction, plays an important role in the accumulation of Z-bodies to form mature Z-disks and in the regulation of sarcomeric length during maturation.

  6. Arbitrary-scan imaging for two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Botcherby, Edward; Smith, Christopher; Booth, Martin; Juskaitis, Rimas; Wilson, Tony

    2010-02-01

    In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic scan rate. This means that the focal spot can be scanned at high speed along any direction in the specimen, without introducing systematic aberrations. We present experimental point spread function measurements for this system using an Olympus 0.8 NA 40X water dipping objective lens that demonstrates an axial range of operation greater than 200 μm. We give details of a novel actuator device used to displace the focusing element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present a bioscience application of this system to image dendritic processes that follow non-linear paths in three-dimensional space. The focal spot was scanned along one such process at 400 Hz with an axial range of more than 90 μm.

  7. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    NASA Astrophysics Data System (ADS)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  8. Nanoscopy for nanoscience: how super-resolution microscopy extends imaging for nanotechnology.

    PubMed

    Johnson, Sam A

    2015-01-01

    Imaging methods have presented scientists with powerful means of investigation for centuries. The ability to resolve structures using light microscopes is though limited to around 200 nm. Fluorescence-based super-resolution light microscopy techniques of several principles and methods have emerged in recent years and offer great potential to extend the capabilities of microscopy. This resolution improvement is especially promising for nanoscience where the imaging of nanoscale structures is inherently restricted by the resolution limit of standard forms of light microscopy. Resolution can be improved by several distinct approaches including structured illumination microscopy, stimulated emission depletion, and single-molecule positioning methods such as photoactivated localization microscopy and stochastic optical reconstruction microscopy and several derivative variations of each of these. These methods involve substantial differences in the resolutions achievable in the different axes, speed of acquisition, compatibility with different labels, ease of use, hardware complexity, and compatibility with live biological samples. The field of super-resolution imaging and its application to nanotechnology is relatively new and still rapidly developing. An overview of how these methods may be used with nanomaterials is presented with some examples of pioneering uses of these approaches.

  9. Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

    NASA Astrophysics Data System (ADS)

    Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

    2013-02-01

    The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

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

    PubMed Central

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

    2016-01-01

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

  11. Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001.

    PubMed

    Steele, A; Goddard, D; Beech, I B; Tapper, R C; Stapleton, D; Smith, J R

    1998-01-01

    A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

  12. Segmentation of scanning electron microscopy images from natural rubber samples with gold nanoparticles using starlet wavelets.

    PubMed

    de Siqueira, Alexandre Fioravante; Cabrera, Flávio Camargo; Pagamisse, Aylton; Job, Aldo Eloizo

    2014-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our method showed accuracy greater than 85% for all test images. Results given by this method will be used in future studies, to computationally estimate the density distribution of gold nanoparticles in natural rubber samples and to predict reduction kinetics of gold nanoparticles at different time periods.

  13. Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001

    NASA Technical Reports Server (NTRS)

    Steele, A.; Goddard, D.; Beech, I. B.; Tapper, R. C.; Stapleton, D.; Smith, J. R.

    1998-01-01

    A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy-dispersive X-ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars--possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924-930) of NASA who used SEM imaging of gold-coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.

  14. Evaluation of noise limits to improve image processing in soft X-ray projection microscopy.

    PubMed

    Jamsranjav, Erdenetogtokh; Kuge, Kenichi; Ito, Atsushi; Kinjo, Yasuhito; Shiina, Tatsuo

    2017-03-03

    Soft X-ray microscopy has been developed for high resolution imaging of hydrated biological specimens due to the availability of water window region. In particular, a projection type microscopy has advantages in wide viewing area, easy zooming function and easy extensibility to computed tomography (CT). The blur of projection image due to the Fresnel diffraction of X-rays, which eventually reduces spatial resolution, could be corrected by an iteration procedure, i.e., repetition of Fresnel and inverse Fresnel transformations. However, it was found that the correction is not enough to be effective for all images, especially for images with low contrast. In order to improve the effectiveness of image correction by computer processing, we in this study evaluated the influence of background noise in the iteration procedure through a simulation study. In the study, images of model specimen with known morphology were used as a substitute for the chromosome images, one of the targets of our microscope. Under the condition that artificial noise was distributed on the images randomly, we introduced two different parameters to evaluate noise effects according to each situation where the iteration procedure was not successful, and proposed an upper limit of the noise within which the effective iteration procedure for the chromosome images was possible. The study indicated that applying the new simulation and noise evaluation method was useful for image processing where background noises cannot be ignored compared with specimen images.

  15. A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

    PubMed

    Wu, Weilin; Lin, Jinyong; Wang, Shu; Li, Yan; Liu, Mingyu; Liu, Gaoqiang; Cai, Jianyong; Chen, Guannan; Chen, Rong

    2016-04-19

    Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness.

  16. Tripling the maximum imaging depth with third-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Yildirim, Murat; Durr, Nicholas; Ben-Yakar, Adela

    2015-09-01

    The growing interest in performing high-resolution, deep-tissue imaging has galvanized the use of longer excitation wavelengths and three-photon-based techniques in nonlinear imaging modalities. This study presents a threefold improvement in maximum imaging depth of ex vivo porcine vocal folds using third-harmonic generation (THG) microscopy at 1552-nm excitation wavelength compared to two-photon microscopy (TPM) at 776-nm excitation wavelength. The experimental, analytical, and Monte Carlo simulation results reveal that THG improves the maximum imaging depth observed in TPM significantly from 140 to 420 μm in a highly scattered medium, reaching the expected theoretical imaging depth of seven extinction lengths. This value almost doubles the previously reported normalized imaging depths of 3.5 to 4.5 extinction lengths using three-photon-based imaging modalities. Since tissue absorption is substantial at the excitation wavelength of 1552 nm, this study assesses the tissue thermal damage during imaging by obtaining the depth-resolved temperature distribution through a numerical simulation incorporating an experimentally obtained thermal relaxation time (τ). By shuttering the laser for a period of 2τ, the numerical algorithm estimates a maximum temperature increase of ˜2°C at the maximum imaging depth of 420 μm. The paper demonstrates that THG imaging using 1552 nm as an illumination wavelength with effective thermal management proves to be a powerful deep imaging modality for highly scattering and absorbing tissues, such as scarred vocal folds.

  17. Real-time tracking mitochondrial dynamic remodeling with two-photon phosphorescent iridium (III) complexes.

    PubMed

    Huang, Huaiyi; Yang, Liang; Zhang, Pingyu; Qiu, Kangqiang; Huang, Juanjuan; Chen, Yu; Diao, JiaJie; Liu, Jiankang; Ji, Liangnian; Long, Jiangang; Chao, Hui

    2016-03-01

    Mitochondrial fission and fusion control the shape, size, number, and function of mitochondria in the cells of organisms from yeast to mammals. The disruption of mitochondrial fission and fusion is involved in severe human diseases such as Parkinson's disease, Alzheimer's disease, metabolic diseases, and cancers. Agents that can real-time track the mitochondrial dynamics are of great importance. However, the short excitation wavelengths and rapidly photo-bleaching properties of commercial mitochondrial dyes render them unsuitable for tracking mitochondrial dynamics. Thus, mitochondrial targeting agents that exhibit superior photo-stability under continual light irradiation, deep tissue penetration and at intrinsically high three-dimensional resolutions are urgently needed. Two-photon-excited compounds employ low-energy near-infrared light and have emerged as a non-invasive tool for real-time cell imaging. Here, cyclometalated Ir(III) complexes (Ir1-Ir5) are demonstrated as one- and two-photon phosphorescent probes for the real-time imaging and tracking of mitochondrial fission and fusion. The results indicate that Ir2 is well suited for two-photon phosphorescent tracking of mitochondrial fission and fusion in living cells and in Caenorhabditis elegans (C. elegans). This study provides a practical use for mitochondrial targeting two-photon phosphorescent Ir(III) complexes.

  18. Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

    PubMed

    Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

    2016-03-15

    To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

  19. Imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy.

    PubMed

    Schouten, Marijn; De Luca, Giulia M R; Alatriste González, Diana K; de Jong, Babette E; Timmermans, Wendy; Xiong, Hui; Krugers, Harm; Manders, Erik M M; Fitzsimons, Carlos P

    2014-05-04

    Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm. Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the

  20. Adult Human Neurogenesis: From Microscopy to Magnetic Resonance Imaging

    PubMed Central

    Sierra, Amanda; Encinas, Juan M.; Maletic-Savatic, Mirjana

    2011-01-01

    Neural stem cells reside in well-defined areas of the adult human brain and are capable of generating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases. PMID:21519376

  1. Adult human neurogenesis: from microscopy to magnetic resonance imaging.

    PubMed

    Sierra, Amanda; Encinas, Juan M; Maletic-Savatic, Mirjana

    2011-01-01

    Neural stem cells reside in well-defined areas of the adult human brain and are capable of generating new neurons throughout the life span. In rodents, it is well established that the new born neurons are involved in olfaction as well as in certain forms of memory and learning. In humans, the functional relevance of adult human neurogenesis is being investigated, in particular its implication in the etiopathology of a variety of brain disorders. Adult neurogenesis in the human brain was discovered by utilizing methodologies directly imported from the rodent research, such as immunohistological detection of proliferation and cell-type specific biomarkers in postmortem or biopsy tissue. However, in the vast majority of cases, these methods do not support longitudinal studies; thus, the capacity of the putative stem cells to form new neurons under different disease conditions cannot be tested. More recently, new technologies have been specifically developed for the detection and quantification of neural stem cells in the living human brain. These technologies rely on the use of magnetic resonance imaging, available in hospitals worldwide. Although they require further validation in rodents and primates, these new methods hold the potential to test the contribution of adult human neurogenesis to brain function in both health and disease. This review reports on the current knowledge on adult human neurogenesis. We first review the different methods available to assess human neurogenesis, both ex vivo and in vivo and then appraise the changes of adult neurogenesis in human diseases.

  2. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    PubMed Central

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-01-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02–04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity. PMID:26926390

  3. Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging

    NASA Astrophysics Data System (ADS)

    Li, Yan; Forbrich, Alex; Wu, Jiahui; Shao, Peng; Campbell, Robert E.; Zemp, Roger

    2016-03-01

    A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which are promising probe molecules for use in photoacoustic imaging and as acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Typical approaches for fluorescent protein optimization by screening of large libraries of variants cannot be effectively applied to chromoproteins due to their characteristic lack of fluorescence. To address this challenge, we have developed a directed evolution method to iteratively screen large libraries of protein variants on the basis of their photoacoustic signal levels. By applying this procedure to the promising Ultramarine and cjBlue chromoprotein templates, we were able to identify improved variants with a 02–04 fold increase in photoacoustic signal-to-noise ratio after only a few evolutionary steps. These improved variants enable more accurate spectral de-mixing and localization of protein-producing bacteria in vivo and serve as effective FRET acceptors for both fluorescence- and photoacoustic-based detection of protease activity.

  4. Molecular Fluorescence, Phosphorescence, and Chemiluminescence Spectrometry

    SciTech Connect

    Powe, Aleeta; Das, Susmita; Lowry, Mark; El-Zahab, Bilal; Fakayode, Sayo; Geng, Maxwell; Baker, Gary A; Wang, Lin; McCarroll, Matthew; Patonay, Gabor; Li, Min; Aljarrah, Mohannad; Neal, Sharon; Warner, Isiah M

    2010-01-01

    This review covers the 2 year period since our last review (1) from January 2008 through December 2009. A computer search of Chemical Abstracts provided most of the references for this review. A search for documents written in English containing the terms fluorescence or phosphorescence or chemiluminescence published in 2008-2009 resulted in more than 100 000 hits. An initial screening reduced this number to approximately 23 000 publications that were considered for inclusion in this review. Key word searches of this subset provided subtopics of manageable size. Other citations were found through individual searches by the various authors who wrote a particular section of this review.

  5. Photocatalytic reaction characteristics of the titanium dioxide supported on the long phosphorescent phosphor by a low pressure chemical vapor deposition.

    PubMed

    Kim, Jung-Sik; Kim, Seung-Woo; Jung, Sang-Chul

    2014-10-01

    This study investigated the photocatalytic behavior of titanium dioxide (TiO2)-supported on the long phosphorescent materials. Nanocrystalline TiO2 was directly deposited on the plate of alkaline earth aluminate phosphor, CaAl2O4: Eu2+, Nd3+ by a low pressure chemical vapor deposition (LPCVD). Photocatalytic reaction performance was examined with the decomposition of benzene gas by using a gas chromatography (GC) system under ultraviolet and visible light (λ > 410 nm) irradiations. The LPCVD TiO2-coated phosphors showed active photocatalytic reaction under visible irradiation. The mechanism of the photocatalytic reactivity for the TiO,-coated phosphorescent phosphor was discussed in terms of the energy band structure and phosphorescence. The coupling of TiO2 with phosphor may result in energy band bending in the junction region, which makes the TiO, crystal at the interface to be photo-reactive under visible light irradiation. The fastest degradation of ben- zene gas occurred for the TiO,-coated phosphor prepared with 1 min deposition time (-150 nm thickness). The LPCVD TiO,-coated phosphor is also photo-reactive under darkness through the light photons emitted from the CaAl2O4 phosphor. In addition, the TiO2-coated phosphorescent phosphors were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM).

  6. Scanning probe microscopy of atoms and molecules on insulating films: from imaging to molecular manipulation.

    PubMed

    Meyer, Gerhard; Gross, Leo; Mohn, Fabian; Repp, Jascha

    2012-01-01

    Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) of single atoms and molecules on ultrathin insulating films have led to a wealth of novel observations and insights. Based on the reduced electronic coupling to the metallic substrate, these techniques allow the charge state of individual atoms to be controlled, orbitals of individual molecules to be imaged and metal-molecule complexes to be built up. Near-contact AFM adds the unique capabilities of imaging and probing the chemical structure of single molecules with atomic resolution. With the help of atomic/molecular manipulation techniques, chemical binding processes and molecular switches can be studied in detail.

  7. Quantitative imaging of cellular adhesion by total internal reflection holographic microscopy.

    PubMed

    Ash, William M; Krzewina, Leo; Kim, Myung K

    2009-12-01

    Total internal reflection (TIR) holographic microscopy uses a prism in TIR as a near-field imager to perform quantitative phase microscopy of cell-substrate interfaces. The presence of microscopic organisms, cell-substrate interfaces, adhesions, and tissue structures on the prism's TIR face causes relative index of refraction and frustrated TIR to modulate the object beam's evanescent wave phase front. We present quantitative phase images of test specimens such as Amoeba proteus and cells such as SKOV-3 and 3T3 fibroblasts.

  8. Imaging via complete cantilever dynamic detection: general dynamic mode imaging and spectroscopy in scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Somnath, Suhas; Collins, Liam; Matheson, Michael A.; Sukumar, Sreenivas R.; Kalinin, Sergei V.; Jesse, Stephen

    2016-10-01

    We develop and implement a multifrequency spectroscopy and spectroscopic imaging mode, referred to as general dynamic mode (GDM), that captures the complete spatially- and stimulus dependent information on nonlinear cantilever dynamics in scanning probe microscopy (SPM). GDM acquires the cantilever response including harmonics and mode mixing products across the entire broadband cantilever spectrum as a function of excitation frequency. GDM spectra substitute the classical measurements in SPM, e.g. amplitude and phase in lock-in detection. Here, GDM is used to investigate the response of a purely capacitively driven cantilever. We use information theory techniques to mine the data and verify the findings with governing equations and classical lock-in based approaches. We explore the dependence of the cantilever dynamics on the tip–sample distance, AC and DC driving bias. This approach can be applied to investigate the dynamic behavior of other systems within and beyond dynamic SPM. GDM is expected to be useful for separating the contribution of different physical phenomena in the cantilever response and understanding the role of cantilever dynamics in dynamic AFM techniques.

  9. Imaging via complete cantilever dynamic detection: General dynamic mode imaging and spectroscopy in scanning probe microscopy

    SciTech Connect

    Somnath, Suhas; Collins, Liam; Matheson, Michael A.; Sukumar, Sreenivas R.; Kalinin, Sergei V.; Jesse, Stephen

    2016-09-08

    We develop and implement a multifrequency spectroscopy and spectroscopic imaging mode, referred to as general dynamic mode (GDM), that captures the complete spatially- and stimulus dependent information on nonlinear cantilever dynamics in scanning probe microscopy (SPM). GDM acquires the cantilever response including harmonics and mode mixing products across the entire broadband cantilever spectrum as a function of excitation frequency. GDM spectra substitute the classical measurements in SPM, e.g. amplitude and phase in lock-in detection. Here, GDM is used to investigate the response of a purely capacitively driven cantilever. We use information theory techniques to mine the data and verify the findings with governing equations and classical lock-in based approaches. We explore the dependence of the cantilever dynamics on the tip–sample distance, AC and DC driving bias. This approach can be applied to investigate the dynamic behavior of other systems within and beyond dynamic SPM. In conclusion, GDM is expected to be useful for separating the contribution of different physical phenomena in the cantilever response and understanding the role of cantilever dynamics in dynamic AFM techniques.

  10. Imaging via complete cantilever dynamic detection: General dynamic mode imaging and spectroscopy in scanning probe microscopy

    DOE PAGES

    Somnath, Suhas; Collins, Liam; Matheson, Michael A.; ...

    2016-09-08

    We develop and implement a multifrequency spectroscopy and spectroscopic imaging mode, referred to as general dynamic mode (GDM), that captures the complete spatially- and stimulus dependent information on nonlinear cantilever dynamics in scanning probe microscopy (SPM). GDM acquires the cantilever response including harmonics and mode mixing products across the entire broadband cantilever spectrum as a function of excitation frequency. GDM spectra substitute the classical measurements in SPM, e.g. amplitude and phase in lock-in detection. Here, GDM is used to investigate the response of a purely capacitively driven cantilever. We use information theory techniques to mine the data and verify themore » findings with governing equations and classical lock-in based approaches. We explore the dependence of the cantilever dynamics on the tip–sample distance, AC and DC driving bias. This approach can be applied to investigate the dynamic behavior of other systems within and beyond dynamic SPM. In conclusion, GDM is expected to be useful for separating the contribution of different physical phenomena in the cantilever response and understanding the role of cantilever dynamics in dynamic AFM techniques.« less

  11. Sub-cellular resolution imaging with Gabor domain optical coherence microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

    Optical Coherence Microscopy (OCM) utilizes a high NA microscope objective in the sample arm to achieve an axially and laterally high resolution OCT image. An increase in NA, however, leads to a dramatically decreased depth of focus (DOF), and hence shortens the imaging depth range so that high lateral resolution is maintained only within a small depth region around the focal plane. One solution to increase the depth of imaging while keeping a high lateral resolution is dynamic-focusing. Utilizing the voltage controlled refocus capability of a liquid lens, we have recently presented a solution for invariant high resolution imaging using the liquid lens embedded within a fixed optics hand-held custom microscope designed specifically for optical imaging systems using a broadband light source at 800 nm center wavelength. Subsequently, we have developed a Gabor-Domain Optical Coherence Microscopy (GD-OCM) that utilizes the high speed imaging of spectral domain OCT, the high lateral resolution of OCM, and the ability of real time refocusing of our custom design variable focus objective. In this paper we demonstrate in detail how portions of the infocus cross-sectional images can be extracted and fused to form an invariant lateral resolution image with multiple crosssectional images acquired corresponding to a discrete refocusing step along depth enabled by the varifocal probe. We demonstrate sub-cellular resolution imaging of an African frog tadpole (Xenopus Laevis) taken from a 500 μm x 500 μm cross-section.

  12. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

    PubMed

    Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

    2010-03-19

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  13. Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

    NASA Astrophysics Data System (ADS)

    Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

    2010-03-01

    The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

  14. Improved localization accuracy in stochastic super-resolution fluorescence microscopy by K-factor image deshadowing.

    PubMed

    Ilovitsh, Tali; Meiri, Amihai; Ebeling, Carl G; Menon, Rajesh; Gerton, Jordan M; Jorgensen, Erik M; Zalevsky, Zeev

    2013-12-16

    Localization of a single fluorescent particle with sub-diffraction-limit accuracy is a key merit in localization microscopy. Existing methods such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) achieve localization accuracies of single emitters that can reach an order of magnitude lower than the conventional resolving capabilities of optical microscopy. However, these techniques require a sparse distribution of simultaneously activated fluorophores in the field of view, resulting in larger time needed for the construction of the full image. In this paper we present the use of a nonlinear image decomposition algorithm termed K-factor, which reduces an image into a nonlinear set of contrast-ordered decompositions whose joint product reassembles the original image. The K-factor technique, when implemented on raw data prior to localization, can improve the localization accuracy of standard existing methods, and also enable the localization of overlapping particles, allowing the use of increased fluorophore activation density, and thereby increased data collection speed. Numerical simulations of fluorescence data with random probe positions, and especially at high densities of activated fluorophores, demonstrate an improvement of up to 85% in the localization precision compared to single fitting techniques. Implementing the proposed concept on experimental data of cellular structures yielded a 37% improvement in resolution for the same super-resolution image acquisition time, and a decrease of 42% in the collection time of super-resolution data with the same resolution.

  15. Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

    PubMed

    Fan, Jiadong; Sun, Zhibin; Zhang, Jian; Huang, Qingjie; Yao, Shengkun; Zong, Yunbing; Kohmura, Yoshiki; Ishikawa, Tetsuya; Liu, Hong; Jiang, Huaidong

    2015-06-16

    Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyβ-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.

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

    PubMed

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

    2014-11-30

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

  17. Rapid volumetric temporal focusing multiphoton microscopy of neural activity: theory, image processing, and experimental realization

    NASA Astrophysics Data System (ADS)

    Dana, Hod; Marom, Anat; Kruger, Nimrod; Ellman, Aviv; Shoham, Shy

    2012-03-01

    The development of rapid volumetric imaging systems for functional multiphoton microscopy is essential for dynamical imaging of large-scale neuronal network activity. Here, we introduce a line-illuminating temporal-focusing microscope capable of rapid three-dimensional imaging at 10-20 volumes/sec, and study the system's characteristics both theoretically and experimentally. We demonstrate that our system is capable of functional volumetric calcium imaging of distributed neuronal activity patterns, and introduce a computational strategy for activity reconstruction in strongly scattering media.

  18. C-scan photoacoustic microscopy for invivo imaging of Drosophila pupae

    NASA Astrophysics Data System (ADS)

    Xi, Lei; Zhou, Lei; Jiang, Huabei

    2012-07-01

    In this study, C-scan based high resolution photoacoustic microscopy (CPAM) is devised and used to invivo image the DsRed-expressing cells in the central nervous system (CNS) of Drosophila pupae. The quality of out-of-focus images was improved significantly from both the phantom and invivo experiments. Organs containing DsRed-expressing cells in Drosophila pupae can be imaged using our CPAM and agreed well with the histological sections. This technology allows rapid imaging and monitoring of organismal development in mesoscopic-scale animals as well as the generation of a detailed atlas of CNS development in the pupal stage.

  19. The Open Microscopy Environment: open image informatics for the biological sciences

    NASA Astrophysics Data System (ADS)

    Blackburn, Colin; Allan, Chris; Besson, Sébastien; Burel, Jean-Marie; Carroll, Mark; Ferguson, Richard K.; Flynn, Helen; Gault, David; Gillen, Kenneth; Leigh, Roger; Leo, Simone; Li, Simon; Lindner, Dominik; Linkert, Melissa; Moore, Josh; Moore, William J.; Ramalingam, Balaji; Rozbicki, Emil; Rustici, Gabriella; Tarkowska, Aleksandra; Walczysko, Petr; Williams, Eleanor; Swedlow, Jason R.

    2016-07-01

    Despite significant advances in biological imaging and analysis, major informatics challenges remain unsolved: file formats are proprietary, storage and analysis facilities are lacking, as are standards for sharing image data and results. While the open FITS file format is ubiquitous in astronomy, astronomical imaging shares many challenges with biological imaging, including the need to share large image sets using secure, cross-platform APIs, and the need for scalable applications for processing and visualization. The Open Microscopy Environment (OME) is an open-source software framework developed to address these challenges. OME tools include: an open data model for multidimensional imaging (OME Data Model); an open file format (OME-TIFF) and library (Bio-Formats) enabling free access to images (5D+) written in more than 145 formats from many imaging domains, including FITS; and a data management server (OMERO). The Java-based OMERO client-server platform comprises an image metadata store, an image repository, visualization and analysis by remote access, allowing sharing and publishing of image data. OMERO provides a means to manage the data through a multi-platform API. OMERO's model-based architecture has enabled its extension into a range of imaging domains, including light and electron microscopy, high content screening, digital pathology and recently into applications using non-image data from clinical and genomic studies. This is made possible using the Bio-Formats library. The current release includes a single mechanism for accessing image data of all types, regardless of original file format, via Java, C/C++ and Python and a variety of applications and environments (e.g. ImageJ, Matlab and R).

  20. Confocal scanning laser microscopy and quantitative image analysis: application to cream cheese microstructure investigation.

    PubMed

    Fenoul, F; Le Denmat, M; Hamdi, F; Cuvelier, G; Michon, C

    2008-04-01

    The naked eye observation of cream cheese confocal scanning laser microscopy images only provides qualitative information about its microstructure. Because those products are dense dairy gels, confocal scanning laser microscopy images of 2 different cream cheeses may appear close. Quantitative image analysis is then necessary to compensate for human eye deficiency (e.g., lack of precision, subjectivity). Two kinds of quantitative image analysis were performed in this study: high-order statistical methods and grayscale mathematical morphology. They were applied to study the microstructure of 3 different cream cheeses (same manufacturing process, same dry matter content, but different fat and protein contents). Advantages and drawbacks of both methods are reviewed. The way they may be used to describe cream cheese microstructure is also presented.

  1. Volumetric imaging of erythrocytes using label-free multiphoton photoacoustic microscopy.

    PubMed

    Shelton, Ryan L; Mattison, Scott P; Applegate, Brian E

    2014-10-01

    Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high-resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes.

  2. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Trabelsi, Heykel; Soumillion, Patrice; Dufrêne, Yves F.

    2013-12-01

    Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues.

  3. Strip mosaicing confocal microscopy for rapid imaging over large areas of excised tissue

    NASA Astrophysics Data System (ADS)

    Abeytunge, Sanjee; Li, Yongbiao; Larson, Bjorg; Peterson, Gary; Toledo-Crow, Ricardo; Rajadhyaksha, Milind

    2012-03-01

    Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in fresh tissue, without the processing that is required for conventional pathology. Previously, basal cell carcinoma margins were detected by mosaicing of confocal images of 12 x 12 mm2 of excised tissue from Mohs surgery. This mosaicing took 9 minutes. Recently we reported the initial feasibility of a faster approach called "strip mosaicing" on 10 x 10 mm2 of tissue that was demonstrated in 3 minutes. In this paper we report further advances in instrumentation and software. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Thus, strip mosaicing confocal microscopy may serve as an adjunct to pathology for imaging tumor margins to guide surgery.

  4. 4Pi microscopy of type A with 1-photon excitation in biological fluorescence imaging

    NASA Astrophysics Data System (ADS)

    Lang, Marion; Müller, Tobias; Engelhardt, Johann; Hell, Stefan W.

    2007-03-01

    We demonstrate that oil immersion lenses with a semi-aperture angle ≥ 74° enable 4Pi confocal fluorescence microscopy of type A with 1 photon excitation. The axial sidelobes amount to < 50 % of the main diffraction maximum, implying that lobe induced artifacts can be removed from the image data. The advancement reported herein enables a relative inexpensive implementation of 4Pi microscopy, providing axially superresolved 3D-imaging in transparent samples. As an example, we show dual-color 4Pi images of double stained Golgi stacks in a mammalian cell with 110 nm axial resolution. The resolution can be further enhanced to values slightly below 100 nm by image deconvolution.

  5. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    SciTech Connect

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  6. Picometre-precision analysis of scanning transmission electron microscopy images of platinum nanocatalysts.

    PubMed

    Yankovich, Andrew B; Berkels, Benjamin; Dahmen, W; Binev, P; Sanchez, S I; Bradley, S A; Li, Ao; Szlufarska, Izabela; Voyles, Paul M

    2014-06-11

    Measuring picometre-scale shifts in the positions of individual atoms in materials provides new insight into the structure of surfaces, defects and interfaces that influence a broad variety of materials' behaviour. Here we demonstrate sub-picometre precision measurements of atom positions in aberration-corrected Z-contrast scanning transmission electron microscopy images based on the non-rigid registration and averaging of an image series. Non-rigid registration achieves five to seven times better precision than previous methods. Non-rigidly registered images of a silica-supported platinum nanocatalyst show pm-scale contraction of atoms at a (111)/(111) corner towards the particle centre and expansion of a flat (111) facet. Sub-picometre precision and standardless atom counting with <1 atom uncertainty in the same scanning transmission electron microscopy image provide new insight into the three-dimensional atomic structure of catalyst nanoparticle surfaces, which contain the active sites controlling catalytic reactions.

  7. Quantitative analysis of Scanning Tunneling Microscopy images for surface structure determination: Sulfur on Re(0001)

    SciTech Connect

    Ogletree, D.F.; Dunphy, J.C.; Salmeron, M.B.; Sautet, P. |

    1993-02-01

    Scanning Tunneling Microscopy (STM) images of adsorbed atoms and molecules on single crystal substrates provide important information on surface structure and order. In many cases images are interpreted qualitatively based on other information on the system. To obtain quantitative information, a theoretical analysis of the STM image is required. A new method of calculating STM images is presented that includes a full description of the STM tip and surface structure. This method is applied to experimental STM images of sulfur adsorbed on Re(0001). Effects of adsorption site, adsorbate geometry, tip composition and tunnel gap resistance on STM image contrast are analyzed. The chemical identity of tip apex atom and substrate subsurface structure are both shown to significantly affect STM image contrast.

  8. High-contrast imaging of mycobacterium tuberculosis using third-harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Bo Ram; Lee, Eungjang; Park, Seung-Han

    2015-07-01

    Nonlinear optical microcopy has become an important tool in investigating biomaterials due to its various advantages such as label-free imaging capabilities. In particular, it has been shown that third-harmonic generation (THG) signals can be produced at interfaces between an aqueous medium (e.g. cytoplasm, interstitial fluid) and a mineralized lipidic surface. In this work, we have demonstrated that label-free high-contrast THG images of the mycobacterium tuberculosis can be obtained using THG microscopy.

  9. Fluorescence microscopy studies of a peripheral-benzodiazepine-receptor-targeted molecular probe for brain tumor imaging

    NASA Astrophysics Data System (ADS)

    Marcu, Laura; Vernier, P. Thomas; Manning, H. Charles; Salemi, Sarah; Li, Aimin; Craft, Cheryl M.; Gundersen, Martin A.; Bornhop, Darryl J.

    2003-10-01

    This study investigates the potential of a new multi-modal lanthanide chelate complex for specifically targeting brain tumor cells. We report here results from ongoing studies of up-take, sub-cellular localization and binding specificity of this new molecular imaging probe. Fluorescence microscopy investigations in living rat C6 glioma tumor cells demonstrate that the new imaging agent has affinity for glioma cells and binds to mitochondria.

  10. Imaging fully hydrated whole cells by coherent x-ray diffraction microscopy.

    PubMed

    Nam, Daewoong; Park, Jaehyun; Gallagher-Jones, Marcus; Kim, Sangsoo; Kim, Sunam; Kohmura, Yoshiki; Naitow, Hisashi; Kunishima, Naoki; Yoshida, Takashi; Ishikawa, Tetsuya; Song, Changyong

    2013-03-01

    Nanoscale imaging of biological specimens in their native condition is of long-standing interest, in particular with direct, high resolution views of internal structures of intact specimens, though as yet progress has been limited. Here we introduce wet coherent x-ray diffraction microscopy capable of imaging fully hydrated and unstained biological specimens. Whole cell morphologies and internal structures better than 25 nm can be clearly visualized without contrast degradation.

  11. Adaptive and robust statistical methods for processing near-field scanning microwave microscopy images.

    PubMed

    Coakley, K J; Imtiaz, A; Wallis, T M; Weber, J C; Berweger, S; Kabos, P

    2015-03-01

    Near-field scanning microwave microscopy offers great potential to facilitate characterization, development and modeling of materials. By acquiring microwave images at multiple frequencies and amplitudes (along with the other modalities) one can study material and device physics at different lateral and depth scales. Images are typically noisy and contaminated by artifacts that can vary from scan line to scan line and planar-like trends due to sample tilt errors. Here, we level images based on an estimate of a smooth 2-d trend determined with a robust implementation of a local regression method. In this robust approach, features and outliers which are not due to the trend are automatically downweighted. We denoise images with the Adaptive Weights Smoothing method. This method smooths out additive noise while preserving edge-like features in images. We demonstrate the feasibility of our methods on topography images and microwave |S11| images. For one challenging test case, we demonstrate that our method outperforms alternative methods from the scanning probe microscopy data analysis software package Gwyddion. Our methods should be useful for massive image data sets where manual selection of landmarks or image subsets by a user is impractical.

  12. Integrated optical coherence tomography and optical coherence microscopy imaging of human pathology

    NASA Astrophysics Data System (ADS)

    Lee, Hsiang-Chieh; Zhou, Chao; Wang, Yihong; Aquirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-02-01

    Excisional biopsy is the current gold standard for disease diagnosis; however, it requires a relatively long processing time and it may also suffer from unacceptable false negative rates due to sampling errors. Optical coherence tomography (OCT) is a promising imaging technique that provide real-time, high resolution and three-dimensional (3D) images of tissue morphology. Optical coherence microscopy (OCM) is an extension of OCT, combining both the coherence gating and the confocal gating techniques. OCM imaging achieves cellular resolution with deeper imaging depth compared to confocal microscopy. An integrated OCT/OCM imaging system can provide co-registered multiscale imaging of tissue morphology. 3D-OCT provides architectural information with a large field of view and can be used to find regions of interest; while OCM provides high magnification to enable cellular imaging. The integrated OCT/OCM system has an axial resolution of <4um and transverse resolutions of 14um and <2um for OCT and OCM, respectively. In this study, a wide range of human pathologic specimens, including colon (58), thyroid (43), breast (34), and kidney (19), were imaged with OCT and OCM within 2 to 6 hours after excision. The images were compared with H & E histology to identify characteristic features useful for disease diagnosis. The feasibility of visualizing human pathology using integrated OCT/OCM was demonstrated in the pathology laboratory settings.

  13. Fibre optic confocal imaging (FOCI) for subsurface microscopy of the colon in vivo.

    PubMed Central

    Delaney, P M; King, R G; Lambert, J R; Harris, M R

    1994-01-01

    Fibre optic confocal imaging (FOCI) is a new type of microscopy which has been recently developed (Delaney et al. 1993). In contrast to conventional light microscopy, FOCI and other confocal techniques allow clear imaging of subsurface structures within translucent objects. However, unlike conventional confocal microscopes which are bulky (because of a need for accurate alignment of large components) FOCI allows the imaging end to be miniaturised and relatively mobile. FOCI is thus particularly suited for clear subsurface imaging of structures within living animals or subjects. The aim of the present study was to assess the suitability of using FOCI for imaging of subsurface structures within the colon, both in vitro (human and rat biopsies) and in vivo (in rats). Images were obtained in fluorescence mode (excitation 488 nm, detection above 515 nm) following topical application of fluorescein. By this technique the glandular structure of the colon was imaged. FOCI is thus suitable for subsurface imaging of the colon in vivo. Images Fig. 2 Fig. 3 PMID:8157487

  14. Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

    PubMed Central

    Kim, Dahan; Hess, Samuel T.

    2013-01-01

    Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension

  15. Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

    PubMed

    Curthoys, Nikki M; Mlodzianoski, Michael J; Kim, Dahan; Hess, Samuel T

    2013-12-09

    Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of

  16. [Analysis of reticulocytes: manual microscopy, flow analyzers or image analyzers? (analytical review)].

    PubMed

    Piatnitskiĭ, A M; Medovyĭ, V S; Parpara, A A

    2007-10-01

    Three procedures for determining the quantity and composition of reticulocytes (visual analysis during manual microscopy; flow cytometry, semi-automatic analysis using a computerized microscopy unit) are compared. The errors characteristics for each procedure and the reasons why manual analysis still remains most used are discussed. The paper points out that it is impossible to draw a conclusion on the accuracy of the procedures, by using the values of the correlation coefficient. Evidence is provided for the thesis that the statistical accuracy may be achieved only when the image analyzer of a computerized microscopy unit is employed, therefore this procedure is recommended for use as a reference one. The characteristics of a procedure for analyzing reticulocytes by means of a MEKOS-Ts2 computerized microscopy unit are presented.

  17. Imaging of surgical margin in pancreatic metastasis using two-photon excited fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Jing; Hong, Zhipeng; Chen, Hong; Chen, Youting; Xu, Yahao; Zhu, Xiaoqin; Zhuo, Shuangmu; Shi, Zheng; Chen, Jianxin

    2014-09-01

    Two-photon excited fluorescence (TPEF) microscopy, has become a powerful tool for imaging unstained tissue samples at subcellular level in biomedical research. The purpose of this study was to determine whether TPEF imaging of histological sections without H-E staining can be used to identify the boundary between normal pancreas and pancreatic metastasis from renal cell carcinoma (RCC). The typical features such as the significant increase of cancerous nests, the absence of pancreatic ductal, the appearance of cancer cells were observed to present the boundary between normal pancreas and pancreatic metastasis from RCC. These results correlated well with the corresponding histological outcomes. With the advent of clinically miniaturized TPEF microscopy and integrative endoscopy, TPEF microscopy has the potential application on surgical location of pancreatic metastasis from RCC in the near future.

  18. Combination of widefield fluorescence imaging and nonlinear optical microscopy of oral epithelial neoplasia

    NASA Astrophysics Data System (ADS)

    Pal, Rahul; Edward, Kert; Brown, Tyra; Ma, Liang; Yang, Jinping; McCammon, Susan; Motamedi, Massoud; Vargas, Gracie

    2013-03-01

    Multiphoton Autofluorescence Microscopy (MPAM) and Second Harmonic Generation Microscopy (SHGM) have shown the potential for noninvasive assessment of oral precancers and cancers. We have explored a combination of these nonlinear optical microscopic imaging techniques with widefield fluorescence imaging to assess morphometry similar to that of pathologic evaluation as well as information from endogenous fluorophores, which are altered with neoplastic transformation. Widefield fluorescence revealed areas of interest corresponding to sites with precancers or early tumors, generally resulting in a decrease in green emission or increase in red emission. Subsequent microscopy revealed significant differences in morphology between normal, dysplastic/neoplastic mucosa for all layers. Combination of a widefield and a microscopic technique provides a novel approach for tissue morphometric analysis along with large area assessment of tissue autofluorescence properties.

  19. Multimodal imaging of human cerebellum - merging X-ray phase microtomography, magnetic resonance microscopy and histology

    NASA Astrophysics Data System (ADS)

    Schulz, Georg; Waschkies, Conny; Pfeiffer, Franz; Zanette, Irene; Weitkamp, Timm; David, Christian; Müller, Bert

    2012-11-01

    Imaging modalities including magnetic resonance imaging and X-ray computed tomography are established methods in daily clinical diagnosis of human brain. Clinical equipment does not provide sufficient spatial resolution to obtain morphological information on the cellular level, essential for applying minimally or non-invasive surgical interventions. Therefore, generic data with lateral sub-micrometer resolution have been generated from histological slices post mortem. Sub-cellular spatial resolution, lost in the third dimension as a result of sectioning, is obtained using magnetic resonance microscopy and micro computed tomography. We demonstrate that for human cerebellum grating-based X-ray phase tomography shows complementary contrast to magnetic resonance microscopy and histology. In this study, the contrast-to-noise values of magnetic resonance microscopy and phase tomography were comparable whereas the spatial resolution in phase tomography is an order of magnitude better. The registered data with their complementary information permit the distinct segmentation of tissues within the human cerebellum.

  20. Multi-modal digital holographic microscopy for wide-field fluorescence and 3D phase imaging

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Xia, Peng; Matoba, Osamu; Nitta, Koichi; Awatsuji, Yasuhiro

    2016-03-01

    Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

  1. Microwave and digital imaging technology reduce turnaround times for diagnostic electron microscopy.

    PubMed

    Giberson, Richard T; Austin, Ronald L; Charlesworth, Jon; Adamson, Grete; Herrera, Guillermo A

    2003-01-01

    The contributions of microwave methods and digital imaging techniques, when taken together, can reduce routine specimen processing and evaluation for diagnostic electron microscopy to a time frame never thought possible. Significant improvements in both technologies over the last 5 years led the authors to evaluate their combined attributes as the most likely candidate to provide a realistic solution in the reduction of turnaround times for diagnostic electron microscopy. For diagnostic electron microscopy to compete favorably with immunohistochemistry and other ancillary diagnostic techniques, it must improve its turnaround time. To evaluate this hypothesis the microwave-assisted processing results of over 2,000 diagnostic cases were evaluated as was a digital image administration system used for the acquisition and dissemination of diagnostic results. The incorporation of both technologies resulted in turnaround times being reduced to 4 h or less.

  2. Low-cost fluorescence microscopy for point-of-care cell imaging

    NASA Astrophysics Data System (ADS)

    Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

    2010-02-01

    Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

  3. Live imaging of nervous system development and function using light-sheet microscopy.

    PubMed

    Lemon, William C; Keller, Philipp J

    2015-01-01

    In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

  4. Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications

    PubMed Central

    Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W.; Dokmeci, Mehmet Remzi; Boyden, Edward S.; Khademhosseini, Ali

    2016-01-01

    To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such “hybrid microscopy” methods—combining physical and optical magnifications—can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes (“mini-microscopes”), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics—a process we refer to as Expansion Mini-Microscopy (ExMM)—is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places. PMID:26975883

  5. Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications

    NASA Astrophysics Data System (ADS)

    Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W.; Dokmeci, Mehmet Remzi; Boyden, Edward S.; Khademhosseini, Ali

    2016-03-01

    To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such “hybrid microscopy” methods—combining physical and optical magnifications—can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes (“mini-microscopes”), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics—a process we refer to as Expansion Mini-Microscopy (ExMM)—is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

  6. Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

    PubMed

    Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

    2015-01-01

    Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

  7. Cryogenic-temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids.

    PubMed

    Kleinerman, O; Parra-Vasquez, A Nicholas G; Green, M J; Behabtu, N; Schmidt, J; Kesselman, E; Young, C C; Cohen, Y; Pasquali, M; Talmon, Y

    2015-07-01

    Cryogenic electron microscopy (cryo-EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo-TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo-SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo-EM involve either water or organic components. In this paper, we introduce the use of novel cryo-TEM and cryo-SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single-walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo-TEM and cryo-SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid-crystalline CNT phases, used as the 'dope' for all-carbon-fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self-assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.

  8. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging.

    PubMed

    Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

    2015-11-26

    Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non

  9. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging

    NASA Astrophysics Data System (ADS)

    Errico, Claudia; Pierre, Juliette; Pezet, Sophie; Desailly, Yann; Lenkei, Zsolt; Couture, Olivier; Tanter, Mickael

    2015-11-01

    Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents—inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non

  10. Virtual spectral multiplexing for applications in in-situ imaging microscopy of transient phenomena

    NASA Astrophysics Data System (ADS)

    Deglint, Jason; Kazemzadeh, Farnoud; Shafiee, Mohammad Javad; Li, Edward; Khodadad, Iman; Saini, Simarjeet S.; Wong, Alexander; Clausi, David A.

    2015-09-01

    Multispectral sensing is specifically designed to provide quantitative spectral information about various materials or scenes. Using spectral information, various properties of objects can be measured and analysed. Microscopy, the observing and imaging of objects at the micron- or nano-scale, is one application where multispectral sensing can be advantageous, as many fields of science and research that use microscopy would benefit from observing a specimen in multiple wavelengths. Multispectral microscopy is available, but often requires the operator of the device to switch filters which is a labor intensive process. Furthermore, the need for filter switching makes such systems particularly limiting in cases where the sample contains live species that are constantly moving or exhibit transient phenomena. Direct methods for capturing multispectral data of a live sample simultaneously can also be challenging for microscopy applications as it requires an elaborate optical systems design which uses beamsplitters and a number of detectors proportional to the number of bands sought after. Such devices can therefore be quite costly to build and difficult to maintain, particularly for microscopy. In this paper, we present the concept of virtual spectral demultiplexing imaging (VSDI) microscopy for low-cost in-situ multispectral microscopy of transient phenomena. In VSDI microscopy, the spectral response of a color detector in the microscope is characterized and virtual spectral demultiplexing is performed on the simultaneously-acquired broadband detector measurements based on the developed spectral characterization model to produce microscopic imagery at multiple wavelengths. The proposed VSDI microscope was used to observe colorful nanowire arrays at various wavelengths simultaneously to illustrate its efficacy.

  11. Optical coherence microscopy for deep tissue imaging of the cerebral cortex with intrinsic contrast

    PubMed Central

    Srinivasan, Vivek J.; Radhakrishnan, Harsha; Jiang, James Y.; Barry, Scott; Cable, Alex E.

    2012-01-01

    In vivo optical microscopic imaging techniques have recently emerged as important tools for the study of neurobiological development and pathophysiology. In particular, two-photon microscopy has proved to be a robust and highly flexible method for in vivo imaging in highly scattering tissue. However, two-photon imaging typically requires extrinsic dyes or contrast agents, and imaging depths are limited to a few hundred microns. Here we demonstrate Optical Coherence Microscopy (OCM) for in vivo imaging of neuronal cell bodies and cortical myelination up to depths of ~1.3 mm in the rat neocortex. Imaging does not require the administration of exogenous dyes or contrast agents, and is achieved through intrinsic scattering contrast and image processing alone. Furthermore, using OCM we demonstrate in vivo, quantitative measurements of optical properties (index of refraction and attenuation coefficient) in the cortex, and correlate these properties with laminar cellular architecture determined from the images. Lastly, we show that OCM enables direct visualization of cellular changes during cell depolarization and may therefore provide novel optical markers of cell viability. PMID:22330462

  12. Extending the fundamental imaging-depth limit of multi-photon microscopy by imaging with photo-activatable fluorophores.

    PubMed

    Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei

    2012-08-13

    It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.

  13. Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.

    PubMed

    Udan, Ryan S; Piazza, Victor G; Hsu, Chih-Wei; Hadjantonakis, Anna-Katerina; Dickinson, Mary E

    2014-11-01

    Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

  14. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    SciTech Connect

    Tittmann, B. R.; Xi, X.

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  15. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-03-01

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved.

  16. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy

    PubMed Central

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-01-01

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved. PMID:28317915

  17. Deep-brain imaging via epi-fluorescence Computational Cannula Microscopy.

    PubMed

    Kim, Ganghun; Nagarajan, Naveen; Pastuzyn, Elissa; Jenks, Kyle; Capecchi, Mario; Shepherd, Jason; Menon, Rajesh

    2017-03-20

    Here we demonstrate widefield (field diameter = 200 μm) fluorescence microscopy and video imaging inside the rodent brain at a depth of 2 mm using a simple surgical glass needle (cannula) of diameter 0.22 mm as the primary optical element. The cannula guides excitation light into the brain and the fluorescence signal out of the brain. Concomitant image-processing algorithms are utilized to convert the spatially scrambled images into fluorescent images and video. The small size of the cannula enables minimally invasive imaging, while the long length (>2 mm) allow for deep-brain imaging with no additional complexity in the optical system. Since no scanning is involved, widefield fluorescence video at the native frame rate of the camera can be achieved.

  18. Cell depth imaging by point laser scanning fluorescence microscopy with an optical disk pickup head

    NASA Astrophysics Data System (ADS)

    Tsai, Rung-Ywan; Chen, Jung-Po; Lee, Yuan-Chin; Chiang, Hung-Chih; Cheng, Chih-Ming; Huang, Chun-Chieh; Huang, Tai-Ting; Cheng, Chung-Ta; Tiao, Golden

    2015-09-01

    A compact, cost-effective, and position-addressable digital laser scanning microscopy (DLSM) instrument is made using a commercially available Blu-ray disc read-only memory (BD-ROM) pickup head. Fluorescent cell images captured by DLSM have resolutions of 0.38 µm. Because of the position-addressable function, multispectral fluorescence cell images are captured using the same sample slide with different excitation laser sources. Specially designed objective lenses with the same working distance as the image-capturing beam are used for the different excitation laser sources. By accurately controlling the tilting angles of the sample slide or by moving the collimator lens of the image-capturing beam, the fluorescence cell images along different depth positions of the sample are obtained. Thus, z-section images with micrometer-depth resolutions are achievable.

  19. Image formation in phase-shifting digital holography and applications to microscopy.

    PubMed

    Yamaguchi, I; Kato, J; Ohta, S; Mizuno, J

    2001-12-01

    We discuss image formation in phase-shifting digital holography by developing an analytical formulation based on the Fresnel-Kirchhoff diffraction theory. Image-plane position and imaging magnification are derived for general configurations in which a spherical reference is employed. The influences of discrete sampling of the resulting interference patterns by a CCD and numerical reconstruction on qualities of point images are investigated. Dependence of the point images on the ratio of the minimum fringe spacing to pixel pitch of the CCD is numerically analyzed. Two-point resolution and magnification are also investigated as a function of pixel numbers by a simulation using a one-dimensional model. In experiments magnified images of biological objects and a resolution target were reconstructed with the same quality as by conventional microscopy.

  20. High speed optical coherence microscopy with autofocus adjustment and a miniaturized endoscopic imaging probe

    PubMed Central

    Aguirre, Aaron D.; Sawinski, Juergen; Huang, Shu-Wei; Zhou, Chao; Denk, Winfried; Fujimoto, James G.

    2010-01-01

    Optical coherence microscopy (OCM) is a promising technique for high resolution cellular imaging in human tissues. An OCM system for high-speed en face cellular resolution imaging was developed at 1060 nm wavelength at frame rates up to 5 Hz with resolutions of < 4 µm axial and < 2 µm transverse. The system utilized a novel polarization compensation method to combat wavelength dependent source polarization and achieve broadband electro-optic phase modulation compatible with ultrahigh axial resolution. In addition, the system incorporated an auto-focusing feature that enables precise, near real-time alignment of the confocal and coherence gates in tissue, allowing user-friendly optimization of image quality during the imaging procedure. Ex vivo cellular images of human esophagus, colon, and cervix as well as in vivo results from human skin are presented. Finally, the system design is demonstrated with a miniaturized piezoelectric fiber-scanning probe which can be adapted for laparoscopic and endoscopic imaging applications. PMID:20389435

  1. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy.

    PubMed

    Pawliczek, Piotr; Romanowska-Pawliczek, Anna; Soltys, Zbigniew

    2010-03-01

    Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson-Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines.

  2. Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ

    PubMed Central

    Müller, Marcel; Mönkemöller, Viola; Hennig, Simon; Hübner, Wolfgang; Huser, Thomas

    2016-01-01

    Super-resolved structured illumination microscopy (SR-SIM) is an important tool for fluorescence microscopy. SR-SIM microscopes perform multiple image acquisitions with varying illumination patterns, and reconstruct them to a super-resolved image. In its most frequent, linear implementation, SR-SIM doubles the spatial resolution. The reconstruction is performed numerically on the acquired wide-field image data, and thus relies on a software implementation of specific SR-SIM image reconstruction algorithms. We present fairSIM, an easy-to-use plugin that provides SR-SIM reconstructions for a wide range of SR-SIM platforms directly within ImageJ. For research groups developing their own implementations of super-resolution structured illumination microscopy, fairSIM takes away the hurdle of generating yet another implementation of the reconstruction algorithm. For users of commercial microscopes, it offers an additional, in-depth analysis option for their data independent of specific operating systems. As a modular, open-source solution, fairSIM can easily be adapted, automated and extended as the field of SR-SIM progresses. PMID:26996201

  3. Coherence-controlled holographic microscopy for live-cell quantitative phase imaging

    NASA Astrophysics Data System (ADS)

    Slabý, TomáÅ.¡; Křížová, Aneta; Lošt'ák, Martin; Čolláková, Jana; Jůzová, Veronika; Veselý, Pavel; Chmelík, Radim

    2015-03-01

    In this paper we present coherence-controlled holographic microscopy (CCHM) and various examples of observations of living cells including combination of CCHM with fluorescence microscopy. CCHM is a novel technique of quantitative phase imaging (QPI). It is based on grating off-axis interferometer, which is fully adapted for the use of incoherent illumination. This enables high-quality QPI free from speckles and parasitic interferences and lateral resolution of classical widefield microscopes. Label-free nature of QPI makes CCHM a useful tool for long-term observations of living cells. Moreover, coherence-gating effect induced by the use of incoherent illumination enables QPI of cells even in scattering media. Combination of CCHM with common imaging techniques brings the possibility to exploit advantages of QPI while simultaneously identifying the observed structures or processes by well-established imaging methods. We used CCHM for investigation of general parameters of cell life cycles and for research of cells reactions to different treatment. Cells were also visualized in 3D collagen gel with the use of CCHM. It was found that both the cell activity and movement of the collagen fibers can be registered. The method of CCHM in combination with fluorescence microscopy was used in order to obtain complementary information about cell morphology and identify typical morphological changes associated with different types of cell death. This combination of CCHM with common imaging technique has a potential to provide new knowledge about various processes and simultaneously their confirmation by comparison with known imaging method.

  4. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  5. Shadow scanning lens-free microscopy with tomographic reconstruction of 2D images

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Blushtein, Eugeny A.; Morev, Vladislav S.

    2016-04-01

    Shadow Scanning Lens-free Microscopy (SSLM) is a possible method for optical imaging that can potentially achieve high spatial resolution. At present work we discuss the SSLM and analyse the resolution limit conditioned by the light scattering from the edge scanning imaging system that uses a shadow from moving knife edge or wire to collect the sets of tomographic projection data of two-dimensional objects. The results of numerical estimation of the SSLM resolution for reconstruction of 2D object image are presented. The experimental setup of SSLM with wire scanning element was developed. The developed device works in a UV band range and shows the spatial resolution about 90 nm.

  6. Live cell imaging based on surface plasmon-enhanced fluorescence microscopy using random nanostructures

    NASA Astrophysics Data System (ADS)

    Oh, Youngjin; Lee, Wonju; Son, Taehwang; Kim, Sook Young; Shin, Jeon-Soo; Kim, Donghyun

    2014-02-01

    Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

  7. Imaging Single ZnO Vertical Nanowire Laser Cavities using UV-Laser Scanning Confocal Microscopy

    SciTech Connect

    Gargas, D.J.; Toimil-Molares, M.E.; Yang, P.

    2008-11-17

    We report the fabrication and optical characterization of individual ZnO vertical nanowire laser cavities. Dilute nanowire arrays with interwire spacing>10 ?m were produced by a modified chemical vapor transport (CVT) method yielding an ideal platform for single nanowire imaging and spectroscopy. Lasing characteristics of a single vertical nanowire are presented, as well as high-resolution photoluminescence imaging by UV-laser scanning confocal microscopy. In addition, three-dimensional (3D) mapping of the photoluminescence emission performed in both planar and vertical dimensions demonstrates height-selective imaging useful for vertical nanowires and heteronanostructures emerging in the field of optoelectronics and nanophotonics.

  8. Imaging of buried phosphorus nanostructures in silicon using scanning tunneling microscopy

    SciTech Connect

    Oberbeck, Lars; Reusch, Thilo C. G.; Hallam, Toby; Simmons, Michelle Y. E-mail: michelle.simmons@unsw.edu.au; Schofield, Steven R.; Curson, Neil J. E-mail: michelle.simmons@unsw.edu.au

    2014-06-23

    We demonstrate the locating and imaging of single phosphorus atoms and phosphorus dopant nanostructures, buried beneath the Si(001) surface using scanning tunneling microscopy. The buried dopant nanostructures have been fabricated in a bottom-up approach using scanning tunneling microscope lithography on Si(001). We find that current imaging tunneling spectroscopy is suited to locate and image buried nanostructures at room temperature and with residual surface roughness present. From these studies, we can place an upper limit on the lateral diffusion during encapsulation with low-temperature Si molecular beam epitaxy.

  9. Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

    PubMed Central

    Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge

    2015-01-01

    Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color. PMID:26153696

  10. Origin and compensation of imaging artefacts in localization-based super-resolution microscopy.

    PubMed

    Erdélyi, M; Sinkó, J; Kákonyi, R; Kelemen, A; Rees, E; Varga, D; Szabó, G

    2015-10-15

    Interpretation of high resolution images provided by localization-based microscopy techniques is a challenge due to imaging artefacts that can be categorized by their origin. They can be introduced by the optical system, by the studied sample or by the applied algorithms. Some artefacts can be eliminated via precise calibration procedures, others can be reduced only below a certain value. Images studied both theoretically and experimentally are qualified either by pattern specific metrics or by a more general metric based on fluorescence correlation spectroscopy.

  11. Perfect optical vortex enhanced surface plasmon excitation for plasmonic structured illumination microscopy imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Chonglei; Min, Changjun; Du, Luping; Yuan, X.-C.

    2016-05-01

    We propose an all-optical technique for plasmonic structured illumination microscopy (PSIM) with perfect optical vortex (POV). POV can improve the efficiency of the excitation of surface plasma and reduce the background noise of the excited fluorescence. The plasmonic standing wave patterns are excited by POV with fractional topological charges for accurate phase shift of {-2π/3, 0, and 2π/3}. The imaging resolution of less than 200 nm was produced. This PSIM technique is expected to be used as a wide field, super resolution imaging technique in dynamic biological imaging.

  12. Decoupling indirect topographic cross-talk in band excitation piezoresponse force microscopy imaging and spectroscopy

    DOE PAGES

    Mazet, Lucie; Jesse, Stephen; Niu, Gang; ...

    2016-06-20

    Here, all scanning probe microscopies are subjected to topographic cross-talk, meaning the topography-related contrast in functional images. Here, we investigate the signatures of indirect topographic cross-talk in piezoresponse force microscopy (PFM) imaging and spectroscopy and its decoupling using band excitation (BE) method in ferroelectric BaTiO3 deposited on the Si substrates with free standing nanopillars of diameter 50 nm. Comparison between the single-frequency PFM and BE-PFM results shows that the measured signal can be significantly distorted by topography-induced shifts in the contact resonance frequency and cantilever transfer function. However, with proper correction, such shifts do not affect PFM imaging and hysteresismore » loop measurements. This suggests the necessity of an advanced approach, such as BE-PFM, for detection of intrinsic sample piezoresponse on the topographically non-uniform surfaces.« less

  13. Tip radius preservation for high resolution imaging in amplitude modulation atomic force microscopy

    SciTech Connect

    Ramos, Jorge R.

    2014-07-28

    The acquisition of high resolution images in atomic force microscopy (AFM) is correlated to the cantilever's tip shape, size, and imaging conditions. In this work, relative tip wear is quantified based on the evolution of a direct experimental observable in amplitude modulation atomic force microscopy, i.e., the critical amplitude. We further show that the scanning parameters required to guarantee a maximum compressive stress that is lower than the yield/fracture stress of the tip can be estimated via experimental observables. In both counts, the optimized parameters to acquire AFM images while preserving the tip are discussed. The results are validated experimentally by employing IgG antibodies as a model system.

  14. Imaging of dense cell cultures by multiwavelength lens-free video microscopy.

    PubMed

    Allier, C; Morel, S; Vincent, R; Ghenim, L; Navarro, F; Menneteau, M; Bordy, T; Hervé, L; Cioni, O; Gidrol, X; Usson, Y; Dinten, J-M

    2017-02-27

    They present results for lens-free microscopy for the imaging of dense cell culture. With this aim, they use a multiwavelength LED illumination with well separated wavelengths, together with the implementation of an appropriate holographic reconstruction algorithm. This allows for a fast and efficient reconstruction of the phase image of densely packed cells (up to 700 cells/mm(2) ) over a large field of view of 29.4 mm(2) . Combined with the compactness of the system which fits altogether inside an incubator, lens-free microscopy becomes a unique tool to monitor cell cultures over several days. The high contrast phase shift images provide robust cell segmentation and tracking, and enable high throughput monitoring of individual cell dimensions, dry mass, and motility. They tested the multiwavelength lens-free video-microscope over a broad range of cell lines, including mesenchymal, endothelial, and epithelial cells. © 2017 International Society for Advancement of Cytometry.

  15. In vivo volumetric fluorescence sectioning microscopy with mechanical-scan-free hybrid illumination imaging

    PubMed Central

    Lin, Chen-Yen; Lin, Wei-Hsin; Chien, Ju-Hsuan; Tsai, Jui-Chang; Luo, Yuan

    2016-01-01

    Optical sectioning microscopy in wide-field fashion has been widely used to obtain three-dimensional images of biological samples; however, it requires scanning in depth and considerable time to acquire multiple depth information of a volumetric sample. In this paper, in vivo optical sectioning microscopy with volumetric hybrid illumination, with no mechanical moving parts, is presented. The proposed system is configured such that the optical sectioning is provided by hybrid illumination using a digital micro-mirror device (DMD) for uniform and non-uniform pattern projection, while the depth of imaging planes is varied by using an electrically tunable-focus lens with invariant magnification and resolution. We present and characterize the design, implementation, and experimentally demonstrate the proposed system’s ability through 3D imaging of in vivo Canenorhabditis elegans’ growth cones. PMID:27867708

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

    PubMed Central

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

    2015-01-01

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

  17. Decoupling indirect topographic cross-talk in band excitation piezoresponse force microscopy imaging and spectroscopy

    SciTech Connect

    Mazet, Lucie; Jesse, Stephen; Niu, Gang; Schroeder, Thomas; Schamm-Chardon, Sylvie; Dubourdieu, Catherine; Baddorf, Arthur P.; Kalinin, Sergei V.; Yang, Sang Mo; Okatan, M. Baris

    2016-06-20

    Here, all scanning probe microscopies are subjected to topographic cross-talk, meaning the topography-related contrast in functional images. Here, we investigate the signatures of indirect topographic cross-talk in piezoresponse force microscopy (PFM) imaging and spectroscopy and its decoupling using band excitation (BE) method in ferroelectric BaTiO3 deposited on the Si substrates with free standing nanopillars of diameter 50 nm. Comparison between the single-frequency PFM and BE-PFM results shows that the measured signal can be significantly distorted by topography-induced shifts in the contact resonance frequency and cantilever transfer function. However, with proper correction, such shifts do not affect PFM imaging and hysteresis loop measurements. This suggests the necessity of an advanced approach, such as BE-PFM, for detection of intrinsic sample piezoresponse on the topographically non-uniform surfaces.

  18. Selective-plane illumination microscopy for high-content volumetric biological imaging

    NASA Astrophysics Data System (ADS)

    McGorty, Ryan; Huang, Bo

    2016-03-01

    Light-sheet microscopy, also named selective-plane illumination microscopy, enables optical sectioning with minimal light delivered to the sample. Therefore, it allows one to gather volumetric datasets of developing embryos and other light-sensitive samples over extended times. We have configured a light-sheet microscope that, unlike most previous designs, can image samples in formats compatible with high-content imaging. Our microscope can be used with multi-well plates or with microfluidic devices. In designing our optical system to accommodate these types of sample holders we encounter large optical aberrations. We counter these aberrations with both static optical components in the imaging path and with adaptive optics. Potential applications of this microscope include studying the development of a large number of embryos in parallel and over long times with subcellular resolution and doing high-throughput screens on organisms or cells where volumetric data is necessary.

  19. Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Zhongjiang; Li, Bingbing; Yang, Sihua

    2014-09-01

    A label-free high resolution photoacoustic microscopy (PAM) system for imaging cellular malformation is presented. The carbon fibers were used to testify the lateral resolution of the PAM. Currently, the lateral resolution is better than 2.7 μm. The human normal red blood cells (RBCs) were used to prove the imaging capability of the system, and a single red blood cell was mapped with high contrast. Moreover, the iron deficiency anemia RBCs were clearly distinguished from the cell morphology by using the PAM. The experimental results demonstrate that the photoacoustic microscopy system can accomplish label-free photoacoustic imaging and that it has clinical potential for use in the detection of erythrocytes and blood vessels malformation.

  20. Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy.

    PubMed

    McIntire, T M; Brant, D A

    1999-12-01

    Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.

  1. Fluorescence microscopy imaging with a Fresnel zone plate array based optofluidic microscope

    PubMed Central

    Han, Chao; Lee, Lap Man; Yang, Changhuei

    2013-01-01

    We report the implementation of an on-chip microscope system, termed fluorescence optofluidic microscope (FOFM), which is capable of fluorescence microscopy imaging of samples in fluid media. The FOFM employs an array of Fresnel zone plates (FZP) to generate an array of focused light spots within a microfluidic channel. As a sample flows through the channel and across the array of focused light spots, the fluorescence emissions are collected by a filter-coated CMOS sensor, which serves as the channel's floor. The collected data can then be processed to render fluorescence microscopy images at a resolution determined by the focused light spot size (experimentally measured as 0.65 μm FWHM). In our experiments, our established resolution was 1.0 μm due to Nyquist criterion consideration. As a demonstration, we show that such a system can be used to image the cell nuclei stained by Acridine Orange and cytoplasm labeled by Qtracker®. PMID:21935556

  2. Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

    DOE PAGES

    Wang, Xueju; Pan, Zhipeng; Fan, Feifei; ...

    2015-09-10

    We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for themore » analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.« less

  3. Imaging and manipulation of adatoms on an alumina surface by noncontact atomic force microscopy.

    PubMed

    Simon, G H; Heyde, M; Freund, H-J

    2012-02-29

    Noncontact atomic force microscopy (NC-AFM) has been performed on an aluminum oxide film grown on NiAl(110) in ultrahigh vacuum (UHV) at low temperature (5 K). Results reproduce the topography of the structural model, unlike scanning tunnelling microscopy (STM) images. Equipped with this extraordinary contrast the network of extended defects, which stems from domain boundaries intersecting the film surface, can be analysed in atomic detail. The knowledge of occurring surface structures opens up the opportunity to determine adsorption sites of individual adsorbates on the alumina film. The level of difficulty for such imaging depends on the imaging characteristics of the substrate and the interaction which can be maintained above the adsorbate. Positions of single adsorbed gold atoms within the unit cell have been determined despite their easy removal at slightly higher interaction strength. Preliminary manipulation experiments indicate a pick-up process for the vanishing of the gold adatoms from the film surface.

  4. Imaging and control of domain structures in ferroelectric thin films via scanning force microscopy.

    SciTech Connect

    Gruverman, A.; Auciello, O.; Tokumoto, H.; Materials Science Division; Joint Research Center for Atom Tech.; National Inst. for Advanced Interdisciplinary Research

    1998-01-01

    Scanning force microscopy (SFM) is becoming a powerful technique with great potential both for imaging and for control of domain structures in ferroelectric materials at the nanometer scale. Application of SFM to visualization of domain structures in ferroelectric thin films is described. Imaging methods of ferroelectric domains are based on the detection of surface charges in the noncontact mode of SFM and on the measurement of the piezoelectric response of a ferroelectric film to an external field applied by the tip in the SFM contact mode. This latter mode can be used for nondestructive evaluation of local ferroelectric and piezoelectric properties and for manipulation of domains of less than 50 nm in diameter. The effect of the film thickness and crystallinity on the imaging resolution is discussed. Scanning force microscopy is shown to be a technique well suited for nanoscale investigation of switching processes and electrical degradation effects in ferroelectric thin films.

  5. Total three-dimensional imaging of phase objects using defocusing microscopy: Application to red blood cells

    NASA Astrophysics Data System (ADS)

    Roma, P. M. S.; Siman, L.; Amaral, F. T.; Agero, U.; Mesquita, O. N.

    2014-06-01

    We introduce Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total three-dimensional (3D) imaging of transparent objects. By total 3D imaging, we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic, and hypertonic solutions. For each situation, the shapes of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of red blood cell (RBC) surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine the volume, surface area, sphericity index, and RBC refractive index for each osmotic condition.

  6. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

    PubMed

    Hartshorn, Christopher M; Lee, Young Jong; Camp, Charles H; Liu, Zhen; Heddleston, John; Canfield, Nicole; Rhodes, Timothy A; Hight Walker, Angela R; Marsac, Patrick J; Cicerone, Marcus T

    2013-09-03

    We compare a coherent Raman imaging modality, broadband coherent anti-Stokes Raman scattering (BCARS) microscopy, with spontaneous Raman microscopy for quantitative and qualitative assessment of multicomponent pharmaceuticals. Indomethacin was used as a model active pharmaceutical ingredient (API) and was analyzed in a tabulated solid dosage form, embedded within commonly used excipients. In comparison with wide-field spontaneous Raman chemical imaging, BCARS acquired images 10× faster, at higher spatiochemical resolution and with spectra of much higher SNR, eliminating the need for multivariate methods to identify chemical components. The significant increase in spatiochemical resolution allowed identification of an unanticipated API phase that was missed by the spontaneous wide-field method and bulk Raman spectroscopy. We confirmed the presence of the unanticipated API phase using confocal spontaneous Raman, which provided spatiochemical resolution similar to BCARS but at 100× slower acquisition times.

  7. Ultrafast nanoscale imaging of surface charges by scanning resistive probe microscopy.

    SciTech Connect

    Ko, H.; Ryu, K.; Park, H.; Park, C.; Jeon, D.; Kim, Y. K.; Jung, J.; Min, D-K.; Kim, Y.; Lee, H. N.; Park, Y.; Shin, H.; Hong, S.

    2011-01-01

    Nanoscale manipulation of surface charges and their imaging are essential for understanding local electronic behaviors of polar materials and advanced electronic devices. Electrostatic force microscopy and Kelvin probe force microscopy have been extensively used to probe and image local surface charges responsible for electrodynamics and transport phenomena. However, they rely on the weak electric force modulation of cantilever that limits both spatial and temporal resolutions. Here we present a field effect transistor embedded probe that can directly image surface charges on a length scale of 25 nm and a time scale of less than 125 {mu}s. On the basis of the calculation of net surface charges in a 25 nm diameter ferroelectric domain, we could estimate the charge density resolution to be as low as 0.08 {mu}C/cm{sup 2}, which is equivalent to 1/20 electron per nanometer square at room temperature.

  8. Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

    DOEpatents

    Xie, Xiaoliang Sunney; Freudiger, Christian; Min, Wei

    2011-09-27

    A microscopy imaging system includes a first light source for providing a first train of pulses at a first center optical frequency .omega..sub.1, a second light source for providing a second train of pulses at a second center optical frequency .omega..sub.2, a modulator system, an optical detector, and a processor. The modulator system is for modulating a beam property of the second train of pulses at a modulation frequency f of at least 100 kHz. The optical detector is for detecting an integrated intensity of substantially all optical frequency components of the first train of pulses from the common focal volume by blocking the second train of pulses being modulated. The processor is for detecting, a modulation at the modulation frequency f, of the integrated intensity of the optical frequency components of the first train of pulses to provide a pixel of an image for the microscopy imaging system.

  9. Stochastic Optical Reconstruction Microscopy Imaging of Microtubule Arrays in Intact Arabidopsis thaliana Seedling Roots

    PubMed Central

    Dong, Bin; Yang, Xiaochen; Zhu, Shaobin; Bassham, Diane C.; Fang, Ning

    2015-01-01

    Super-resolution fluorescence microscopy has generated tremendous success in revealing detailed subcellular structures in animal cells. However, its application to plant cell biology remains extremely limited due to numerous technical challenges, including the generally high fluorescence background of plant cells and the presence of the cell wall. In the current study, stochastic optical reconstruction microscopy (STORM) imaging of intact Arabidopsis thaliana seedling roots with a spatial resolution of 20–40 nm was demonstrated. Using the super-resolution images, the spatial organization of cortical microtubules in different parts of a whole Arabidopsis root tip was analyzed quantitatively, and the results show the dramatic differences in the density and spatial organization of cortical microtubules in cells of different differentiation stages or types. The method developed can be applied to plant cell biological processes, including imaging of additional elements of the cytoskeleton, organelle substructure, and membrane domains. PMID:26503365

  10. Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

    SciTech Connect

    Wang, Xueju; Pan, Zhipeng; Fan, Feifei; Wang, Jiangwei; Liu, Yang; Mao, Scott X.; Zhu, Ting; Xia, Shuman

    2015-09-10

    We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for the analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.

  11. Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

    NASA Astrophysics Data System (ADS)

    Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

    2016-12-01

    Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

  12. Development of a combined atomic force microscopy and side-view imaging system for mechanotransduction research

    NASA Astrophysics Data System (ADS)

    Beicker, Kellie N.

    Key metrics for understanding cell response to mechanical stimuli include rearrangement of the cytoskeletal and nucleoskeletal structure, induced strains and biochemical distributions; however, structural information during applied stress is limited by our ability to image cells under load. In order to study the mechanics of single cells and subcellular components under load, I have developed a unique imaging system that combines an atomic force microscope (AFM) with both vertical light-sheet (VLS) illumination and a new imaging technique called PRISM - Pathway Rotated Imaging for Sideways Microscopy. The combined AFM and PRISM system facilitates the imaging of cell deformation in the direction of applied force with accompanying pico-Newton resolution force measurements. The additional inclusion of light-sheet microscopy improves the signal-to-noise ratio achieved by illumination of only a thin layer of the cell. This system is capable of pico-newton resolution force measurements with simultaneous side-view high frame rate imaging for single-molecule and single-cell force studies. Longer-term goals for this instrument are to investigate how external mechanical stimuli, specifically single-molecule interactions, alter gene expression, motility, and differentiation. The overall goal of my dissertation work is to design a tool useful for mechanobiology studies of single cells. This requires the design and implementation of PRISM and VLS systems that can be coupled to the standard Asylum AFM on inverted optical microscope. I also examine the strategy and implementation of experimental procedures and data analysis pipelines for single-cell and single-molecule force spectroscopy. These goals can be broken down as follows: • Performed single-molecule force spectroscopy experiments. • Performed single-cell force spectroscopy experiments. • Constructed and characterized the side-view microscopy system. • Applied combined AFM and side-vew microscopy system.

  13. Biocompatible hybrid nanomaterials involving polymers and hydrogels interfaced with phosphorescent complexes and toxin-free metallic nanoparticles for biomedical applications

    NASA Astrophysics Data System (ADS)

    Marpu, Sreekar B.

    The major topics discussed are all relevant to interfacing brightly phosphorescent and non-luminescent coinage metal complexes of [Ag(I) and Au(I)] with biopolymers and thermoresponsive gels for making hybrid nanomaterials with an explanation on syntheses, characterization and their significance in biomedical fields. Experimental results and ongoing work on determining outreaching consequences of these hybrid nanomaterials for various biomedical applications like cancer therapy, bio-imaging and antibacterial abilities are described. In vitro and in vivo studies have been performed on majority of the discussed hybrid nanomaterials and determined that the cytotoxicity or antibacterial activity are comparatively superior when compared to analogues in literature. Consequential differences are noticed in photoluminescence enhancement from hybrid phosphorescent hydrogels, phosphorescent complex ability to physically crosslink, Au(I) sulfides tendency to form NIR (near-infrared) absorbing AuNPs compared to any similar work in literature. Syntheses of these hybrid nanomaterials has been thoroughly investigated and it is determined that either metallic nanoparticles syntheses or syntheses of phosphorescent hydrogels can be carried in single step without involving any hazardous reducing agents or crosslinkers or stabilizers that are commonly employed during multiple step syntheses protocols for syntheses of similar materials in literature. These astounding results that have been discovered within studies of hybrid nanomaterials are an asset to applications ranging from materials development to health science and will have striking effect on environmental and green chemistry approaches.

  14. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis.

    PubMed

    Skytte, Jacob L; Ghita, Ovidiu; Whelan, Paul F; Andersen, Ulf; Møller, Flemming; Dahl, Anders B; Larsen, Rasmus

    2015-06-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermented dairy products. When studying such networks, hundreds of images can be obtained, and here image analysis methods are essential for using the images in statistical analysis. Previously, methods including gray level co-occurrence matrix analysis and fractal analysis have been used with success. However, a range of other image texture characterization methods exists. These methods describe an image by a frequency distribution of predefined image features (denoted textons). Our contribution is an investigation of the choice of image analysis methods by performing a comparative study of 7 major approaches to image texture description. Here, CSLM images from a yogurt fermentation study are investigated, where production factors including fat content, protein content, heat treatment, and incubation temperature are varied. The descriptors are evaluated through nearest neighbor classification, variance analysis, and cluster analysis. Our investigation suggests that the texton-based descriptors provide a fuller description of the images compared to gray-level co-occurrence matrix descriptors and fractal analysis, while still being as applicable and in some cases as easy to tune.

  15. Imaging stability in force-feedback high-speed atomic force microscopy.

    PubMed

    Kim, Byung I; Boehm, Ryan D

    2013-02-01

    We studied the stability of force-feedback high-speed atomic force microscopy (HSAFM) by imaging soft, hard, and biological sample surfaces at various applied forces. The HSAFM images showed sudden topographic variations of streaky fringes with a negative applied force when collected on a soft hydrocarbon film grown on a grating sample, whereas they showed stable topographic features with positive applied forces. The instability of HSAFM images with the negative applied force was explained by the transition between contact and noncontact regimes in the force-distance curve. When the grating surface was cleaned, and thus hydrophilic by removing the hydrocarbon film, enhanced imaging stability was observed at both positive and negative applied forces. The higher adhesive interaction between the tip and the surface explains the improved imaging stability. The effects of imaging rate on the imaging stability were tested on an even softer adhesive Escherichia coli biofilm deposited onto the grating structure. The biofilm and planktonic cell structures in HSAFM images were reproducible within the force deviation less than ∼0.5 nN at the imaging rate up to 0.2s per frame, suggesting that the force-feedback HSAFM was stable for various imaging speeds in imaging softer adhesive biological samples.

  16. Label-free imaging of human breast tissues using coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Yaliang; Gao, Liang; Wang, Zhiyong; Thrall, Michael J.; Luo, Pengfei; Wong, Kelvin K.; Wong, Stephen T.

    2011-03-01

    Breast cancer is a common disease in women. Current imaging and diagnostic methods for breast cancer confront several limitations, like time-consuming, invasive and with a high cost. Alternative strategies are in high demand to alleviate patients' trauma and lower medical expenses. Coherent anti-Stokes Raman scattering (CARS) imaging technique offers many advantages, including label-free, sub-wavelength spatial resolution and video-rate imaging speed. Therefore, it has been demonstrated as a powerful tool for various biomedical applications. In this study, we present a label-free fast imaging method to identify breast cancer and its subtypes using CARS microscopy. Human breast tissues, including normal, benign and invasive carcinomas, were imaged ex vivo using a custom-built CARS microscope. Compared with results from corresponding hematoxylin and eosin (H&E) stains, the CARS technique has demonstrated its capability in identifying morphological features in a similar way as in H&E stain. These features can be used to distinguish breast cancer from normal and benign tissues, and further separate cancer subtypes from each other. Our pilot study suggests that CARS microscopy could be used as a routine examination tool to characterize breast cancer ex vivo. Moreover, its label-free and fast imaging properties render this technique as a promising approach for in vivo and real-time imaging and diagnosis of breast cancer.

  17. Limited-memory scaled gradient projection methods for real-time image deconvolution in microscopy

    NASA Astrophysics Data System (ADS)

    Porta, F.; Zanella, R.; Zanghirati, G.; Zanni, L.

    2015-04-01

    Gradient projection methods have given rise to effective tools for image deconvolution in several relevant areas, such as microscopy, medical imaging and astronomy. Due to the large scale of the optimization problems arising in nowadays imaging applications and to the growing request of real-time reconstructions, an interesting challenge to be faced consists in designing new acceleration techniques for the gradient schemes, able to preserve their simplicity and low computational cost of each iteration. In this work we propose an acceleration strategy for a state-of-the-art scaled gradient projection method for image deconvolution in microscopy. The acceleration idea is derived by adapting a step-length selection rule, recently introduced for limited-memory steepest descent methods in unconstrained optimization, to the special constrained optimization framework arising in image reconstruction. We describe how important issues related to the generalization of the step-length rule to the imaging optimization problem have been faced and we evaluate the improvements due to the acceleration strategy by numerical experiments on large-scale image deconvolution problems.

  18. SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

    PubMed Central

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron A.; Charbon, Edoardo

    2017-01-01

    sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm. PMID:28287122

  19. Multiphoton imaging microscopy at deeper layers with adaptive optics control of spherical aberration.

    PubMed

    Bueno, Juan M; Skorsetz, Martin; Palacios, Raquel; Gualda, Emilio J; Artal, Pablo

    2014-01-01

    Despite the inherent confocality and optical sectioning capabilities of multiphoton microscopy, three-dimensional (3-D) imaging of thick samples is limited by the specimen-induced aberrations. The combination of immersion objectives and sensorless adaptive optics (AO) techniques has been suggested to overcome this difficulty. However, a complex plane-by-plane correction of aberrations is required, and its performance depends on a set of image-based merit functions. We propose here an alternative approach to increase penetration depth in 3-D multiphoton microscopy imaging. It is based on the manipulation of the spherical aberration (SA) of the incident beam with an AO device while performing fast tomographic multiphoton imaging. When inducing SA, the image quality at best focus is reduced; however, better quality images are obtained from deeper planes within the sample. This is a compromise that enables registration of improved 3-D multiphoton images using nonimmersion objectives. Examples on ocular tissues and nonbiological samples providing different types of nonlinear signal are presented. The implementation of this technique in a future clinical instrument might provide a better visualization of corneal structures in living eyes.

  20. SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking.

    PubMed

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron A; Charbon, Edoardo

    2017-03-13

    sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.

  1. SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking

    NASA Astrophysics Data System (ADS)

    Antolovic, Ivan Michel; Burri, Samuel; Bruschini, Claudio; Hoebe, Ron A.; Charbon, Edoardo

    2017-03-01

    sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.

  2. Quantitative sub-surface and non-contact imaging using scanning microwave microscopy

    NASA Astrophysics Data System (ADS)

    Gramse, Georg; Brinciotti, Enrico; Lucibello, Andrea; Patil, Samadhan B.; Kasper, Manuel; Rankl, Christian; Giridharagopal, Rajiv; Hinterdorfer, Peter; Marcelli, Romolo; Kienberger, Ferry

    2015-03-01

    The capability of scanning microwave microscopy for calibrated sub-surface and non-contact capacitance imaging of silicon (Si) samples is quantitatively studied at broadband frequencies ranging from 1 to 20 GHz. Calibrated capacitance images of flat Si test samples with varying dopant density (1015-1019 atoms cm-3) and covered with dielectric thin films of SiO2 (100-400 nm thickness) are measured to demonstrate the sensitivity of scanning microwave microscopy (SMM) for sub-surface imaging. Using standard SMM imaging conditions the dopant areas could still be sensed under a 400 nm thick oxide layer. Non-contact SMM imaging in lift-mode and constant height mode is quantitatively demonstrated on a 50 nm thick SiO2 test pad. The differences between non-contact and contact mode capacitances are studied with respect to the main parameters influencing the imaging contrast, namely the probe tip diameter and the tip-sample distance. Finite element modelling was used to further analyse the influence of the tip radius and the tip-sample distance on the SMM sensitivity. The understanding of how the two key parameters determine the SMM sensitivity and quantitative capacitances represents an important step towards its routine application for non-contact and sub-surface imaging.

  3. A correlative method for imaging identical regions of samples by micro-CT, light microscopy, and electron microscopy: imaging adipose tissue in a model system.

    PubMed

    Sengle, Gerhard; Tufa, Sara F; Sakai, Lynn Y; Zulliger, Martin A; Keene, Douglas R

    2013-04-01

    We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content.

  4. Phosphorescent organic light emitting diodes with high efficiency and brightness

    DOEpatents

    Forrest, Stephen R; Zhang, Yifan

    2015-11-12

    An organic light emitting device including a) an anode; b) a cathode; and c) an emissive layer disposed between the anode and the cathode, the emissive layer comprising an organic host compound and a phosphorescent compound exhibiting a Stokes Shift overlap greater than 0.3 eV. The organic light emitting device may further include a hole transport layer disposed between the emissive layer and the anode; and an electron transport layer disposed between the emissive layer and the cathode. In some embodiments, the phosphorescent compound exhibits a phosphorescent lifetime of less than 10 .mu.s. In some embodiments, the concentration of the phosphorescent compound ranges from 0.5 wt. % to 10 wt. %.

  5. Bright afterglow illuminator made of phosphorescent material and fluorescent fibers.

    PubMed

    Saito, M; Yamamoto, K

    2000-08-20

    Phosphorescent oxides and fluorescent dyes were used together to create a fiber-type illuminator that glows in the dark without the need for electric power. Dye-doped polymer fibers, which were bundled at one end, were linearly arrayed in a polysiloxane resin that contained phosphorescent oxide particles. The phosphorescent resin continued to glow for a long time even after the excitation light was removed. Organic dyes in a polymer fiber were excited by the phosphorescence and emitted fluorescence toward the fiber end. Fluorescence from a number of dyes was combined in the long fiber, and, consequently a bright light beam emerged from the fiber end. Useful performance, i.e., high power density, narrow beam divergence, and long afterglow, is demonstrated by the prototype fiber illuminator.

  6. Real-time three-dimensional imaging of cell division by differential interference contrast microscopy.

    PubMed

    Tsunoda, M; Isailovic, D; Yeung, E S

    2008-11-01

    Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub-diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer-controlled vertical scanning of the microscope stage.

  7. Interferometric backward third harmonic generation microscopy for axial imaging with accuracy beyond the diffraction limit.

    PubMed

    Sandkuijl, Daaf; Kontenis, Lukas; Coelho, Nuno M; McCulloch, Christopher; Barzda, Virginijus

    2014-01-01

    A new nonlinear microscopy technique based on interference of backward-reflected third harmonic generation (I-THG) from multiple interfaces is presented. The technique is used to measure height variations or changes of a layer thickness with an accuracy of up to 5 nm. Height variations of a patterned glass surface and thickness variations of fibroblasts are visualized with the interferometric epi-THG microscope with an accuracy at least two orders of magnitude better than diffraction limit. The microscopy technique can be broadly applied for measuring distance variations between membranes or multilayer structures inside biological tissue and for surface height variation imaging.

  8. Novel Smart Windows Based on Transparent Phosphorescent OLEDs

    SciTech Connect

    Brian D'Andrade; Stephen Forest

    2006-09-15

    In this program, Universal Display Corporation (UDC) and Princeton University developed the use of white transparent phosphorescent organic light emitting devices (PHOLEDs{trademark}) to make low-cost ''transparent OLED (TOLED) smart windows'', that switch rapidly from being a highly efficient solid-state light source to being a transparent window. PHOLEDs are ideal for large area devices, and the UDC-Princeton team has demonstrated white PHOLEDs with efficiencies of >24 lm/W at a luminance of 1,000 cd/m{sup 2}. TOLEDs have transparencies >70% over the visible wavelengths of light, but their transparency drops to less than 5% for wavelengths shorter than 350 nm, so they can also be used as ultraviolet (UV) light filters. In addition to controlling the flow of UV radiation, TOLEDs coupled with an electromechanical or electrically activated reflecting shutter on a glass window can be employed to control the flow of heat from infrared (IR) radiation by varying the reflectance/transparency of the glass for wavelengths greater than 800nm. One particularly attractive shutter technology is reversible electrochromic mirrors (REM). Our goal was therefore to integrate two innovative concepts to meet the U.S. Department of Energy goals: high power efficiency TOLEDs, plus electrically controlled reflectors to produce a ''smart window''. Our efforts during this one year program have succeeded in producing a prototype smart window shown in the Fig. I, below. The four states of the smart window are pictured: reflective with lamp on, reflective with lamp off, transparent with lamp on, and transparent with lamp off. In the transparent states, the image is an outdoor setting viewed through the window. In the reflective states, the image is an indoor setting viewed via reflection off the window. We believe that the integration of our high efficiency white phosphorescent TOLED illumination source, with electrically activated shutters represents an innovative low-cost approach to

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-02-01

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

  11. Status and potential for phosphorescent OLED technology

    NASA Astrophysics Data System (ADS)

    Hack, M.; Weaver, M. S.; Adamovich, V.; Kwong, R. C.; Lu, M. H.; Brown, J. J.

    2005-07-01

    As organic light emitting device (OLED) technology is building up momentum in the commercial marketplace, phosphorescent OLEDs (PHOLEDsTM) are proving themselves to be an ideal display medium for a wide range of product applications: from small mobile displays to large area TVs. As part of this work we continue to advance PHOLED technology by new materials design and device architectures. For example a green PHOLED with 4.3 V, 70 cd/A, 50 lm/W and > 10,000 hours lifetime at 1,000 cd/m2 is reported. PHOLEDs enable very low power consumption displays with low display operating temperatures, and can be deposited by a range of different deposition techniques. Along with state-of-the-art device performance we report results on the ruggedness of PHOLED materials in high volume manufacturing environments.

  12. A Comparison of Image Quality Evaluation Techniques for Transmission X-Ray Microscopy

    SciTech Connect

    Bolgert, Peter J; /Marquette U. /SLAC

    2012-08-31

    Beamline 6-2c at Stanford Synchrotron Radiation Lightsource (SSRL) is capable of Transmission X-ray Microscopy (TXM) at 30 nm resolution. Raw images from the microscope must undergo extensive image processing before publication. Since typical data sets normally contain thousands of images, it is necessary to automate the image processing workflow as much as possible, particularly for the aligning and averaging of similar images. Currently we align images using the 'phase correlation' algorithm, which calculates the relative offset of two images by multiplying them in the frequency domain. For images containing high frequency noise, this algorithm will align noise with noise, resulting in a blurry average. To remedy this we multiply the images by a Gaussian function in the frequency domain, so that the algorithm ignores the high frequency noise while properly aligning the features of interest (FOI). The shape of the Gaussian is manually tuned by the user until the resulting average image is sharpest. To automatically optimize this process, it is necessary for the computer to evaluate the quality of the average image by quantifying its sharpness. In our research we explored two image sharpness metrics, the variance method and the frequency threshold method. The variance method uses the variance of the image as an indicator of sharpness while the frequency threshold method sums up the power in a specific frequency band. These metrics were tested on a variety of test images, containing both real and artificial noise. To apply these sharpness metrics, we designed and built a MATLAB graphical user interface (GUI) called 'Blur Master.' We found that it is possible for blurry images to have a large variance if they contain high amounts of noise. On the other hand, we found the frequency method to be quite reliable, although it is necessary to manually choose suitable limits for the frequency band. Further research must be performed to design an algorithm which

  13. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

    PubMed Central

    Thompson, Rebecca F.; Walker, Matt; Siebert, C. Alistair; Muench, Stephen P.; Ranson, Neil A.

    2016-01-01

    Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a ‘resolution revolution’, owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM. PMID:26931652

  14. Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality

    PubMed Central

    Bittel, Amy M.; Nickerson, Andrew; Saldivar, Isaac S.; Dolman, Nick J.; Nan, Xiaolin; Gibbs, Summer L.

    2016-01-01

    Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance. PMID:27412307

  15. Dual tree complex wavelet transform based denoising of optical microscopy images.

    PubMed

    Bal, Ufuk

    2012-12-01

    Photon shot noise is the main noise source of optical microscopy images and can be modeled by a Poisson process. Several discrete wavelet transform based methods have been proposed in the literature for denoising images corrupted by Poisson noise. However, the discrete wavelet transform (DWT) has disadvantages such as shift variance, aliasing, and lack of directional selectivity. To overcome these problems, a dual tree complex wavelet transform is used in our proposed denoising algorithm. Our denoising algorithm is based on the assumption that for the Poisson noise case threshold values for wavelet coefficients can be estimated from the approximation coefficients. Our proposed method was compared with one of the state of the art denoising algorithms. Better results were obtained by using the proposed algorithm in terms of image quality metrics. Furthermore, the contrast enhancement effect of the proposed method on collagen fıber images is examined. Our method allows fast and efficient enhancement of images obtained under low light intensity conditions.

  16. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    PubMed Central

    Doktycz, M. J.; Qi, H.; Morrell-Falvey, J. L.

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction. Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors. PMID:23165043

  17. Methodology for Quantitative Characterization of Fluorophore Photoswitching to Predict Superresolution Microscopy Image Quality

    NASA Astrophysics Data System (ADS)

    Bittel, Amy M.; Nickerson, Andrew; Saldivar, Isaac S.; Dolman, Nick J.; Nan, Xiaolin; Gibbs, Summer L.

    2016-07-01

    Single-molecule localization microscopy (SMLM) image quality and resolution strongly depend on the photoswitching properties of fluorophores used for sample labeling. Development of fluorophores with optimized photoswitching will considerably improve SMLM spatial and spectral resolution. Currently, evaluating fluorophore photoswitching requires protein-conjugation before assessment mandating specific fluorophore functionality, which is a major hurdle for systematic characterization. Herein, we validated polyvinyl alcohol (PVA) as a single-molecule environment to efficiently quantify the photoswitching properties of fluorophores and identified photoswitching properties predictive of quality SMLM images. We demonstrated that the same fluorophore photoswitching properties measured in PVA films and using antibody adsorption, a protein-conjugation environment analogous to labeled cells, were significantly correlated to microtubule width and continuity, surrogate measures of SMLM image quality. Defining PVA as a fluorophore photoswitching screening platform will facilitate SMLM fluorophore development and optimal image buffer assessment through facile and accurate photoswitching property characterization, which translates to SMLM fluorophore imaging performance.

  18. Online quantitative phase imaging of vascular endothelial cells under fluid shear stress utilizing digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Odenthal-Schnittler, Maria; Schnittler, Hans Joachim; Kemper, Björn

    2016-03-01

    We have explored the utilization of quantitative phase imaging with digital holographic microscopy (DHM) as a novel tool for quantifying the dynamics of morphologic parameters (morphodynamics) of confluent endothelial cell layers under fluid shear stress conditions. Human umbilical vein endothelial cells (HUVECs) were exposed to fluid shear stress in a transparent cone/plate flow device (BioTech-Flow-System) and imaged with a modular setup for quantitative DHM phase imaging for up to 48 h. The resulting series of quantitative phase image sequences were analyzed for the average surface roughness of the cell layers and cell alignment. Our results demonstrate that quantitative phase imaging is a powerful and reliable tool to quantify the dynamics of morphological adaptation of endothelial cells to fluid shear stress.

  19. Automated Analysis of Fluorescence Microscopy Images to Identify Protein-Protein Interactions

    DOE PAGES

    Venkatraman, S.; Doktycz, M. J.; Qi, H.; ...

    2006-01-01

    The identification of protein interactions is important for elucidating biological networks. One obstacle in comprehensive interaction studies is the analyses of large datasets, particularly those containing images. Development of an automated system to analyze an image-based protein interaction dataset is needed. Such an analysis system is described here, to automatically extract features from fluorescence microscopy images obtained from a bacterial protein interaction assay. These features are used to relay quantitative values that aid in the automated scoring of positive interactions. Experimental observations indicate that identifying at least 50% positive cells in an image is sufficient to detect a protein interaction.more » Based on this criterion, the automated system presents 100% accuracy in detecting positive interactions for a dataset of 16 images. Algorithms were implemented using MATLAB and the software developed is available on request from the authors.« less

  20. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  1. Enhanced light element imaging in atomic resolution scanning transmission electron microscopy.

    PubMed

    Findlay, S D; Kohno, Y; Cardamone, L A; Ikuhara, Y; Shibata, N

    2014-01-01

    We show that an imaging mode based on taking the difference between signals recorded from the bright field (forward scattering region) in atomic resolution scanning transmission electron microscopy provides an enhancement of the detectability of light elements over existing techniques. In some instances this is an enhancement of the visibility of the light element columns relative to heavy element columns. In all cases explored it is an enhancement in the signal-to-noise ratio of the image at the light column site. The image formation mechanisms are explained and the technique is compared with earlier approaches. Experimental data, supported by simulation, are presented for imaging the oxygen columns in LaAlO₃. Case studies looking at imaging hydrogen columns in YH₂ and lithium columns in Al₃Li are also explored through simulation, particularly with respect to the dependence on defocus, probe-forming aperture angle and detector collection aperture angles.

  2. Objective for EUV microscopy, EUV lithography, and x-ray imaging

    DOEpatents

    Bitter, Manfred; Hill, Kenneth W.; Efthimion, Philip

    2016-05-03

    Disclosed is an imaging apparatus for EUV spectroscopy, EUV microscopy, EUV lithography, and x-ray imaging. This new imaging apparatus could, in particular, make significant contributions to EUV lithography at wavelengths in the range from 10 to 15 nm, which is presently being developed for the manufacturing of the next-generation integrated circuits. The disclosure provides a novel adjustable imaging apparatus that allows for the production of stigmatic images in x-ray imaging, EUV imaging, and EUVL. The imaging apparatus of the present invention incorporates additional properties compared to previously described objectives. The use of a pair of spherical reflectors containing a concave and convex arrangement has been applied to a EUV imaging system to allow for the image and optics to all be placed on the same side of a vacuum chamber. Additionally, the two spherical reflector segments previously described have been replaced by two full spheres or, more precisely, two spherical annuli, so that the total photon throughput is largely increased. Finally, the range of permissible Bragg angles and possible magnifications of the objective has been largely increased.

  3. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  4. Deblurring of Class-Averaged Images in Single-Particle Electron Microscopy

    PubMed Central

    Park, Wooram; Madden, Dean R.; Rockmore, Daniel N.; Chirikjian, Gregory S.

    2010-01-01

    This paper proposes a method for deblurring of class-averaged images in single-particle electron microscopy (EM). Since EM images of biological samples are very noisy, the images which are nominally identical projection images are often grouped, aligned and averaged in order to cancel or reduce the background noise. However, the noise in the individual EM images generates errors in the alignment process, which creates an inherent limit on the accuracy of the resulting class averages. This inaccurate class average due to the alignment errors can be viewed as the result of a convolution of an underlying clear image with a blurring function. In this work, we develop a deconvolution method that gives an estimate for the underlying clear image from a blurred class-averaged image using precomputed statistics of misalignment. Since this convolution is over the group of rigid body motions of the plane, SE(2), we use the Fourier transform for SE(2) in order to convert the convolution into a matrix multiplication in the corresponding Fourier space. For practical implementation we use a Hermite-function-based image modeling technique, because Hermite expansions enable lossless Cartesian-polar coordinate conversion using the Laguerre-Fourier expansions, and Hermite expansion and Laguerre-Fourier expansion retain their structures under the Fourier transform. Based on these mathematical properties, we can obtain the deconvolution of the blurred class average using simple matrix multiplication. Tests of the proposed deconvolution method using synthetic and experimental EM images confirm the performance of our method. PMID:20221416

  5. Vibrational Spectroscopic Microscopy: Raman, Near-Infrared and Mid-Infrared Imaging Techniques

    NASA Astrophysics Data System (ADS)

    Lewis, E. Neil; Levin, Ira W.

    1995-02-01

    New instrumental approaches for performing vibrational Raman, near-infrared and mid-infrared spectroscopic imaging microscopy are described. The instruments integrate imaging quality filters such as acousto-optic tunable filters (AOTFs), with visible charge-coupled device (CCD) and infrared focal-plane array detectors. These systems are used in conjunction with infinity-corrected, refractive microscopes for operation in the visible and near-infrared spectral regions and with Cassegrainian reflective optics for operation in the mid-infrared spectral interval. Chemically specific images at moderate spectral resolution (2 nm) and high spatial resolution (1 [mu]m) can be collected rapidly and noninvasively. Image data are presented containing 128 × 128 pixels, although significantly larger format images can be collected in approximately the same time. The instruments can be readily configured for both absorption and reflectance spectroscopies. We present Raman emission images of polystyrene microspheres and a lipid/amino acid mixture and near-infrared images of onion epidermis and a hydrated phospholipid dispersion. Images generated from mid-infrared spectral data are presented for a KBr disk containing nonhomogeneous domains of lipid and for 50-[mu]m slices of monkey cerebellum. These are the first results illustrating the use of infrared focal-plane array detectors as chemically specific spectroscopic imaging devices and demonstrating their application in biomolecular areas. Extensions and future applications of the various vibrational spectroscopic imaging techniques are discussed.

  6. High-Resolution Solid Modeling of Biological Samples Imaged with 3D Fluorescence Microscopy

    PubMed Central

    Ferko, Michael C.; Patterson, Brian W.; Butler, Peter J.

    2011-01-01

    Optical-sectioning, digital fluorescence microscopy provides images representing temporally- and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling. PMID:16758474

  7. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    PubMed Central

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  8. Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Kremers, Gert-Jan; van Munster, Erik B.; Goedhart, Joachim; Gadella, Theodorus W. J.

    2008-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins. PMID:18359789

  9. Deep Voting: A Robust Approach Toward Nucleus Localization in Microscopy Images.

    PubMed

    Xie, Yuanpu; Kong, Xiangfei; Xing, Fuyong; Liu, Fujun; Su, Hai; Yang, Lin

    2015-10-01

    Robust and accurate nuclei localization in microscopy image can provide crucial clues for accurate computer-aid diagnosis. In this paper, we propose a convolutional neural network (CNN) based hough voting method to localize nucleus centroids with heavy cluttering and morphologic variations in microscopy images. Our method, which we name as deep voting, mainly consists of two steps. (1) Given an input image, our method assigns each local patch several pairs of voting offset vectors which indicate the positions it votes to, and the corresponding voting confidence (used to weight each votes), our model can be viewed as an implicit hough-voting codebook. (2) We collect the weighted votes from all the testing patches and compute the final voting density map in a way similar to Parzen-window estimation. The final nucleus positions are identified by searching the local maxima of the density map. Our method only requires a few annotation efforts (just one click near the nucleus center). Experiment results on Neuroendocrine Tumor (NET) microscopy images proves the proposed method to be state-of-the-art.

  10. Global error minimization in image mosaicing using graph connectivity and its applications in microscopy

    PubMed Central

    Khurd, Parmeshwar; Grady, Leo; Oketokoun, Rafiou; Sundar, Hari; Gajera, Tejas; Gibbs-Strauss, Summer; Frangioni, John V.; Kamen, Ali

    2011-01-01

    Several applications such as multiprojector displays and microscopy require the mosaicing of images (tiles) acquired by a camera as it traverses an unknown trajectory in 3D space. A homography relates the image coordinates of a point in each tile to those of a reference tile provided the 3D scene is planar. Our approach in such applications is to first perform pairwise alignment of the tiles that have imaged common regions in order to recover a homography relating the tile pair. We then find the global set of homographies relating each individual tile to a reference tile such that the homographies relating all tile pairs are kept as consistent as possible. Using these global homographies, one can generate a mosaic of the entire scene. We derive a general analytical solution for the global homographies by representing the pair-wise homographies on a connectivity graph. Our solution can accommodate imprecise prior information regarding the global homographies whenever such information is available. We also derive equations for the special case of translation estimation of an X-Y microscopy stage used in histology imaging and present examples of stitched microscopy slices of specimens obtained after radical prostatectomy or prostate biopsy. In addition, we demonstrate the superiority of our approach over tree-structured approaches for global error minimization. PMID:22811964

  11. Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

    NASA Astrophysics Data System (ADS)

    Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

    2015-03-01

    Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

  12. Integrated microscopy for real-time imaging of mechanotransduction studies in live cells

    NASA Astrophysics Data System (ADS)

    Trache, Andreea; Lim, Soon-Mi

    2009-05-01

    Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.

  13. Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

    PubMed Central

    Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

    2016-01-01

    Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

  14. Chemical imaging by single pulse interferometric coherent anti-stokes Raman scattering microscopy.

    PubMed

    Lim, Sang-Hyun; Caster, Allison G; Nicolet, Olivier; Leone, Stephen R

    2006-03-23

    A single pulse interferometric coherent anti-Stokes Raman (CARS) spectroscopy method is used to obtain broadband CARS spectra and microscopy images of liquid and polymer samples. The pump, Stokes, and probe pulses are all selected inside a single broadband ultrafast pulse by a phase- and polarization-controlled pulse shaping technique and used to generate two spectral interference CARS signals simultaneously. The normalized difference of these two signals provides an amplified background-free broadband resonant CARS spectrum over the 400-1500 cm(-1) range with 35 cm(-1) spectral resolution. Chemically selective microscopy images of multicomponent polymer and liquid samples are investigated with this new CARS method. Multiplex CARS spectra at 10,000 spatial points are measured within a few minutes, and used to construct chemically selective microscopy images with a spatial resolution of 400 nm. The spectral bandwidth limits, sensitivity, homodyne amplification advantages, spatial resolution, depolarization, chromatic aberration, and chemical imaging aspects of this new technique are discussed in detail.

  15. Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

    PubMed Central

    Rupprecht, Peter; Prevedel, Robert; Groessl, Florian; Haubensak, Wulf E.; Vaziri, Alipasha

    2015-01-01

    A number of questions in system biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organisms. However, the broader applicability of light-sculpting microcopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important parameters and their trade-offs are experimentally demonstrated by performing calcium-imaging in acute mouse-brain slices. We further show that synchronization of line-scanning techniques with rolling-shutter read-out of the camera can reduce scattering effects and enhance image contrast at depth. PMID:25780729

  16. Development of carbon electrodes for electrochemistry, solid-state electronics and multimodal atomic force microscopy imaging

    NASA Astrophysics Data System (ADS)

    Morton, Kirstin Claire

    Carbon is one of the most remarkable elements due to its wide abundance on Earth and its many allotropes, which include diamond and graphite. Many carbon allotropes are conductive and in recent decades scientists have discovered and synthesized many new forms of carbon, including graphene and carbon nanotubes. The work in this thesis specifically focuses on the fabrication and characterization of pyrolyzed parylene C (PPC), a conductive pyrocarbon, as an electrode material for diodes, as a conductive coating for atomic force microscopy (AFM) probes and as an ultramicroelectrode (UME) for the electrochemical interrogation of cellular systems in vitro. Herein, planar and three-dimensional (3D) PPC electrodes were microscopically, spectroscopically and electrochemically characterized. First, planar PPC films and PPC-coated nanopipettes were utilized to detect a model redox species, Ru(NH3) 6Cl3. Then, free-standing PPC thin films were chemically doped, with hydrazine and concentrated nitric acid, to yield p- and n-type carbon films. Doped PPC thin films were positioned in conjunction with doped silicon to create Schottky and p-n junction diodes for use in an alternating current half-wave rectifier circuit. Pyrolyzed parylene C has found particular merit as a 3D electrode coating of AFM probes. Current sensing-atomic force microscopy imaging in air of nanoscale metallic features was undertaken to demonstrate the electronic imaging applicability of PPC AFM probes. Upon further insulation with parylene C and modification with a focused ion beam, a PPC UME was microfabricated near the AFM probe apex and utilized for electrochemical imaging. Subsequently, scanning electrochemical microscopy-atomic force microscopy imaging was undertaken to electrochemically quantify and image the spatial location of dopamine exocytotic release, elicited mechanically via the AFM probe itself, from differentiated pheochromocytoma 12 cells in vitro.

  17. X-ray microscopy and imaging of Escherichia coli, LPS and DNA.

    PubMed

    Rajyaguru, J M; Kado, M; Torres, D; Richardson, M; Muszynski, M J

    1997-11-01

    Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X-ray microscopy can take instant images of specimens but is mostly restricted to a few synchrotron X-ray sources. We have utilized a bench-top nanosecond laser-plasma to produce a single-shot source of nanosecond X-rays tuned for maximum contrast with carbon-rich material. To examine the ultrastructure by absorption profiles, we utilized a laser-produced plasma generated by a single-shot laser (1.06 microns wavelength, 5 x 10(12) W cm-2 intensity) focused on to a silicon target as an X-ray source for high-resolution X-ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)-coated grids (resist). This resist was exposed to X-rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10-ns burst of laser energy (at 20J) radiates strong emission lines in the region of 300 eV. The X-rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E.coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 A level, produces images similar to the space-filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

  18. Detecting and segmenting cell nuclei in two-dimensional microscopy images

    PubMed Central

    Liu, Chi; Shang, Fei; Ozolek, John A.; Rohde, Gustavo K.

    2016-01-01

    Introduction: Cell nuclei are important indicators of cellular processes and diseases. Segmentation is an essential stage in systems for quantitative analysis of nuclei extracted from microscopy images. Given the wide variety of nuclei appearance in different organs and staining procedures, a plethora of methods have been described in the literature to improve the segmentation accuracy and robustness. Materials and Methods: In this paper, we propose an unsupervised method for cell nuclei detection and segmentation in two-dimensional microscopy images. The nuclei in the image are detected automatically using a matching-based method. Next, edge maps are generated at multiple image blurring levels followed by edge selection performed in polar space. The nuclei contours are refined iteratively in the constructed edge pyramid. The validation study was conducted over two cell nuclei datasets with manual labeling, including 25 hematoxylin and eosin-stained liver histopathology images and 35 Papanicolaou-stained thyroid images. Results: The nuclei detection accuracy was measured by miss rate, and the segmentation accuracy was evaluated by two types of error metrics. Overall, the nuclei detection efficiency of the proposed method is similar to the supervised template matching method. In comparison to four existing state-of-the-art segmentation methods, the proposed method performed the best with average segmentation error 10.34% and 0.33 measured by area error rate and normalized sum of distances (×10). Conclusion: Quantitative analysis showed that the method is automatic and accurate when segmenting cell nuclei from microscopy images with noisy background and has the potential to be used in clinic settings. PMID:28066682

  19. The application of on-chip optofluidic microscopy for imaging Giardia lamblia trophozoites and cysts.

    PubMed

    Lee, Lap Man; Cui, Xiquan; Yang, Changhuei

    2009-10-01

    The optofluidic microscope (OFM) is a lensless, low-cost and highly compact on-chip device that can enable high-resolution microscopy imaging. The OFM performs imaging by flowing/scanning the target objects across a slanted hole array; by measuring the time-varying light transmission changes through the holes, we can then render images of the target objects at a resolution that is comparable to the holes' size. This paper reports the adaptation of the OFM for imaging Giardia lamblia trophozoites and cysts, a disease-causing parasite species that is commonly found in poor-quality water sources. We also describe our study of the impact of pressure-based flow and DC electrokinetic-based flow in controlling the flow motion of Giardia cysts--rotation-free translation of the parasite is important for good OFM image acquisition. Finally, we report the successful microscopy imaging of both Giardia trophozoites and cysts with an OFM that has a focal plane resolution of 0.8 microns.

  20. Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

    DOE PAGES

    Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

    2016-08-09

    In this paper, we present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional datamore » cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. Finally, we demonstrate this 'big data' approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.« less

  1. Imaging nanoscale lattice variations by machine learning of x-ray diffraction microscopy data

    SciTech Connect

    Laanait, Nouamane; Zhang, Zhan; Schlepütz, Christian M.

    2016-08-09

    In this paper, we present a novel methodology based on machine learning to extract lattice variations in crystalline materials, at the nanoscale, from an x-ray Bragg diffraction-based imaging technique. By employing a full-field microscopy setup, we capture real space images of materials, with imaging contrast determined solely by the x-ray diffracted signal. The data sets that emanate from this imaging technique are a hybrid of real space information (image spatial support) and reciprocal lattice space information (image contrast), and are intrinsically multidimensional (5D). By a judicious application of established unsupervised machine learning techniques and multivariate analysis to this multidimensional data cube, we show how to extract features that can be ascribed physical interpretations in terms of common structural distortions, such as lattice tilts and dislocation arrays. Finally, we demonstrate this 'big data' approach to x-ray diffraction microscopy by identifying structural defects present in an epitaxial ferroelectric thin-film of lead zirconate titanate.

  2. Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscopy

    SciTech Connect

    De Jonge, Niels; Peckys, Diana B; Veith, Gabriel M; Joy, David Charles

    2009-01-01

    Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

  3. Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces.

    PubMed Central

    Möller, C; Allen, M; Elings, V; Engel, A; Müller, D J

    1999-01-01

    Compared to contact-mode atomic force microscopy (CMAFM), tapping-mode atomic force microscopy (TMAFM) has the advantage of allowing imaging surfaces of macromolecules, even when they are only weakly attached to the support. In this study, TMAFM is applied to two different regular protein layers whose structures are known to great detail, the purple membrane from Halobacterium salinarum and the hexagonally packed intermediate (HPI) layer from Deinococcus radiodurans, to assess the faithfulness of high-resolution TMAFM images. Topographs exhibited a lateral resolution between 1.1 and 1. 5 nm and a vertical resolution of approximately 0.1 nm. For all protein surfaces, TMAFM and CMAFM topographs were in excellent agreement. TMAFM was capable of imaging the fragile polypeptide loop connecting the transmembrane alpha-helices E and F of bacteriorhodopsin in its native extended conformation. The standard deviation (SD) of averages calculated from TMAFM topographs exhibited an enhanced minimum (between 0.1 and 0.9 nm) that can be assigned to the higher noise of the raw data. However, the SD difference, indicating the flexibility of protein subunits, exhibited an excellent agreement between the two imaging modes. This demonstrates that the recently invented imaging-mode TMAFM has the ability to faithfully record high-resolution images and has sufficient sensitivity to contour individual peptide loops without detectable deformations. PMID:10423460

  4. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with <4 μm axial resolution (OCT and OCM), and 14 μm (OCT) and <2 μm (OCM) transverse resolution. The system allows seamless switching between low and high magnifications in a way similar to traditional microscopy. Good correspondence is observed between optical images and histological sections. Characteristic features that suggest malignant lesions, such as complex papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  5. Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

    DOE PAGES

    Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; ...

    2016-09-16

    We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yieldmore » can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.« less

  6. Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

    SciTech Connect

    Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; Scrymgeour, David A.; Maria, Jon-Paul; Paisley, Elizabeth A.; Kitahara, Andrew R.

    2016-09-16

    We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yield can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.

  7. Comparison of rotational imaging optical coherence tomography and selective plane illumination microscopy for embryonic study

    NASA Astrophysics Data System (ADS)

    Wu, Chen; Ran, Shihao; Le, Henry H.; Singh, Manmohan; Larina, Irina V.; Mayerich, David; Dickinson, Mary E.; Larin, Kirill V.

    2016-03-01

    The mouse is a common model for studying developmental diseases. Different optical techniques have been developed to investigate mouse embryos, but each has its own set of limitations and restrictions. In this study, we imaged the same E9.5 mouse embryo with rotational imaging Optical Coherence Tomography (RI-OCT) and Selective Plane Illumination Microscopy (SPIM), and compared the two techniques. Results demonstrate that both methods can provide images with micrometer-scale spatial resolution. The RI-OCT technique was developed to increase imaging depth of OCT by performing traditional OCT imaging at multiple sides and co-registering the images. In SPIM, optical sectioning is achieved by illuminating the sample with a sheet of light. In this study, the images acquired from both techniques are compared with each other to evaluate the benefits and drawbacks of each technique for embryonic imaging. Since 3D stacks can be obtained by SPIM from different angles by rotating the sample, it might be possible to build a hybrid setup of two imaging modalities to combine the advantages of each technique.

  8. Optimizing Imaging Conditions for Demanding Multi-Color Super Resolution Localization Microscopy

    PubMed Central

    Nahidiazar, Leila; Agronskaia, Alexandra V.; Broertjes, Jorrit; van den Broek, Bram; Jalink, Kees

    2016-01-01

    Single Molecule Localization super-resolution Microscopy (SMLM) has become a powerful tool to study cellular architecture at the nanometer scale. In SMLM, single fluorophore labels are made to repeatedly switch on and off (“blink”), and their exact locations are determined by mathematically finding the centers of individual blinks. The image quality obtainable by SMLM critically depends on efficacy of blinking (brightness, fraction of molecules in the on-state) and on preparation longevity and labeling density. Recent work has identified several combinations of bright dyes and imaging buffers that work well together. Unfortunately, different dyes blink optimally in different imaging buffers, and acquisition of good quality 2- and 3-color images has therefore remained challenging. In this study we describe a new imaging buffer, OxEA, that supports 3-color imaging of the popular Alexa dyes. We also describe incremental improvements in preparation technique that significantly decrease lateral- and axial drift, as well as increase preparation longevity. We show that these improvements allow us to collect very large series of images from the same cell, enabling image stitching, extended 3D imaging as well as multi-color recording. PMID:27391487

  9. An imaging technique using rotational polarization microscopy for the microstructure analysis of carbon/carbon composites.

    PubMed

    Miaoling, Li; Lehua, Qi; Hejun, Li

    2012-01-01

    A novel image analysis technique was proposed for microstructure investigation of carbon/carbon (C/C) composites. The rotational polarization microscopy was developed to meet the special imaging requirements. The samples of C/C composites were observed in reflection polarized light microscope, where the analyzer was rotated instead of the stage, and the polarizer was taken out. The bireflectance of like-graphite negative uniaxial crystal was analyzed. It was the theoretic foundation of image collection and data processing. The analyzer was rotated through 36 × 10° intervals without any movement of the specimen. The polished cross-section of C/C composites took micrographs at each analyzer orientation. All image data collected from the same field of view were processed by image registration and image fusion. The synthesized images were obtained by calculating the maximum and minimum gray values and their differences at each point of the million pixels at 18 orientations of the analyzer. They are unique and quite reliable to be applied to analyze the microstructure of C/C composites. Subsequently, image segmentation was performed, and the feature parameters of each component were calculated. Good agreement was found between the results from image analysis and experimental data.

  10. Imaging latex–carbon nanotube composites by subsurface electrostatic force microscopy

    SciTech Connect

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee; Loyola, Bryan; O’Bryan, Greg; Friddle, Raymond William

    2016-09-08

    Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface. Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.

  11. Imaging latex-carbon nanotube composites by subsurface electrostatic force microscopy

    NASA Astrophysics Data System (ADS)

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen; Loyola, Bryan; O'Bryan, Greg; Friddle, Raymond W.

    2016-10-01

    Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle’s depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film-air interface. This result suggests that water-mediated charge build-up at the film-air interface may be the source of electrostatic phase contrast in ambient conditions.

  12. High-Resolution Imaging of Dendrimers Used in Drug Delivery via Scanning Probe Microscopy.

    PubMed

    Shi, Lifang; Fleming, Christopher J; Riechers, Shawn L; Yin, Nai-Ning; Luo, Juntao; Lam, Kit S; Liu, Gang-Yu

    2011-01-01

    Dendrimers and telodendrimer micelles represent two new classes of vehicles for drug delivery that have attracted much attention recently. Their structural characterization at the molecular and submolecular level remains a challenge due to the difficulties in reaching high resolution when imaging small particles in their native media. This investigation offers a new approach towards this challenge, using scanning tunneling microscopy (STM) and atomic force microscopy (AFM). By using new sample preparation protocols, this work demonstrates that (a) intramolecular features such as drug molecules and dendrimer termini can be resolved; and (b) telodendrimer micelles can be immobilized on the surface without compromising structural integrity, and as such, high resolution AFM imaging may be performed to attain 3D information. This high-resolution structural information should enhance our knowledge of the nanocarrier structure and nanocarrier-drug interaction and, therefore, facilitate design and optimization of the efficiency in drug delivery.

  13. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Lu, Feng; Streets, Aaron M.; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-05-01

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr00308f

  14. High-Speed Nonlinear Interferometric Vibrational Imaging of Biological Tissue With Comparison to Raman Microscopy

    PubMed Central

    Benalcazar, Wladimir A.; Chowdary, Praveen D.; Jiang, Zhi; Marks, Daniel L.; Chaney, Eric J.; Gruebele, Martin; Boppart, Stephen A.

    2011-01-01

    Vibrational contrast imaging of the distribution of complex biological molecules requires the use of techniques that provide broadband spectra with sufficient resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy is currently limited in meeting these requirements due to the presence of a nonresonant background and its inability to target multiple resonances simultaneously. We present nonlinear interferometric vibrational imaging (NIVI), a technique based on CARS that uses femtosecond pump and Stokes pulses to retrieve broadband vibrational spectra over 200 cm–1 (full-width at half maximum). By chirping the pump and performing spectral interferometric detection, the anti-Stokes pulses are resolved in time. This phase-sensitive detection allows suppression of not only the nonresonant background, but also of the real part of the nonlinear susceptibility χ(3), improving the spectral resolution and features to make them comparable to those acquired with spontaneous Raman microscopy, as shown for a material sample and mammary tissue. PMID:22058432

  15. Imaging latex–carbon nanotube composites by subsurface electrostatic force microscopy

    DOE PAGES

    Patel, Sajan; Petty, Clayton W.; Krafcik, Karen Lee; ...

    2016-09-08

    Electrostatic modes of atomic force microscopy have shown to be non-destructive and relatively simple methods for imaging conductors embedded in insulating polymers. Here we use electrostatic force microscopy to image the dispersion of carbon nanotubes in a latex-based conductive composite, which brings forth features not observed in previously studied systems employing linear polymer films. A fixed-potential model of the probe-nanotube electrostatics is presented which in principle gives access to the conductive nanoparticle's depth and radius, and the polymer film dielectric constant. Comparing this model to the data results in nanotube depths that appear to be slightly above the film–air interface.more » Furthermore, this result suggests that water-mediated charge build-up at the film–air interface may be the source of electrostatic phase contrast in ambient conditions.« less

  16. High-Speed Nonlinear Interferometric Vibrational Imaging of Biological Tissue With Comparison to Raman Microscopy.

    PubMed

    Benalcazar, Wladimir A; Chowdary, Praveen D; Jiang, Zhi; Marks, Daniel L; Chaney, Eric J; Gruebele, Martin; Boppart, Stephen A

    2009-12-04

    Vibrational contrast imaging of the distribution of complex biological molecules requires the use of techniques that provide broadband spectra with sufficient resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy is currently limited in meeting these requirements due to the presence of a nonresonant background and its inability to target multiple resonances simultaneously. We present nonlinear interferometric vibrational imaging (NIVI), a technique based on CARS that uses femtosecond pump and Stokes pulses to retrieve broadband vibrational spectra over 200 cm(-1) (full-width at half maximum). By chirping the pump and performing spectral interferometric detection, the anti-Stokes pulses are resolved in time. This phase-sensitive detection allows suppression of not only the nonresonant background, but also of the real part of the nonlinear susceptibility χ((3)), improving the spectral resolution and features to make them comparable to those acquired with spontaneous Raman microscopy, as shown for a material sample and mammary tissue.

  17. Chip-based optical microscopy for imaging membrane sieve plates of liver scavenger cells

    NASA Astrophysics Data System (ADS)

    Helle, Øystein I.; Øie, Cristina I.; McCourt, Peter; Ahluwalia, Balpreet S.

    2015-08-01

    The evanescent field on top of optical waveguides is used to image membrane network and sieve-plates of liver endothelial cells. In waveguide excitation, the evanescent field is dominant only near the surface (~100-150 nm) providing a default optical sectioning by illuminating fluorophores in close proximity to the surface and thus benefiting higher signal-to-noise ratio. The sieve plates of liver sinusoidal endothelial cells are present on the cell membrane, thus near-field waveguide chip-based microscopy configuration is preferred over epi-fluorescence. The waveguide chip is compatible with optical fiber components allowing easy multiplexing to different wavelengths. In this paper, we will discuss the challenges and opportunities provided by integrated optical microscopy for imaging cell membranes.

  18. Comparative three-dimensional imaging of living neurons with confocal and atomic force microscopy.

    PubMed

    McNally, Helen A; Rajwa, Bartek; Sturgis, Jennie; Robinson, J Paul

    2005-03-30

    Atomic force microscopy applications extend across a number of fields; however, limitations have reduced its effectiveness in live cell analysis. This report discusses the use of AFM to evaluate the three-dimensional (3-D) architecture of living chick dorsal root ganglia and sympathetic ganglia. These data sets were compared to similar images acquired with confocal laser scanning microscopy of identical cells. For this comparison we made use of visualization techniques which were applicable to both sets of data and identified several issues when coupling these technologies. These direct comparisons offer quantitative validation and confirmation of the character of novel images acquired by AFM. This paper is one in a series emphasizing various new applications of AFM in neurobiology.

  19. Image contrast mechanisms in dynamic friction force microscopy: Antimony particles on graphite

    NASA Astrophysics Data System (ADS)

    Mertens, Felix; Göddenhenrich, Thomas; Dietzel, Dirk; Schirmeisen, Andre

    2017-01-01

    Dynamic Friction Force Microscopy (DFFM) is a technique based on Atomic Force Microscopy (AFM) where resonance oscillations of the cantilever are excited by lateral actuation of the sample. During this process, the AFM tip in contact with the sample undergoes a complex movement which consists of alternating periods of sticking and sliding. Therefore, DFFM can give access to dynamic transition effects in friction that are not accessible by alternative techniques. Using antimony nanoparticles on graphite as a model system, we analyzed how combined influences of friction and topography can effect different experimental configurations of DFFM. Based on the experimental results, for example, contrast inversion between fractional resonance and band excitation imaging strategies to extract reliable tribological information from DFFM images are devised.

  20. Energy transfer and device performance in phosphorescent dye doped polymer light emitting diodes

    NASA Astrophysics Data System (ADS)

    Noh, Yong-Young; Lee, Chang-Lyoul; Kim, Jang-Joo; Yase, Kiyoshi

    2003-02-01

    Singlet and triplet-triplet energy transfer in phosphorescent dye doped polymer light emitting devices were investigated. Poly(N-vinylcarbazol) and poly[9,9'-di-n-hexyl-2,7-fluorene-alt- 1,4-(2,5-di-n-hexyloxy)phenylene] (PFHP) were selected as the host polymer for the phosphorescent dopants fac-tris(2-phenylpyridine) iridium(III) [Ir(ppy)3] and 2,3,7,8,12,13, 17,18-octaethyl-21H,23H-porphyrin platinum(II) (PtOEP) because of their high triplet energy levels and long phosphorescence lifetimes. In case of PVK film, efficient triplet energy transfers to both PtOEP and Ir(ppy)3 were observed. In contrast, the triplet energy transfer did not occur or was very weak from PFHP to both PtOEP and Ir(ppy)3 although usual requirements for triplet energy transfer were satisfied. Furthermore, the singlet-singlet energy transfer did not take place from PFHP to Ir(ppy)3 in doped films even though the Förster radius is more than 30 Å. However, the blended film of Ir(ppy)3 with PFHP and PMMA showed the green emission from Ir(ppy)3 via singlet energy transfer. In addition, the solution of PFHP and Ir(ppy)3 (8 wt. %) in p-xylene also showed green emission. The blocking of the energy transfers in the phosphorescent dye doped PFHP films is found to be originated from the formation of aggregates which is evident from the microscopic images taken by transmission electron microscope, atomic force microscope, and fluorescence microscope. The formation of aggregates prevents dopant molecules from being in close proximity with host molecules thereby inhibiting energy transfer processes. The phase separation deteriorates the device performance also. Therefore, the chemical compatibility of a dopant with a host polymer as well as conventional requirements for energy transfers must be significantly considered to fabricate efficient phosphorescent dye doped polymer light emitting devices.