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Sample records for generation imaging microscopy

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

    Science.gov (United States)

    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.

  2. Reconstruction of complementary images in second harmonic generation microscopy

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. Imaging theory of nonlinear second harmonic and third harmonic generations in confocal microscopy

    Institute of Scientific and Technical Information of China (English)

    TANG; Zhilie; XING; Da; LIU; Songhao

    2004-01-01

    The imaging theory of nonlinear second harmonic generation (SHG) and third harmonic generation (THG) in confocal microscopy is presented in this paper. The nonlinear effect of SHG and THG on the imaging properties of confocal microscopy has been analyzed in detail by the imaging theory. It is proved that the imaging process of SHG and THG in confocal microscopy, which is different from conventional coherent imaging or incoherent imaging, can be divided into two different processes of coherent imaging. The three-dimensional point spread functions (3D-PSF) of SHG and THG confocal microscopy are derived based on the nonlinear principles of SHG and THG. The imaging properties of SHG and THG confocal microscopy are discussed in detail according to its 3D-PSF. It is shown that the resolution of SHG and THG confocal microscopy is higher than that of single-and two-photon confocal microscopy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    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.

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

    Science.gov (United States)

    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.

  7. Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy.

    Science.gov (United States)

    Rivard, Maxime; Couture, Charles-André; Miri, Amir K; Laliberté, Mathieu; Bertrand-Grenier, Antony; Mongeau, Luc; Légaré, François

    2013-01-01

    We report that combining interferometry with Second Harmonic Generation (SHG) microscopy provides valuable information about the relative orientation of noncentrosymmetric structures composing tissues. This is confirmed through the imaging of rat medial gastrocnemius muscle. The inteferometric Second Harmonic Generation (ISHG) images reveal that each side of the myosin filaments composing the A band of the sarcomere generates π phase shifted SHG signal which implies that the myosin proteins at each end of the filaments are oriented in opposite directions. This highlights the bipolar structural organization of the myosin filaments and shows that muscles can be considered as a periodically poled biological structure.

  8. Second harmonic generation microscopy

    DEFF Research Database (Denmark)

    Brüggemann, Dagmar Adeline; Brewer, Jonathan R.; Risbo, Jens

    2010-01-01

    Myofibers and collagen show non-linear optical properties enabling imaging using second harmonic generation (SHG) microscopy. The technique is evaluated for use as a tool for real-time studies of thermally induced changes in thin samples of unfixed and unstained pork. The forward and the backward......-temperature endotherm peak observable in the differential scanning calorimetry (DSC) thermograms. DSC analysis of epimysium, the connective tissue layer that enfold skeletal muscles, produces one large endotherm starting at 57 °C and peaking at 59.5 °C. SHG microscopy of collagen fibers reveals a variability of thermal...

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

    Science.gov (United States)

    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.

  10. Interferometric backward third harmonic generation microscopy for axial imaging with accuracy beyond the diffraction limit.

    Directory of Open Access Journals (Sweden)

    Daaf Sandkuijl

    Full Text Available 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.

  11. Nonlinear chemical imaging microscopy: near-field third harmonic generation imaging of human red blood cells.

    Science.gov (United States)

    Schaller, R D; Johnson, J C; Saykally, R J

    2000-11-01

    Third harmonic generation (THG) imaging using a near-field scanning optical microscope (NSOM) is demonstrated for the first time. A femtosecond, tunable near-infrared laser was used to generate both nonresonant and resonantly enhanced third harmonic radiation in human red blood cells. We show that resonantly enhanced THG is a chemically specific bulk probe in NSOM imaging by tuning the excitation source onto and off of resonance with the Soret transition of oxyhemoglobin. Additionally, we provide evidence that tightly focused, nonresonant, far-field THG imaging experiments do not produce contrast that is truly surface specific.

  12. Label-free live brain imaging and targeted patching with third-harmonic generation microscopy

    Science.gov (United States)

    Witte, Stefan; Negrean, Adrian; Lodder, Johannes C.; de Kock, Christiaan P. J.; Testa Silva, Guilherme; Mansvelder, Huibert D.; Louise Groot, Marie

    2011-01-01

    The ability to visualize neurons inside living brain tissue is a fundamental requirement in neuroscience and neurosurgery. Especially the development of a noninvasive probe of brain morphology with micrometer-scale resolution is highly desirable, as it would provide a noninvasive approach to optical biopsies in diagnostic medicine. Two-photon laser-scanning microscopy (2PLSM) is a powerful tool in this regard, and has become the standard for minimally invasive high-resolution imaging of living biological samples. However, while 2PLSM-based optical methods provide sufficient resolution, they have been hampered by the requirement for fluorescent dyes to provide image contrast. Here we demonstrate high-contrast imaging of live brain tissue at cellular resolution, without the need for fluorescent probes, using optical third-harmonic generation (THG). We exploit the specific geometry and lipid content of brain tissue at the cellular level to achieve partial phase matching of THG, providing an alternative contrast mechanism to fluorescence. We find that THG brain imaging allows rapid, noninvasive label-free imaging of neurons, white-matter structures, and blood vessels simultaneously. Furthermore, we exploit THG-based imaging to guide micropipettes towards designated neurons inside live tissue. This work is a major step towards label-free microscopic live brain imaging, and opens up possibilities for the development of laser-guided microsurgery techniques in the living brain. PMID:21444784

  13. Imaging Collagen in Scar Tissue: Developments in Second Harmonic Generation Microscopy for Biomedical Applications.

    Science.gov (United States)

    Mostaço-Guidolin, Leila; Rosin, Nicole L; Hackett, Tillie-Louise

    2017-08-15

    The ability to respond to injury with tissue repair is a fundamental property of all multicellular organisms. The extracellular matrix (ECM), composed of fibrillar collagens as well as a number of other components is dis-regulated during repair in many organs. In many tissues, scaring results when the balance is lost between ECM synthesis and degradation. Investigating what disrupts this balance and what effect this can have on tissue function remains an active area of research. Recent advances in the imaging of fibrillar collagen using second harmonic generation (SHG) imaging have proven useful in enhancing our understanding of the supramolecular changes that occur during scar formation and disease progression. Here, we review the physical properties of SHG, and the current nonlinear optical microscopy imaging (NLOM) systems that are used for SHG imaging. We provide an extensive review of studies that have used SHG in skin, lung, cardiovascular, tendon and ligaments, and eye tissue to understand alterations in fibrillar collagens in scar tissue. Lastly, we review the current methods of image analysis that are used to extract important information about the role of fibrillar collagens in scar formation.

  14. Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer.

    Science.gov (United States)

    Tilbury, Karissa; Campagnola, Paul J

    2015-01-01

    In this perspective, we discuss how the nonlinear optical technique of second-harmonic generation (SHG) microscopy has been used to greatly enhance our understanding of the tumor microenvironment (TME) of breast and ovarian cancer. Striking changes in collagen architecture are associated with these epithelial cancers, and SHG can image these changes with great sensitivity and specificity with submicrometer resolution. This information has not historically been exploited by pathologists but has the potential to enhance diagnostic and prognostic capabilities. We summarize the utility of image processing tools that analyze fiber morphology in SHG images of breast and ovarian cancer in human tissues and animal models. We also describe methods that exploit the SHG physical underpinnings that are effective in delineating normal and malignant tissues. First we describe the use of polarization-resolved SHG that yields metrics related to macromolecular and supramolecular structures. The coherence and corresponding phase-matching process of SHG results in emission directionality (forward to backward), which is related to sub-resolution fibrillar assembly. These analyses are more general and more broadly applicable than purely morphology-based analyses; however, they are more computationally intensive. Intravital imaging techniques are also emerging that incorporate all of these quantitative analyses. Now, all these techniques can be coupled with rapidly advancing miniaturization of imaging systems to afford their use in clinical situations including enhancing pathology analysis and also in assisting in real-time surgical determination of tumor margins.

  15. In Vivo Imaging of Myelin in the Vertebrate Central Nervous System Using Third Harmonic Generation Microscopy

    Science.gov (United States)

    Farrar, Matthew J.; Wise, Frank W.; Fetcho, Joseph R.; Schaffer, Chris B.

    2011-01-01

    Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy—a coherent, nonlinear, dye-free imaging modality—provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery. PMID:21354410

  16. Imaging the noncentrosymmetric structural organisation of tissue with Interferometric Second Harmonic Generation microscopy

    CERN Document Server

    Rivard, Maxime; Laliberte, Mathieu; Bertrand-Grenier, Antony; Martin, Francois; Pepin, Henri; Pfeffer, Christian P; Brown, Cameron; Rammuno, Lora; Legare, Francois

    2012-01-01

    We report the imaging of tendon, a connective tissue rich in collagen type I proteins, with Interferometric Second Harmonic Generation (I-SHG) microscopy. We observed that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 {\\mu}m, while in the transverse direction it is ~1-15 {\\mu}m. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nanocylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the fraction occupied by fibrils oriented in one direction is larger than in the other.

  17. Imaging the noncentrosymmetric structural organization of tendon with Interferometric Second Harmonic Generation microscopy.

    Science.gov (United States)

    Rivard, Maxime; Popov, Konstantin; Couture, Charles-André; Laliberté, Mathieu; Bertrand-Grenier, Antony; Martin, François; Pépin, Henri; Pfeffer, Christian P; Brown, Cameron; Ramunno, Lora; Légaré, François

    2014-08-01

    We report the imaging of tendon with Interferometric Second Harmonic Generation microscopy. We observe that the noncentrosymmetric structural organization can be maintained along the fibrillar axis over more than 150 μm, while in the transverse direction it is ∼1-15 μm. Those results are explained by modeling tendon as a heterogeneous distribution of noncentrosymmetric nano-cylinders (collagen fibrils) oriented along the fibrillar axis. The preservation of the noncentrosymmetric structural organization over multiple tens of microns reveals that tendon is made of domains in which the ratio between fibrils with positive and negative polarity is unbalanced. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. In vivo imaging of dermal collagen in skin burn by collagen-sensitive second-harmonic-generation microscopy

    Science.gov (United States)

    Yasui, Takeshi; Tanaka, Ryosuke; Hase, Eiji; Fukushima, Shu-ichiro; Araki, Tsutomu

    2013-02-01

    Optical assessment of skin burns is possible with second-harmonic-generation (SHG) microscopy due to its high sensitivity to thermal denaturation of collagen molecules. In contrast to previous studies that were performed using excised tissue specimens ex vivo, in this study, we demonstrated in vivo observation of dermal collagen fibers in living rat burn models with SHG microscopy. We confirmed that changes in SHG vanishing patterns in the SHG images depended on the burn degree. The results imply that SHG microscopy can be used as a low-invasiveness, highly quantitative tool for skin burn assessment.

  19. Multiphoton Microscopy for Ophthalmic Imaging

    Directory of Open Access Journals (Sweden)

    Emily A. Gibson

    2011-01-01

    Full Text Available We review multiphoton microscopy (MPM including two-photon autofluorescence (2PAF, second harmonic generation (SHG, third harmonic generation (THG, fluorescence lifetime (FLIM, and coherent anti-Stokes Raman Scattering (CARS with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

  20. Hot spots in energetic materials generated by infrared and ultrasound, detected by thermal imaging microscopy.

    Science.gov (United States)

    Chen, Ming-Wei; You, Sizhu; Suslick, Kenneth S; Dlott, Dana D

    2014-02-01

    We have observed and characterized hot spot formation and hot-spot ignition of energetic materials (EM), where hot spots were created by ultrasonic or long-wavelength infrared (LWIR) exposure, and were detected by high-speed thermal microscopy. The microscope had 15-20 μm spatial resolution and 8.3 ms temporal resolution. LWIR was generated by a CO2 laser (tunable near 10.6 μm or 28.3 THz) and ultrasound by a 20 kHz acoustic horn. Both methods of energy input created spatially homogeneous energy fields, allowing hot spots to develop spontaneously due to the microstructure of the sample materials. We observed formation of hot spots which grew and caused the EM to ignite. The EM studied here consisted of composite solids with 1,3,5-trinitroperhydro-1,3,5-triazine crystals and polymer binders. EM simulants based on sucrose crystals in binders were also examined. The mechanisms of hot spot generation were different with LWIR and ultrasound. With LWIR, hot spots were most efficiently generated within the EM crystals at LWIR wavelengths having longer absorption depths of ∼25 μm, suggesting that hot spot generation mechanisms involved localized absorbing defects within the crystals, LWIR focusing in the crystals or LWIR interference in the crystals. With ultrasound, hot spots were primarily generated in regions of the polymer binder immediately adjacent to crystal surfaces, rather than inside the EM crystals.

  1. Photothermal imaging scanning microscopy

    Science.gov (United States)

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

    2006-07-11

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

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

    Science.gov (United States)

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

    2011-01-01

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

  3. Label-free imaging of basement membranes differentiates normal, precancerous, and cancerous colonic tissues by second-harmonic generation microscopy.

    Science.gov (United States)

    Zhuo, Shuangmu; Yan, Jun; Chen, Gang; Shi, Hong; Zhu, Xiaoqin; Lu, Jianping; Chen, Jianxin; Xie, Shusen

    2012-01-01

    Since changes in the basement membranes are the critical indicators for differentiating normal, precancerous, and cancerous colonic tissues, direct visualization of these warning signs is essential for the early diagnosis and treatment of colonic cancer. Here, we present that second harmonic generation (SHG) microscopy can probe the changes of basement membranes in different colonic cancer stages. Our results also show the capability of using the quantitative analyses of images for quantifying these changes in different cancer stages. These results suggest that SHG microscopy has the potential in label-freely imaging the changes of basement membranes for effectively distinguishing between normal, precancerous, and cancerous colonic tissues. To our knowledge, this is the first demonstration of the dynamics of basement membrane changes in different colonic cancer stages using entirely intrinsic source of contrast.

  4. Selective imaging in second-harmonic-generation microscopy by polarization manipulation

    Science.gov (United States)

    Chu, Shi-Wei; Tai, Shih-Peng; Sun, Chi-Kuang; Lin, Chi-Hung

    2007-09-01

    Second-harmonic-generation (SHG) has proved itself as an important contrast mechanism in microscopic applications. Its noninvasiveness, optical sectioning capability, and high-penetrability provide attractive features in observation of thick biological tissues. Fibrous proteins, such as myosin and collagen, are dominant SHG harmonophores in vertebrates. Due to their biophotonic crystal nature, SHGs from these proteins are known to exhibit specific polarization dependencies, reflecting local molecule arrangements. Here the authors demonstrate a scheme to distinguish SHG from myosin-based muscle fibers and intertwined collagenous perimysium through polarization selection, without complicated staining or sample/image processing required.

  5. Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy.

    Science.gov (United States)

    Chaudhary, Rajeev; Campbell, Kirby R; Tilbury, Karissa B; Vanderby, Ray; Block, Walter F; Kijowski, Richard; Campagnola, Paul J

    2015-04-01

    The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. Our results revealed differences in SHG forward-backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella. Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

  6. Alterations of the extracellular matrix in ovarian cancer studied by Second Harmonic Generation imaging microscopy

    Directory of Open Access Journals (Sweden)

    Campagnola Paul J

    2010-03-01

    Full Text Available Abstract Background Remodeling of the extracellular matrix (ECM has been implicated in ovarian cancer, and we hypothesize that these alterations may provide a better optical marker of early disease than currently available imaging/screening methods and that understanding their physical manifestations will provide insight into invasion. Methods For this investigation we use Second Harmonic Generation (SHG imaging microcopy to study changes in the structure of the ovarian ECM in human normal and malignant ex vivo biopsies. This method directly visualizes the type I collagen in the ECM and provides quantitative metrics of the fibrillar assembly. To quantify these changes in collagen morphology we utilized an integrated approach combining 3D SHG imaging measurements and bulk optical parameter measurements in conjunction with Monte Carlo simulations of the experimental data to extract tissue structural properties. Results We find the SHG emission attributes (directionality and relative intensity and bulk optical parameters, both of which are related to the tissue structure, are significantly different in the tumors in a manner that is consistent with the change in collagen assembly. The normal and malignant tissues have highly different collagen fiber assemblies, where collectively, our findings show that the malignant ovaries are characterized by lower cell density, denser collagen, as well as higher regularity at both the fibril and fiber levels. This further suggests that the assembly in cancer may be comprised of newly synthesized collagen as opposed to modification of existing collagen. Conclusions Due to the large structural changes in tissue assembly and the SHG sensitivity to these collagen alterations, quantitative discrimination is achieved using small patient data sets. Ultimately these measurements may be developed as intrinsic biomarkers for use in clinical applications.

  7. Optical imaging. Expansion microscopy.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Chunqiang Li

    2016-01-01

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

  9. Fourier-ring descriptor to characterize rare circulating cells from images generated using immunofluorescence microscopy.

    Science.gov (United States)

    Emerson, Tegan; Kirby, Michael; Bethel, Kelly; Kolatkar, Anand; Luttgen, Madelyn; O'Hara, Stephen; Newton, Paul; Kuhn, Peter

    2015-03-01

    We address the problem of subclassification of rare circulating cells using data driven feature selection from images of candidate circulating tumor cells from patients diagnosed with breast, prostate, or lung cancer. We determine a set of low level features which can differentiate among candidate cell types. We have implemented an image representation based on concentric Fourier rings (FRDs) which allow us to exploit size variations and morphological differences among cells while being rotationally invariant. We discuss potential clinical use in the context of treatment monitoring for cancer patients with metastatic disease.

  10. In vivo time-lapse imaging of skin burn wound healing using second-harmonic generation microscopy

    Science.gov (United States)

    Yasui, Takeshi; Tanaka, Ryosuke; Hase, Eiji; Fukushima, Shu-ichiro; Araki, Tsutomu

    2014-02-01

    Wound healing is a process to repair the damaged tissue caused by thermal burn, incised wound, or stab wound. Although the wound healing has many aspects, it is common for dynamics of collagen fiber, such as decomposition, production, or growth, to be closely related with wound healing. If such the healing process can be visualized as a timelapse image of the collagen fiber in the same subject, one may obtain new findings regarding biological repairing mechanisms in the healing process. In this article, to investigate the temporal modoification of dermal collagen fiber in the burn wound healing, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, minimal invasiveness, deep penetration, the absence of interference from background light, and in vivo measurement without additional staining. Since SHG light arises from a non-centrosymmetric triple helix of three polypeptide chains in the collagen molecule, SHG intensity sensitively reflects the structure maturity of collagen molecule and its aggregates. A series of time-lapse SHG images during the wound healing process of 2 weeks clearly indicated that condensation and melting of dermal collagen fibers by the deep dermal burn, decomposition of the damaged collagen fibers in the inflammation phase, production of new collagen fibers in the proliferation phase, and the growth of the new collagen fibers in the remodeling phase. These results show a high potential of SHG microscopy for optical assessment of the wound healing process in vivo.

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

    Science.gov (United States)

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

    2012-03-01

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

  12. Diagnostic potential of multimodal imaging of ovarian tissue using optical coherence tomography and second-harmonic generation microscopy.

    Science.gov (United States)

    Welge, Weston A; DeMarco, Andrew T; Watson, Jennifer M; Rice, Photini S; Barton, Jennifer K; Kupinski, Matthew A

    2014-07-18

    Ovarian cancer is particularly deadly because it is usually diagnosed after it has metastasized. We have previously identified features of ovarian cancer using optical coherence tomography (OCT) and second-harmonic generation (SHG) microscopy (targeting collagen). OCT provides an image of the ovarian microstructure while SHG provides a high-resolution map of collagen fiber bundle arrangement. Here we investigated the diagnostic potential of dual-modality OCT and SHG imaging. We conducted a fully crossed, multi-reader, multi-case study using seven human observers. Each observer classified 44 ex vivo mouse ovaries (16 normal and 28 abnormal) as normal or abnormal from OCT, SHG, and simultaneously viewed, co-registered OCT and SHG images and provided a confidence rating on a six-point scale. We determined the average receiver operating characteristic (ROC) curves, area under the ROC curves (AUC), and other quantitative figures of merit. The results show that OCT has diagnostic potential with an average AUC of 0.91 ± 0.06. The average AUC for SHG was less promising at 0.71 ± 0.13. The average AUC for simultaneous OCT and SHG was not significantly different from OCT alone, possibly due to the limited SHG field of view. The high performance of OCT and co-registered OCT and SHG warrants further investigation.

  13. Second harmonic generation imaging

    CERN Document Server

    2013-01-01

    Second-harmonic generation (SHG) microscopy has shown great promise for imaging live cells and tissues, with applications in basic science, medical research, and tissue engineering. Second Harmonic Generation Imaging offers a complete guide to this optical modality, from basic principles, instrumentation, methods, and image analysis to biomedical applications. The book features contributions by experts in second-harmonic imaging, including many pioneering researchers in the field. Written for researchers at all levels, it takes an in-depth look at the current state of the art and possibilities of SHG microscopy. Organized into three sections, the book: Provides an introduction to the physics of the process, step-by-step instructions on how to build an SHG microscope, and comparisons with related imaging techniques Gives an overview of the capabilities of SHG microscopy for imaging tissues and cells—including cell membranes, muscle, collagen in tissues, and microtubules in live cells—by summarizing experi...

  14. Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.

    Science.gov (United States)

    Ranjit, Suman; Dobrinskikh, Evgenia; Montford, John; Dvornikov, Alexander; Lehman, Allison; Orlicky, David J; Nemenoff, Raphael; Gratton, Enrico; Levi, Moshe; Furgeson, Seth

    2016-11-01

    All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  15. Color atomic force microscopy: A method to acquire three independent potential parameters to generate a color image

    Science.gov (United States)

    Allain, P. E.; Damiron, D.; Miyazaki, Y.; Kaminishi, K.; Pop, F. V.; Kobayashi, D.; Sasaki, N.; Kawakatsu, H.

    2017-09-01

    Atomic force microscopy has enabled imaging at the sub-molecular level, and 3D mapping of the tip-surface potential field. However, fast identification of the surface still remains a challenging topic for the microscope to enjoy widespread use as a tool with chemical contrast. In this paper, as a step towards implementation of such function, we introduce a control scheme and mathematical treatment of the acquired data that enable retrieval of essential information characterizing this potential field, leading to fast acquisition of images with chemical contrast. The control scheme is based on the tip sample distance modulation at an angular frequency ω, and null-control of the ω component of the measured self-excitation frequency of the oscillator. It is demonstrated that this control is robust, and that effective Morse Parameters that give satisfactory curve fit to the measured frequency shift can be calculated at rates comparable to the scan. Atomic features with similar topography were distinguished by differences in these parameters. The decay length parameter was resolved with a resolution of 10 pm. The method was demonstrated on quenched silicon at a scan rate comparable to conventional imaging.

  16. ImageJ for microscopy.

    Science.gov (United States)

    Collins, Tony J

    2007-07-01

    ImageJ is an essential tool for us that fulfills most of our routine image processing and analysis requirements. The near-comprehensive range of import filters that allow easy access to image and meta-data, a broad suite processing and analysis routine, and enthusiastic support from a friendly mailing list are invaluable for all microscopy labs and facilities-not just those on a budget.

  17. Scanning Electron Microscopy Sample Preparation and Imaging.

    Science.gov (United States)

    Nguyen, Jenny Ngoc Tran; Harbison, Amanda M

    2017-01-01

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

  18. Confocal microscopy imaging of solid tissue

    Science.gov (United States)

    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 ...

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

    Science.gov (United States)

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

    2016-12-01

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

  20. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    Science.gov (United States)

    Filippidis, G.; Kouloumentas, C.; Kapsokalyvas, D.; Voglis, G.; Tavernarakis, N.; Papazoglou, T. G.

    2005-08-01

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  1. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Filippidis, G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kouloumentas, C [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Kapsokalyvas, D [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece); Voglis, G [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Tavernarakis, N [Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Heraklion 71110, Crete (Greece); Papazoglou, T G [Institute of Electronic Structure and Laser, Foundation of Research and Technology-Hellas, PO Box 1527, 71110 Heraklion (Greece)

    2005-08-07

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT) (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  2. Image scanning microscopy with radially polarized light

    Science.gov (United States)

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

    2017-03-01

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

  3. In-vivo and label-free imaging of cellular and tissue structures in mouse ear skin by using second- and third-harmonic generation microscopy

    Science.gov (United States)

    Lee, Eung Jang; Kim, Boram; Ahn, Hong-Gyu; Park, Seung-Han; Cheong, Eunji; Lee, Sangyoup

    2015-02-01

    A video-rate multimodal microscope, which can obtain second- and third- harmonic generation (SHG and THG) images simultaneously, is developed for investigating cellular and tissue structures in mouse ear skin. By utilizing in-vivo video-rate epi-detected SHG and THG microscopy, we successfully demonstrate that combined images of subcutaneous cellular components and peripheral nerve fibers, together with the collagen fiber, in the mouse ear pinna can be obtained without employing fluorescent probes. We also show that the flow of red blood cells and the diameter change of arteriole-like blood vessels can be visualized with femtosecond laser pulses with a wavelength of 1036 nm. In particular, the epi-THG contrast images of the blood-vessel walls display clearly the difference between the arteriole-like and the venule capillary-like blood-vessel types. We should emphasize that our newly-developed microscope system has a unique feature in that it can produce simultaneous in-vivo label-free SHG and THG images in contrast to the conventional confocal and two-photon microscopes.

  4. Real-Space Imaging of Carrier Dynamics of Materials Surfaces by Second-Generation Four-Dimensional Scanning Ultrafast Electron Microscopy

    KAUST Repository

    Sun, Jingya

    2015-09-14

    In the fields of photocatalysis and photovoltaics, ultrafast dynamical processes, including carrier trapping and recombination on material surfaces, are among the key factors that determine the overall energy conversion efficiency. A precise knowledge of these dynamical events on the nanometer (nm) and femtosecond (fs) scales was not accessible until recently. The only way to access such fundamental processes fully is to map the surface dynamics selectively in real space and time. In this study, we establish a second generation of four-dimensional scanning ultrafast electron microscopy (4D S-UEM) and demonstrate the ability to record time-resolved images (snapshots) of material surfaces with 650 fs and ∼5 nm temporal and spatial resolutions, respectively. In this method, the surface of a specimen is excited by a clocking optical pulse and imaged using a pulsed primary electron beam as a probe pulse, generating secondary electrons (SEs), which are emitted from the surface of the specimen in a manner that is sensitive to the local electron/hole density. This method provides direct and controllable information regarding surface dynamics. We clearly demonstrate how the surface morphology, grains, defects, and nanostructured features can significantly impact the overall dynamical processes on the surface of photoactive-materials. In addition, the ability to access two regimes of dynamical probing in a single experiment and the energy loss of SEs in semiconductor-nanoscale materials will also be discussed.

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

    Science.gov (United States)

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

    2017-10-01

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

  6. Fabrication of Ion-Shaped Anisotropic Nanoparticles and their Orientational Imaging by Second-Harmonic Generation Microscopy

    Science.gov (United States)

    Slablab, Abdallah; Isotalo, Tero J.; Mäkitalo, Jouni; Turquet, Léo; Coulon, Pierre-Eugène; Niemi, Tapio; Ulysse, Christian; Kociak, Mathieu; Mailly, Dominique; Rizza, Giancarlo; Kauranen, Martti

    2016-11-01

    Ion beam shaping is a novel and powerful tool to engineer nanocomposites with effective three-dimensional (3D) architectures. In particular, this technique offers the possibility to precisely control the size, shape and 3D orientation of metallic nanoparticles at the nanometer scale while keeping the particle volume constant. Here, we use swift heavy ions of xenon for irradiation in order to successfully fabricate nanocomposites consisting of anisotropic gold nanoparticle that are oriented in 3D and embedded in silica matrix. Furthermore, we investigate individual nanorods using a nonlinear optical microscope based on second-harmonic generation (SHG). A tightly focused linearly or radially-polarized laser beam is used to excite nanorods with different orientations. We demonstrate high sensitivity of the SHG response for these polarizations to the orientation of the nanorods. The SHG measurements are in excellent agreement with the results of numerical modeling based on the boundary element method.

  7. NICHD Microscopy and Imaging Core (MIC)

    Data.gov (United States)

    Federal Laboratory Consortium — The NICHD Microscopy and Imaging Core (MIC) is designed as a multi-user research facility providing training and instrumentation for high resolution microscopy and...

  8. Fast image analysis in polarization SHG microscopy.

    Science.gov (United States)

    Amat-Roldan, Ivan; Psilodimitrakopoulos, Sotiris; Loza-Alvarez, Pablo; Artigas, David

    2010-08-02

    Pixel resolution polarization-sensitive second harmonic generation (PSHG) imaging has been recently shown as a promising imaging modality, by largely enhancing the capabilities of conventional intensity-based SHG microscopy. PSHG is able to obtain structural information from the elementary SHG active structures, which play an important role in many biological processes. Although the technique is of major interest, acquiring such information requires long offline processing, even with current computers. In this paper, we present an approach based on Fourier analysis of the anisotropy signature that allows processing the PSHG images in less than a second in standard single core computers. This represents a temporal improvement of several orders of magnitude compared to conventional fitting algorithms. This opens up the possibility for fast PSHG information with the subsequent benefit of potential use in medical applications.

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

    Science.gov (United States)

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

    2014-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Stephen A. Boppart

    2008-06-01

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

  11. Adaptive nonlinear microscopy for whole tissue imaging

    Science.gov (United States)

    Müllenbroich, M. Caroline; McGhee, Ewan J.; Wright, Amanda J.; Anderson, Kurt I.; Mathieson, Keith

    2013-02-01

    Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system's optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.

  12. Multiphoton microscopy imaging of developing tooth germs

    Directory of Open Access Journals (Sweden)

    Pei-Yu Pan

    2014-01-01

    Conclusion: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

  13. Scanning transmission electron microscopy imaging and analysis

    CERN Document Server

    Pennycook, Stephen J

    2011-01-01

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

  14. Nonlinear optical microscopy for imaging thin films and surfaces

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-03-01

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

  15. Image Formation in Second-Harmonic Near-Field Microscopy

    DEFF Research Database (Denmark)

    Bozhevolnyi, Sergey I.; Lozovski, Valeri Z.; Pedersen, Kjeld

    1999-01-01

    A macroscopic self-consistent approach that enables one to rigorously describe image formation in scanning near-field optical second-harmonic generation microscopy is developed. The self-consistent second-harmonic field is determined by taking into account both the linear and nonlinear contributi......A macroscopic self-consistent approach that enables one to rigorously describe image formation in scanning near-field optical second-harmonic generation microscopy is developed. The self-consistent second-harmonic field is determined by taking into account both the linear and nonlinear...

  16. Transmission Electron Microscopy Physics of Image Formation

    CERN Document Server

    Kohl, Helmut

    2008-01-01

    Transmission Electron Microscopy: Physics of Image Formation presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fifth edition includes discussion of recent progress, especially in the area of aberration correction and energy filtering; moreover, the topics introduced in the fourth edition have been updated. Transmission Electron Microscopy: Physics of Image Formation is written f...

  17. Imaging Cytoskeleton Components by Electron Microscopy

    Science.gov (United States)

    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

  18. Edge detection in microscopy images using curvelets

    OpenAIRE

    Koumoutsakos Petros; Gebäck Tobias

    2009-01-01

    Abstract 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...

  19. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-04-25

    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.

  20. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    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.

  1. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    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.

  2. Microscopy imaging device with advanced imaging properties

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. Quantitative imaging of bilirubin by photoacoustic microscopy

    Science.gov (United States)

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

    2013-03-01

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

  4. Image Correlation Microscopy for Uniform Illumination

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Gregor, Ingo; Ros, Robert; Enderlein, Jörg

    2017-02-01

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

  6. microlith : Image Simulation for Biological Phase Microscopy

    CERN Document Server

    Mehta, Shalin B

    2013-01-01

    Accurate simulation of image formation remains under-exploited for biological phase microscopy methods that employ partially coherent illumination, despite being important for the design of imaging systems and the reconstruction algorithms. We present an open-source MATLAB toolbox, microlith (https://code.google.com/p/microlith), that provides accurate simulation of the 3D image of a thin specimen under any partially coherent imaging system, including coherent or incoherent systems. We demonstrate the accuracy of the microlith toolbox by comparing simulated images and experimental images of a phase-only Siemens star test target using dark field and differential interference contrast microscopes. The comparison leads to intriguing insights about the sensitivity of the dark-field microscope to sub-resolution features and effects of specimen birefringence on differential interference contrast.

  7. Dynamic investigation of Drosophila myocytes with second harmonic generation microscopy

    Science.gov (United States)

    Greenhalgh, Catherine; Stewart, Bryan; Cisek, Richard; Prent, Nicole; Major, Arkady; Barzda, Virginijus

    2006-09-01

    The functional dynamics and structure of both larval and adult Drosophila melanogaster muscle were investigated with a nonlinear multimodal microscope. Imaging was carried out using a home built microscope capable of recording the multiphoton excitation fluorescence, second harmonic generation, and third harmonic generation signals simultaneously at a scanning rate of up to ~12 frames/sec. The sample was excited by a home built femtosecond Ti:Sapphire laser at 840 nm, or by a Yb-ion doped potassium gadolinium tungstate (Yb:KGW) crystal based oscillator at 1042 nm. There was no observable damage detected in the myocyte after prolonged scanning with either of the lasers. Microscopic second harmonic generation (SHG) appears particularly strong in the myocytes. This allows the fast contraction dynamics of the myocytes to be followed. The larger sarcomere size observed in the larvae myocytes is especially well suited for studying the contraction dynamics. Microscopic imaging of muscle contractions showed different relaxation and contraction rates. The SHG intensities were significantly higher in the relaxed state of the myocyte compared to the contracted state. The imaging also revealed disappearance of SHG signal in highly stretched sarcomeres, indicating that SHG diminishes in the disordered structures. The study illustrates that SHG microscopy, combined with other nonlinear contrast mechanisms, can help to elucidate physiological mechanisms of contraction. This study also provides further insight into the mechanisms of harmonic generation in biological tissue and shows that crystalline arrangement of macromolecules has a determining factor for the high efficiency second harmonic generation from the bulk structures.

  8. Spiral phase contrast imaging in microscopy.

    Science.gov (United States)

    Fürhapter, Severin; Jesacher, Alexander; Bernet, Stefan; Ritsch-Marte, Monika

    2005-02-07

    We demonstrate an optical method for edge contrast enhancement in light microscopy. The method is based on holographic Fourier plane filtering of the microscopic image with a spiral phase element (also called vortex phase or helical phase filter) displayed as an off-axis hologram at a computer controlled high resolution spatial light modulator (SLM) in the optical imaging pathway. The phase hologram imprints a helical phase term of the form exp(i phi) on the diffracted light field in its Fourier plane. In the image plane, this results in a strong and isotropic edge contrast enhancement for both amplitude and phase objects.

  9. Spatially Resolved Imaging on Photocarrier Generations and Band Alignments at Perovskite/PbI2 Heterointerfaces of Perovskite Solar Cells by Light-Modulated Scanning Tunneling Microscopy.

    Science.gov (United States)

    Shih, Min-Chuan; Li, Shao-Sian; Hsieh, Cheng-Hua; Wang, Ying-Chiao; Yang, Hung-Duen; Chiu, Ya-Ping; Chang, Chia-Seng; Chen, Chun-Wei

    2017-02-08

    The presence of the PbI2 passivation layers at perovskite crystal grains has been found to considerably affect the charge carrier transport behaviors and device performance of perovskite solar cells. This work demonstrates the application of a novel light-modulated scanning tunneling microscopy (LM-STM) technique to reveal the interfacial electronic structures at the heterointerfaces between CH3NH3PbI3 perovskite crystals and PbI2 passivation layers of individual perovskite grains under light illumination. Most importantly, this technique enabled the first observation of spatially resolved mapping images of photoinduced interfacial band bending of valence bands and conduction bands and the photogenerated electron and hole carriers at the heterointerfaces of perovskite crystal grains. By systematically exploring the interfacial electronic structures of individual perovskite grains, enhanced charge separation and reduced back recombination were observed when an optimal design of interfacial PbI2 passivation layers consisting of a thickness less than 20 nm at perovskite crystal grains was applied.

  10. Imaging acute thermal burns by photoacoustic microscopy

    OpenAIRE

    Zhang, Hao F.; Maslov, Konstantin; Stoica, George; Wang, Lihong V.

    2006-01-01

    The clinical significance of a burn depends on the percentage of total body involved and the depth of the burn. Hence a noninvasive method that is able to evaluate burn depth would be of great help in clinical evaluation. To this end, photoacoustic microscopy is used to determine the depth of acute thermal burns by imaging the total hemoglobin concentration in the blood that accumulates along the boundaries of injuries as a result of thermal damage to the vasculature. We induce acute thermal ...

  11. Edge detection in microscopy images using curvelets

    Directory of Open Access Journals (Sweden)

    Koumoutsakos Petros

    2009-03-01

    Full Text Available Abstract 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.

  12. Classification of microscopy images of Langerhans islets

    Science.gov (United States)

    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. Dynamic force microscopy imaging of native membranes

    Energy Technology Data Exchange (ETDEWEB)

    Kienberger, Ferry; Stroh, Cordula; Kada, Gerald; Moser, Rosita; Baumgartner, Werner; Pastushenko, Vassili; Rankl, Christian; Schmidt, Ute; Mueller, Harald; Orlova, Elena; LeGrimellec, Christian; Drenckhahn, Detlev; Blaas, Dieter; Hinterdorfer, Peter

    2003-10-15

    We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5 nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2 nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20 nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.

  14. Nanoscale imaging of RNA with expansion microscopy.

    Science.gov (United States)

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

    2016-08-01

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

  15. Characterization of muscle contraction with second harmonic generation microscopy

    Science.gov (United States)

    Prent, Nicole

    Muscle cells have the ability to change length and generate force due to orchestrated action of myosin nanomotors that cause sliding of actin filaments along myosin filaments in the sarcomeres, the fundamental contractile units, of myocytes. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. In this study, second harmonic generation (SHG) microscopy is employed for imaging striated myocytes. Myosin filaments in striated myocytes inherently have a nonzero second-order susceptibility, [special characters omitted] and therefore generate efficient SHG. Employing polarization-in polarization-out (PIPO) SHG microscopy allows for the accurate determination of the characteristic ratio, [special characters omitted] in birefringent myocytes, which describes the structure of the myosin filament. Analysis shows that the b value at the centre of the myosin filament, where the nonlinear dipoles are better aligned, is slightly lower than the value at the edges of the filament, where there is more disorder in orientation of the nonlinear dipoles from the myosin heads. Forced stretching of myocytes resulted in an SHG intensity increase with the elongation of the sarcomere. SHG microscopy captured individual sarcomeres during contraction, allowing for the measurement of sarcomere length (SL) and SHG intensity (SI) fluctuations. The fluctuations also revealed higher SHG intensity in elongated sarcomeres. The sarcomere synchronization model (SSM) for contracting and quiescent myocytes was developed, and experimentally verified for three cases (isolated cardiomyocyte, embryonic chicken cardiomyocyte, and larva myocyte). During contraction, the action of SLs and SIs between neighbouring sarcomeres partially correlated, whereas in quiescent myocytes the SLs show an anti-correlation and the SIs have no

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

    Science.gov (United States)

    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.

  17. Identification by force modulation microscopy of nanoparticles generated in vacuum arcs Identification by force modulation microscopy of nanoparticles generated in vacuum arcs

    OpenAIRE

    M. Arroyave Franco

    2006-01-01

    An alternative method based on force modulation microscopy (FMM) for identification of nanoparticles produced in the plasma generated by the cathode spots of vacuum arcs is presented. FMM technique is enabled for the detection of variations in the mechanical properties of a surface with high sensitiveness. Titanium nitride (TiN) coatings deposited on oriented silicon by pulsed vacuum arc process have been analyzed. AFM (Atomic Force Microscopy) and FMM images were simultaneously obtained, and...

  18. Nonlinear Polarimetric Microscopy for Biomedical Imaging

    Science.gov (United States)

    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

  19. Generation and application of bessel beams in electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Grillo, Vincenzo, E-mail: vincenzo.grillo@cnr.it [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); CNR-IMEM, Parco Area delle Scienze 37/A, I-43124 Parma (Italy); Harris, Jérémie [Department of Physics, University of Ottawa, 25 Templeton St., Ottawa, Ontario, Canada K1N 6N5 (Canada); Gazzadi, Gian Carlo [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); Balboni, Roberto [CNR-IMM Bologna, Via P. Gobetti 101, 40129 Bologna (Italy); Mafakheri, Erfan [Dipartimento di Fisica Informatica e Matematica, Università di Modena e Reggio Emilia, via G Campi 213/a, I-41125 Modena (Italy); Dennis, Mark R. [H.H. Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL (United Kingdom); Frabboni, Stefano [CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena (Italy); Dipartimento di Fisica Informatica e Matematica, Università di Modena e Reggio Emilia, via G Campi 213/a, I-41125 Modena (Italy); Boyd, Robert W.; Karimi, Ebrahim [Department of Physics, University of Ottawa, 25 Templeton St., Ottawa, Ontario, Canada K1N 6N5 (Canada)

    2016-07-15

    We report a systematic treatment of the holographic generation of electron Bessel beams, with a view to applications in electron microscopy. We describe in detail the theory underlying hologram patterning, as well as the actual electron-optical configuration used experimentally. We show that by optimizing our nanofabrication recipe, electron Bessel beams can be generated with relative efficiencies reaching 37±3%. We also demonstrate by tuning various hologram parameters that electron Bessel beams can be produced with many visible rings, making them ideal for interferometric applications, or in more highly localized forms with fewer rings, more suitable for imaging. We describe the settings required to tune beam localization in this way, and explore beam and hologram configurations that allow the convergences and topological charges of electron Bessel beams to be controlled. We also characterize the phase structure of the Bessel beams generated with our technique, using a simulation procedure that accounts for imperfections in the hologram manufacturing process. - Highlights: • Bessel beams with different convergence, topological charge, visible fringes are demonstrated. • The relation between the Fresnel hologram and the probe shape is explained by detailed calculations and experiments. • Among the holograms here presented the highest relative efficiency is 37%, the best result ever reached for blazed holograms.

  20. Generation and application of bessel beams in electron microscopy.

    Science.gov (United States)

    Grillo, Vincenzo; Harris, Jérémie; Gazzadi, Gian Carlo; Balboni, Roberto; Mafakheri, Erfan; Dennis, Mark R; Frabboni, Stefano; Boyd, Robert W; Karimi, Ebrahim

    2016-07-01

    We report a systematic treatment of the holographic generation of electron Bessel beams, with a view to applications in electron microscopy. We describe in detail the theory underlying hologram patterning, as well as the actual electron-optical configuration used experimentally. We show that by optimizing our nanofabrication recipe, electron Bessel beams can be generated with relative efficiencies reaching 37±3%. We also demonstrate by tuning various hologram parameters that electron Bessel beams can be produced with many visible rings, making them ideal for interferometric applications, or in more highly localized forms with fewer rings, more suitable for imaging. We describe the settings required to tune beam localization in this way, and explore beam and hologram configurations that allow the convergences and topological charges of electron Bessel beams to be controlled. We also characterize the phase structure of the Bessel beams generated with our technique, using a simulation procedure that accounts for imperfections in the hologram manufacturing process. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Sub- Angstrom microscopy through incoherent imaging and image reconstruction

    Energy Technology Data Exchange (ETDEWEB)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F. (Oak Ridge National Lab., TN (United States)); Ferridge, A.G.; Seddon, M.J. (Wellcome Research Lab., Beckenham (United Kingdom))

    1992-03-01

    Z-contrast scanning transmission electron microscopy (STEM) with a high-angle annular detector breaks the coherence of the imaging process, and provides an incoherent image of a crystal projection. Even in the presence of strong dynamical diffraction, the image can be accurately described as a convolution between an object function, sharply peaked at the projected atomic sites, and the probe intensity profile. Such an image can be inverted intuitively without the need for model structures, and therefore provides the important capability to reveal unanticipated interfacial arrangements. It represents a direct image of the crystal projection, revealing the location of the atomic columns and their relative high-angle scattering power. Since no phase is associated with a peak in the object function or the contrast transfer function, extension to higher resolution is also straightforward. Image restoration techniques such as maximum entropy, in conjunction with the 1.3 {Angstrom} probe anticipated for a 300 kV STEM, appear to provide a simple and robust route to the achievement of sub-{Angstrom} resolution electron microscopy.

  2. Sub-{Angstrom} microscopy through incoherent imaging and image reconstruction

    Energy Technology Data Exchange (ETDEWEB)

    Pennycook, S.J.; Jesson, D.E.; Chisholm, M.F. [Oak Ridge National Lab., TN (United States); Ferridge, A.G.; Seddon, M.J. [Wellcome Research Lab., Beckenham (United Kingdom)

    1992-03-01

    Z-contrast scanning transmission electron microscopy (STEM) with a high-angle annular detector breaks the coherence of the imaging process, and provides an incoherent image of a crystal projection. Even in the presence of strong dynamical diffraction, the image can be accurately described as a convolution between an object function, sharply peaked at the projected atomic sites, and the probe intensity profile. Such an image can be inverted intuitively without the need for model structures, and therefore provides the important capability to reveal unanticipated interfacial arrangements. It represents a direct image of the crystal projection, revealing the location of the atomic columns and their relative high-angle scattering power. Since no phase is associated with a peak in the object function or the contrast transfer function, extension to higher resolution is also straightforward. Image restoration techniques such as maximum entropy, in conjunction with the 1.3 {Angstrom} probe anticipated for a 300 kV STEM, appear to provide a simple and robust route to the achievement of sub-{Angstrom} resolution electron microscopy.

  3. Imaging white adipose tissue with confocal microscopy.

    Science.gov (United States)

    Martinez-Santibañez, Gabriel; Cho, Kae Won; Lumeng, Carey N

    2014-01-01

    Adipose tissue is composed of a variety of cell types that include mature adipocytes, endothelial cells, fibroblasts, adipocyte progenitors, and a range of inflammatory leukocytes. These cells work in concert to promote nutrient storage in adipose tissue depots and vary widely based on location. In addition, overnutrition and obesity impart significant changes in the architecture of adipose tissue that are strongly associated with metabolic dysfunction. Recent studies have called attention to the importance of adipose tissue microenvironments in regulating adipocyte function and therefore require techniques that preserve cellular interactions and permit detailed analysis of three-dimensional structures in fat. This chapter summarizes our experience with the use of laser scanning confocal microscopy for imaging adipose tissue in rodents.

  4. Confocal microscopy imaging of the biofilm matrix.

    Science.gov (United States)

    Schlafer, Sebastian; Meyer, Rikke L

    2017-07-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens. Confocal microscopes are held by many research groups, and a number of methods for qualitative and quantitative imaging of the matrix have emerged in recent years. This review provides an overview and a critical discussion of techniques used to visualize different matrix compounds, to determine the concentration of solutes and the diffusive properties of the biofilm matrix. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Performance evaluation of spot detection algorithms in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2012-10-01

    Full Text Available Detection of messenger Ribonucleic Acid (mRNA) spots in fluorescence microscopy images is of great importance for biologists seeking better understanding of cell functionality. Fluorescence microscopy and specific staining methods make biological...

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Imaging of carbon nanomembranes with helium ion microscopy

    Directory of Open Access Journals (Sweden)

    André Beyer

    2015-08-01

    Full Text Available Carbon nanomembranes (CNMs prepared from aromatic self-assembled monolayers constitute a recently developed class of 2D materials. They are made by a combination of self-assembly, radiation-induced cross-linking and the detachment of the cross-linked SAM from its substrate. CNMs can be deposited on arbitrary substrates, including holey and perforated ones, as well as on metallic (transmission electron microscopy grids. Therewith, freestanding membranes with a thickness of 1 nm and macroscopic lateral dimensions can be prepared. Although free-standing CNMs cannot be imaged by light microscopy, charged particle techniques can visualize them. However, CNMs are electrically insulating, which makes them sensitive to charging. We demonstrate that the helium ion microscope (HIM is a good candidate for imaging freestanding CNMs due to its efficient charge compensation tool. Scanning with a beam of helium ions while recording the emitted secondary electrons generates the HIM images. The advantages of HIM are high resolution, high surface sensitivity and large depth of field. The effects of sample charging, imaging of multilayer CNMs as well as imaging artefacts are discussed.

  8. Nanoscale chemical imaging by photoinduced force microscopy

    Science.gov (United States)

    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

  9. Second-harmonic generation imaging of cancer.

    Science.gov (United States)

    Keikhosravi, Adib; Bredfeldt, Jeremy S; Sagar, Abdul Kader; Eliceiri, Kevin W

    2014-01-01

    The last 30 years has seen great advances in optical microscopy with the introduction of sophisticated fluorescence-based imaging methods such as confocal and multiphoton laser scanning microscopy. There is increasing interest in using these methods to quantitatively examine sources of intrinsic biological contrast including autofluorescent endogenous proteins and light interactions such as second-harmonic generation (SHG) in collagen. In particular, SHG-based microscopy has become a widely used quantitative modality for imaging noncentrosymmetric proteins such as collagen in a diverse range of tissues. Due to the underlying physical origin of the SHG signal, it is highly sensitive to collagen fibril/fiber structure and, importantly, to collagen-associated changes that occur in diseases such as cancer, fibrosis, and connective tissue disorders. An overview of SHG physics background and technologies is presented with a focused review on applications of SHG primarily as applied to cancer. © 2014 Elsevier Inc. All rights reserved.

  10. Multi-modal registration for correlative microscopy using image analogies.

    Science.gov (United States)

    Cao, Tian; Zach, Christopher; Modla, Shannon; Powell, Debbie; Czymmek, Kirk; Niethammer, Marc

    2014-08-01

    Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Polarization-Modulated Second Harmonic Generation Microscopy in Collagen

    Energy Technology Data Exchange (ETDEWEB)

    Stoller, P C

    2002-09-30

    Collagen is a key structural protein in the body; several pathological conditions lead to changes in collagen. Among imaging modalities that can be used in vivo, second harmonic generation (SHG) microscopy has a key advantage: it provides {approx}1 {micro}m resolution information about collagen structure as a function of depth. A new technique--polarization-modulated SHG--is presented: it permits simultaneous measurement of collagen orientation, of a lower bound on the magnitude of the second order nonlinear susceptibility tensor, and of the ratio of the two independent elements in this tensor. It is applied to characterizing SHG in collagen and to determining effects of biologically relevant changes in collagen structure. The magnitude of the second harmonic signal in two dimensional images varies with position even in structurally homogeneous tissue; this phenomenon is due to interference between second harmonic light generated by neighboring fibrils, which are randomly oriented parallel or anti-parallel to each other. Studies in which focal spot size was varied indicated that regions where fibrils are co-oriented are less than {approx}1.5 {micro}m in diameter. A quartz reference was used to determine the spot size as well as a lower limit (d{sub xxx} > 0.3 pm/V) for the magnitude of the second order nonlinear susceptibility. The ratio of the two independent tensor elements ranged between d{sub XYY}/d{sub XXX} = 0.60 and 0.75. SHG magnitude alone was not useful for identifying structural anomalies in collagenous tissue. Instead, changes in the polarization dependence of SHG were used to analyze biologically relevant perturbations in collagen structure. Changes in polarization dependence were observed in dehydrated samples, but not in highly crosslinked samples, despite significant alterations in packing structure. Complete thermal denaturation and collagenase digestion produced samples with no detectable SHG signal. Collagen orientation was measured in thin

  12. Functional cardiac imaging by random access microscopy

    Directory of Open Access Journals (Sweden)

    Claudia eCrocini

    2014-10-01

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

  13. Image recombination transform algorithm for superresolution structured illumination microscopy

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. Hyperglycemia-induced abnormalities in rat and human corneas: the potential of second harmonic generation microscopy.

    Directory of Open Access Journals (Sweden)

    Gaël Latour

    Full Text Available BACKGROUND: Second Harmonic Generation (SHG microscopy recently appeared as an efficient optical imaging technique to probe unstained collagen-rich tissues like cornea. Moreover, corneal remodeling occurs in many diseases and precise characterization requires overcoming the limitations of conventional techniques. In this work, we focus on diabetes, which affects hundreds of million people worldwide and most often leads to diabetic retinopathy, with no early diagnostic tool. This study then aims to establish the potential of SHG microscopy for in situ detection and characterization of hyperglycemia-induced abnormalities in the Descemet's membrane, in the posterior cornea. METHODOLOGY/PRINCIPAL FINDINGS: We studied corneas from age-matched control and Goto-Kakizaki rats, a spontaneous model of type 2 diabetes, and corneas from human donors with type 2 diabetes and without any diabetes. SHG imaging was compared to confocal microscopy, to histology characterization using conventional staining and transmitted light microscopy and to transmission electron microscopy. SHG imaging revealed collagen deposits in the Descemet's membrane of unstained corneas in a unique way compared to these gold standard techniques in ophthalmology. It provided background-free images of the three-dimensional interwoven distribution of the collagen deposits, with improved contrast compared to confocal microscopy. It also provided structural capability in intact corneas because of its high specificity to fibrillar collagen, with substantially larger field of view than transmission electron microscopy. Moreover, in vivo SHG imaging was demonstrated in Goto-Kakizaki rats. CONCLUSIONS/SIGNIFICANCE: Our study shows unambiguously the high potential of SHG microscopy for three-dimensional characterization of structural abnormalities in unstained corneas. Furthermore, our demonstration of in vivo SHG imaging opens the way to long-term dynamical studies. This method should be easily

  17. Generation connected with images

    Directory of Open Access Journals (Sweden)

    Adriana RECAMÁN PAYO

    2011-12-01

    Full Text Available 0 0 1 197 1086 Instituto Universitario de Ciencias de la Educación 9 2 1281 14.0 Normal 0 21 false false false ES JA X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:ES; mso-fareast-language:EN-US;} In learning contexts studying the image as a focus of sensitive knowledge and formative purposes is crucial to achieve high levels of quality and educational excellence. As Renobell (2005 stated, image analysis encourages the development of critical capacity and contributes to developing a personal style for the gradual acquisition of a visual culture. Images educate and consequently, their presence in the field of education should not be a mere accompaniment to the text. They should not be limited to adorn or illustrate a linguistic content but to complement and deepen it, activating the thought and the reflection of the reader. In Internet culture, image as a focus of knowledge, of shared use, of social content and educational purposes, contribute to explain the implications and vivacity around this technological environment, which plays a leading role in the current social changes and movements. The culture of the network has changed our perceptual sensitivity to interpret images, which are now more complex, integrated, multidimensional and dynamic than ever. The interactivity, the strong relationship with the text content, the graphic sequentiality, the associated sound effects or the iconical text design reveal the

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Imaging DNA Structure by Atomic Force Microscopy.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  3. Comparison of two detection algorithms for spot tracking in fluorescence microscopy images

    CSIR Research Space (South Africa)

    Mabaso, M

    2014-11-01

    Full Text Available for spot tracking in fluorescence microscopy images Matsilele Mabaso∗, Daniel Withey‡, Bhekisipho Twala† ∗ ‡MDS(MIAS) Council for Scientific and Industrial Research Pretoria, South Africa, Email: ∗MMabaso@csir.co.za †Department of Electrical Engineering.... The quantitative comparative results demonstrated the importance of spot detection in tracking contexts. I. INTRODUCTION In recent years, the field of fluorescence microscopy has been improved and automated, and a large volume of image data are being generated...

  4. Third-harmonic generation microscopy reveals dental anatomy in ancient fossils.

    Science.gov (United States)

    Chen, Yu-Cheng; Lee, Szu-Yu; Wu, Yana; Brink, Kirstin; Shieh, Dar-Bin; Huang, Timothy D; Reisz, Robert R; Sun, Chi-Kuang

    2015-04-01

    Fossil teeth are primary tools in the study of vertebrate evolution, but standard imaging modalities have not been capable of providing high-quality images in dentin, the main component of teeth, owing to small refractive index differences in the fossilized dentin. Our first attempt to use third-harmonic generation (THG) microscopy in fossil teeth has yielded significant submicrometer level anatomy, with an unexpectedly strong signal contrasting fossilized tubules from the surrounding dentin. Comparison between fossilized and extant teeth of crocodilians reveals a consistent evolutionary signature through time, indicating the great significance of THG microscopy in the evolutionary studies of dental anatomy in fossil teeth.

  5. 3D super-resolution imaging by localization microscopy.

    Science.gov (United States)

    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.

  6. Reconstruction of Undersampled Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Tobias Lindstrøm; Arildsen, Thomas; Østergaard, Jan

    2013-01-01

    . Moreover, it is often required to take several images before a relevant observation region is identified. In this paper we show how to significantly reduce the image acquisition time by undersampling. The reconstruction of an undersampled AFM image can be viewed as an inpainting, interpolating problem...

  7. Organized Aggregation of Porphyrins in Lipid Bilayers for Third Harmonic Generation Microscopy.

    Science.gov (United States)

    Cui, Liyang; Tokarz, Danielle; Cisek, Richard; Ng, Kenneth K; Wang, Fan; Chen, Juan; Barzda, Virginijus; Zheng, Gang

    2015-11-16

    Nonlinear optical microscopy has become a powerful tool for high-resolution imaging of cellular and subcellular composition, morphology, and interactions because of its high spatial resolution, deep penetration, and low photo-damage to tissue. Developing specific harmonic probes is essential for exploiting nonlinear microscopic imaging for biomedical applications. We report an organized aggregate of porphyrins (OAP) that formed within lipidic nanoparticles showing fingerprint spectroscopic properties, structure-associated second harmonic generation, and superradiant third harmonic generation. The OAP facilitated harmonic microscopic imaging of living cells with significantly enhanced contrast. The structure-dependent switch between harmonic (OAP-intact) and fluorescence (OAP-disrupted) generation enabled real-time multi-modality imaging of the cellular fate of nanoparticles. Robustly produced under various conditions and easily incorporated into pre-formed lipid nanovesicles, OAP provides a biocompatible nanoplatform for harmonic imaging. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Image Resolution in Scanning Transmission Electron Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    OpenAIRE

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

    2014-01-01

    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....

  10. Surgical implantation of an abdominal imaging window for intravital microscopy

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Ellenbroek, S.I.; Kranenburg, O.; Rinkes, I.H.; van Rheenen, J.

    2013-01-01

    High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows;

  11. Surgical implantation of an abdominal imaging window for intravital microscopy

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Ellenbroek, S.I.; Kranenburg, O.; Rinkes, I.H.; van Rheenen, J.

    2013-01-01

    High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; howeve

  12. Confocal Microscopy Imaging of the Biofilm Matrix

    DEFF Research Database (Denmark)

    Schlafer, Sebastian; Meyer, Rikke Louise

    2016-01-01

    The extracellular matrix is an integral part of microbial biofilms and an important field of research. Confocal laser scanning microscopy is a valuable tool for the study of biofilms, and in particular of the biofilm matrix, as it allows real-time visualization of fully hydrated, living specimens...... the concentration of solutes and the diffusive properties of the biofilm matrix....

  13. Generative Interpretation of Medical Images

    DEFF Research Database (Denmark)

    Stegmann, Mikkel Bille

    2004-01-01

    This thesis describes, proposes and evaluates methods for automated analysis and quantification of medical images. A common theme is the usage of generative methods, which draw inference from unknown images by synthesising new images having shape, pose and appearance similar to the analysed image...... fraction from 4D cardiac cine MRI, myocardial perfusion in bolus passage cardiac perfusion MRI, corpus callosum shape and area in mid-sagittal brain MRI, and finally, lung, heart, clavicle location and cardiothoracic ratio in anterior-posterior chest radiographs....

  14. Image processing for drift compensation in fluorescence microscopy

    DEFF Research Database (Denmark)

    Petersen, Steffen; Thiagarajan, Viruthachalam; Coutinho, Isabel

    2013-01-01

    Fluorescence microscopy is characterized by low background noise, thus a fluorescent object appears as an area of high signal/noise. Thermal gradients may result in apparent motion of the object, leading to a blurred image. Here, we have developed an image processing methodology that may remove....../reduce blur significantly for any type of microscopy. A total of ~100 images were acquired with a pixel size of 30 nm. The acquisition time for each image was approximately 1second. We can quantity the drift in X and Y using the sub pixel accuracy computed centroid location of an image object in each frame....... We can measure drifts down to approximately 10 nm in size and a drift-compensated image can therefore be reconstructed on a grid of the same size using the “Shift and Add” approach leading to an image of identical size asthe individual image. We have also reconstructed the image using a 3 fold larger...

  15. Image correction in magneto-optical microscopy

    DEFF Research Database (Denmark)

    Paturi, P.; Larsen, B.H.; Jacobsen, B.A.

    2003-01-01

    An image-processing procedure that assures correct determination of the magnetic field distribution of magneto-optical images is presented. The method remedies image faults resulting from sources that are proportional to the incident light intensity, such as different types of defects in the indi......An image-processing procedure that assures correct determination of the magnetic field distribution of magneto-optical images is presented. The method remedies image faults resulting from sources that are proportional to the incident light intensity, such as different types of defects...... in the indicator film and unevenness of light, as well as additive signals from detector bias, external light sources, etc. When properly corrected a better measurement of the local magnetic field can be made, even in the case of heavily damaged films. For superconductors the magnetic field distributions may...

  16. Low energy electron microscopy imaging using Medipix2 detector

    NARCIS (Netherlands)

    Sikharulidze, I.; Gastel, van R.; Schramm, S.; Abrahams, J.P.; Poelsema, B.; Tromp, R.M.; Molen, van der S.J.

    2011-01-01

    Low Energy Electron Microscopy (LEEM) and Photo-Emission Electron Microscopy (PEEM) predominantly use a combination of microchannel plate (MCP), phosphor screen and optical camera to record images formed by 10–20 keV electrons. We have tested the performance of a LEEM/PEEM instrument with a Medipix2

  17. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)

    2012-01-01

    textabstractConfocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in

  18. In Vivo Confocal Microscopy expanding horizons in corneal imaging

    NARCIS (Netherlands)

    T. Hillenaar (Toine)

    2012-01-01

    textabstractConfocal microscopy is an emerging optical technique that allows the living human cornea to be imaged on a cellular level. As such, confocal microscopy enables morphologic and quantitative analysis of corneal resident cells in health and disease and provides an exciting bridge between in

  19. A framework for creating realistic synthetic fluorescence microscopy image sequences

    CSIR Research Space (South Africa)

    Mabaso, M

    2016-02-01

    Full Text Available of the 9th International Joint Conference on Biomedical Engineering Systems and Technologies, Rome, Italy. 21-23 February, 2016 A Framework for Creating Realistic Synthetic Fluorescence Microscopy Image Sequences Matsilele Mabaso1, Daniel Withey1...

  20. Correlative atomic force microscopy and localization-based super-resolution microscopy: revealing labelling and image reconstruction artefacts.

    Science.gov (United States)

    Monserrate, Aitor; Casado, Santiago; Flors, Cristina

    2014-03-17

    Hybrid microscopy: A correlative microscopy tool that combines in situ super-resolution fluorescence microscopy based on single-molecule localization and atomic force microscopy is presented. Direct comparison with high- resolution topography allows the authors to improve fluorescence labeling and image analysis in super-resolution imaging.

  1. Third-harmonic generation imaging of breast tissue biopsies.

    Science.gov (United States)

    Lee, Woowon; Kabir, Mohammad M; Emmadi, Rajyasree; Toussaint, Kimani C

    2016-11-01

    We demonstrate for the first time the imaging of unstained breast tissue biopsies using third-harmonic generation (THG) microscopy. As a label-free imaging technique, THG microscopy is compared to phase contrast and polarized light microscopy which are standard imaging methods for breast tissues. A simple feature detection algorithm is applied to detect tumour-associated lymphocyte rich regions in unstained breast biopsy tissue and compared with corresponding regions identified by a pathologist from bright-field images of hematoxylin and eosin stained breast tissue. Our results suggest that THG imaging holds potential as a complementary technique for analysing breast tissue biopsies. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  2. Computer-generated image hologram

    Institute of Scientific and Technical Information of China (English)

    Takeshi Yamaguchi; Hiroshi Yoshikawa

    2011-01-01

    We investigate the computer-generated hologram with full parallax and which can be reconstructed with white light. The object of the hologram is processed from three-dimensional computer graphics polygon data and has shaded surface for hidden surface removal. The optically reconstructed image from the printed hologram is evaluated.%We investigate the computer-generated hologram with full parallax and which can be reconstructed with white light.The object of the hologram is processed from three-dimensional computer graphics polygon data and has shaded surface for hidden surface removal.The optically reconstructed image from the printed hologram is evaluated.

  3. Super-resolution Microscopy in Plant Cell Imaging.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-01-01

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

  5. Simulating realistic imaging conditions for in situ liquid microscopy

    Energy Technology Data Exchange (ETDEWEB)

    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. Dynamic contrast enhancement in widefield microscopy using projector-generated illumination patterns

    Science.gov (United States)

    Carlo Samson, Edward; Mar Blanca, Carlo

    2007-10-01

    We present a simple and cost-effective optical protocol to realize contrast-enhancement imaging (such as dark-field, optical-staining and oblique illumination microscopy) of transparent samples on a conventional widefield microscope using commercial multimedia projectors. The projector functions as both light source and mask generator implemented by creating slideshows of the filters projected along the illumination planes of the microscope. The projected optical masks spatially modulate the distribution of the incident light to selectively enhance structures within the sample according to spatial frequency thereby increasing the image contrast of translucent biological specimens. Any amplitude filter can be customized and dynamically controlled so that switching from one imaging modality to another involves a simple slide transition and can be executed at a keystroke with no physical filters and no moving optical parts. The method yields an image contrast of 89 96% comparable with standard enhancement techniques. The polarization properties of the projector are then utilized to discriminate birefringent and non-birefringent sites on the sample using single-shot, simultaneous polarization and optical-staining microscopy. In addition to dynamic pattern generation and polarization, the projector also provides high illumination power and spectral excitation selectivity through its red-green-blue (RGB) channels. We exploit this last property to explore the feasibility of using video projectors to selectively excite stained samples and perform fluorescence imaging in tandem with reflectance and polarization reflectance microscopy.

  7. Photobleaching correction in fluorescence microscopy images

    Energy Technology Data Exchange (ETDEWEB)

    Vicente, Nathalie B; Diaz Zamboni, Javier E; Adur, Javier F; Paravani, Enrique V; Casco, Victor H [Microscopy Laboratory, School of Engineering - Bioengineering, National University of Entre Rios (UNER), Ruta 11, Km 10 (3101), Oro Verde, Entre Rios (Argentina)

    2007-11-15

    Fluorophores are used to detect molecular expression by highly specific antigen-antibody reactions in fluorescence microscopy techniques. A portion of the fluorophore emits fluorescence when irradiated with electromagnetic waves of particular wavelengths, enabling its detection. Photobleaching irreversibly destroys fluorophores stimulated by radiation within the excitation spectrum, thus eliminating potentially useful information. Since this process may not be completely prevented, techniques have been developed to slow it down or to correct resulting alterations (mainly, the decrease in fluorescent signal). In the present work, the correction by photobleaching curve was studied using E-cadherin (a cell-cell adhesion molecule) expression in Bufo arenarum embryos. Significant improvements were observed when applying this simple, inexpensive and fast technique.

  8. The RTSS Image Generation System

    NARCIS (Netherlands)

    Alvermann, K.; Graeber, S.; Mager, J.W.L.J.; Smith, M.H.

    1996-01-01

    Main market demands for the visual system of a simulator are photorealism and low latency time. RTSS, a general purpose image generation module developed within the European ESPRIT project HAMLET, can meet these demands through the use of High Performance Computing technology. This technology provid

  9. Magnetic force microscopy/current contrast imaging: A new technique for internal current probing of ICs

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, A.N.; Cole, E.I. Jr.; Dodd, B.A.; Anderson, R.E.

    1993-09-01

    This invited paper describes recently reported work on the application of magnetic force microscopy (MFM) to image currents in IC conductors [1]. A computer model for MFM imaging of IC currents and experimental results demonstrating the ability to determine current direction and magnitude with a resolution of {approximately} 1 mA dc and {approximately} 1 {mu}A ac are presented. The physics of MFM signal generation and applications to current imaging and measurement are described.

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

    Science.gov (United States)

    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.

  11. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1989-01-01

    The aim of this book is to present the theory of image and contrast formation and the analytical modes in transmission electron microscopy The principles of particle and wave optics of electrons are described Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal structure determination and imaging of lattice defects X-ray microanalysis and energy-loss spectroscopy are treated as analytical methods The second edition includes discussion of recent progress, especially in the areas of energy-loss spectroscopy, crystal-lattice imaging and reflection electron microscopy

  12. Simulating realistic imaging conditions for in situ liquid microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Welch, David A., E-mail: dawelch@ucdavis.edu [Department of Chemical Engineering and Materials Science, University of California, Davis, CA (United States); Faller, Roland [Department of Chemical Engineering and Materials Science, University of California, Davis, CA (United States); Evans, James E. [Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, Richland, WA (United States); Browning, Nigel D. [Pacific Northwest National Laboratory, Fundamental Computational Sciences Directorate, Richland, WA (United States)

    2013-12-15

    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. - Highlights: • Image simulation has been performed to understand in situ electron microscopy experiments. • Experimentally observed resolution of in situ grown PbS nanoparticles has been virtually reproduced. • General relationships between image resolution and in situ holder design, defocus, and particle size have been determined. • The presented image simulation technique can predict the obtainable resolution of future experiments.

  13. Stokes vector formalism based second harmonic generation microscopy

    Science.gov (United States)

    Qiu, Jianjun; Mazumder, Nirmal; Tsai, Han-Ruei; Hu, Chih-Wei; Kao, Fu-Jen

    2012-02-01

    In this study, we have developed a four-channel Stokes vector formalism based second harmonic generation (SHG) microscopy to map and analyze SHG signal. A four-channel Stokesmeter setup is calibrated and integrated into a laser scanning microscope to measure and characterize the SH's corresponding Stokes parameters. We are demonstrating the use of SH and its Stokes parameters to visualize the birefringence and crystalline orientation of KDP and collagen. We believe the developed method can reveal unprecedented information for biomedical and biomaterial studies.

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

    Directory of Open Access Journals (Sweden)

    Strandh Christer

    2008-07-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  16. Imaging photothermal microscopy for absorption measurements of optical coatings

    Institute of Scientific and Technical Information of China (English)

    Chunxian Tao; Yuanan Zhao; Hongbo He; Dawei Li; Jianda Shao; Zhengxiu Fan

    2009-01-01

    @@ For absorption measurement of large-aperture optical coatings, a novel method of imaging photothermal microscopy based on image lock-in technique is presented.Detailed theoretical analysis and numerical calculation are made based on the image photothermal technique.The feasibility of this imaging method is proved through the coincidence between the theoretical results of single spot method and multi-channel method.The measuring speed of this imaging method can be increased hundreds of times compared with that of the raster scanning.This technique can expand the applications of photothermal technique.

  17. Third harmonic generation microscopy of cells and tissue organization.

    Science.gov (United States)

    Weigelin, Bettina; Bakker, Gert-Jan; Friedl, Peter

    2016-01-15

    The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra- and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in three-dimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice. © 2016. Published by The Company of Biologists Ltd.

  18. Image analysis and microscopy: a useful combination

    Directory of Open Access Journals (Sweden)

    Pinotti L.

    2009-01-01

    Full Text Available The TSE Roadmap published in 2005 (DG for Health and Consumer Protection, 2005 suggests that short and medium term (2005-2009 amendments to control BSE policy should include “a relaxation of certain measures of the current total feed ban when certain conditions are met”. The same document noted “the starting point when revising the current feed ban provisions should be risk-based but at the same time taking into account the control tools in place to evaluate and ensure the proper implementation of this feed ban”. The clear implication is that adequate analytical methods to detect constituents of animal origin in feedstuffs are required. The official analytical method for the detection of constituents of animal origin in feedstuffs is the microscopic examination technique as described in Commission Directive 2003/126/EC of 23 December 2003 [OJ L 339, 24.12.2003, 78]. Although the microscopic method is usually able to distinguish fish from land animal material, it is often unable to distinguish between different terrestrial animals. Fulfillments of the requirements of Regulation 1774/2002/EC laying down health rules concerning animal by-products not intended for human consumption, clearly implies that it must be possible to identify the origin animal materials, at higher taxonomic levels than in the past. Thus improvements in all methods of detecting constituents of animal origin are required, including the microscopic method. This article will examine the problem of meat and bone meal in animal feeds, and the use of microscopic methods in association with computer image analysis to identify the source species of these feedstuff contaminants. Image processing, integrated with morphometric measurements can provide accurate and reliable results and can be a very useful aid to the analyst in the characterization, analysis and control of feedstuffs.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  1. Imaging diffusion in a microfluidic device by third harmonic microscopy

    Science.gov (United States)

    Petzold, Uwe; Büchel, Andreas; Hardt, Steffen; Halfmann, Thomas

    2012-09-01

    We monitor and characterize near-surface diffusion of miscible, transparent liquids in a microfluidic device by third harmonic microscopy. The technique enables observations even of transparent or index-matched media without perturbation of the sample. In particular, we image concentrations of ethanol diffusing in water and estimate the diffusion coefficient from the third harmonic images. We obtain a diffusion coefficient D = (460 ± 30) μm2/s, which is consistent with theoretical predictions. The investigations clearly demonstrate the potential of harmonic microscopy also under the challenging conditions of transparent fluids.

  2. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1993-01-01

    "Transmission Electron Microscopy" presents the theory of image and contrastformation, and the analytical modes in transmission electron microscopy Theprinciples of particle and wave optics of electrons are described Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast Also analysed are the kinetical and dynamical theories of electron diffraction and their applications for crystal-structure determination and imaging of lattices and their defects X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods The third edition includes a brief discussionof Schottky emission guns, some clarification of minor details, and references to the recent literature

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Henry Pinkard

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

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

    Science.gov (United States)

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

    2016-01-01

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

  6. Three-dimensional volume imaging with electron microscopy toward connectome.

    Science.gov (United States)

    Ohno, Nobuhiko; Katoh, Mitsuhiko; Saitoh, Yurika; Saitoh, Sei; Ohno, Shinichi

    2015-02-01

    Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hongda Mao

    2013-01-01

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

  9. Generation of apodized X-ray illumination and its application to scanning and diffraction microscopy.

    Science.gov (United States)

    Khakurel, Krishna P; Kimura, Takashi; Nakamori, Hiroki; Goto, Takumi; Matsuyama, Satoshi; Sasaki, Tomoya; Takei, Masashi; Kohmura, Yoshiki; Ishikawa, Tetsuya; Yamauchi, Kazuto; Nishino, Yoshinori

    2017-01-01

    X-ray science has greatly benefited from the progress in X-ray optics. Advances in the design and the manufacturing techniques of X-ray optics are key to the success of various microscopic and spectroscopic techniques practiced today. Here the generation of apodized X-ray illumination using a two-stage deformable Kirkpatrick-Baez mirror system is presented. Such apodized illumination is marked by the suppression of the side-lobe intensities of the focused beam. Thus generated apodized illumination was employed to improve the image quality in scanning X-ray fluorescence microscopy. Imaging of a non-isolated object by coherent X-ray diffractive imaging with apodized illumination in a non-scanning mode is also presented.

  10. Improved quantification of collagen anisotropy with polarization-resolved second harmonic generation microscopy.

    Science.gov (United States)

    Hristu, Radu; Stanciu, Stefan G; Tranca, Denis E; Stanciu, George A

    2017-09-01

    Imaging tissue samples by polarization-resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label-free manner. Polarization-resolved second harmonic generation microscopy goes beyond simple intensity-based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization-resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas. SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Sub-micron imaging of buried integrated circuit structures using scanning confocal electron microscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Frigo, S. P.; Levine, Z.; Zaluzec, N. J.; Materials Science Division; Northern Arizona Univ.; NIST

    2002-09-09

    Two-dimensional images of model integrated circuit components were collected using the technique of scanning confocal electron microscopy. For structures embedded about 5 {mu}m below the surface of a silicon oxide dielectric, a lateral resolution of 76{+-}9 nm was measured. Elemental mapping via x-ray emission spectrometry is demonstrated. A parallax analysis of images taken for various tilt angles to the electron beam allowed determination of the spacing between two wiring planes. The results show that scanning confocal electron microscopy is capable of probing buried structures at resolutions that will be necessary for the inspection of next-generation integrated circuit technology.

  12. Integrated structural and functional optical imaging combining spectral-domain optical coherence and multiphoton microscopy

    CERN Document Server

    Vinegoni, C; Luo, W; Marks, D L; Ralston, T; Tan, W

    2005-01-01

    An integrated microscope that combines different optical techniques for simultaneous imaging is demonstrated. The microscope enables spectral-domain optical coherence microscopy based on optical backscatter, and multi-photon microscopy for the detection of two-photon fluorescence and second harmonic generation signals. The unique configuration of this integrated microscope allows for the simultaneous acquisition of both anatomical (structural) and functional imaging information with particular emphasis for applications in the fields of tissue engineering and cell biology. In addition, the contemporary analysis of the spectroscopic features can enhance contrast by differentiating among different tissue components.

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

    Directory of Open Access Journals (Sweden)

    Yajing Shen

    2015-12-01

    Full Text Available 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.

  14. Photon budget analysis for fluorescence lifetime imaging microscopy

    NARCIS (Netherlands)

    Zhao, Q.; Young, I.T.; De Jong, J.G.S.

    2011-01-01

    We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon “budget.” These

  15. Imaging of RNA in situ hybridization by atomic force microscopy

    NARCIS (Netherlands)

    Kalle, W.H.J.; Macville, M.V.E.; van de Corput, M.P.C.; de Grooth, B.G.; Tanke, H.J.; Raap, A.K.

    In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Intermolecular Contrast in Atomic Force Microscopy Images without Intermolecular Bonds

    NARCIS (Netherlands)

    Hämäläinen, Sampsa K.; van der Heijden, N.J. (Nadine); van der Lit, Joost; den Hartog, Stephan; Liljeroth, Peter; Swart, Ingmar

    2014-01-01

    Intermolecular features in atomic force microscopy images of organic molecules have been ascribed to intermolecular bonds. A recent theoretical study [P. Hapala et al., Phys. Rev. B 90, 085421 (2014)] showed that these features can also be explained by the flexibility of molecule-terminated tips. We

  18. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1998-01-01

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

  19. Imaging and manipulation of single viruses by atomic force microscopy

    NARCIS (Netherlands)

    Baclayon, M.; Wuite, G. J. L.; Roos, W. H.

    2010-01-01

    The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these requirem

  20. Imaging and manipulation of single viruses by atomic force microscopy

    NARCIS (Netherlands)

    Baclayon, M.; Wuite, G. J. L.; Roos, W. H.

    2010-01-01

    The recent developments in virus research and the application of functional viral particles in nanotechnology and medicine rely on sophisticated imaging and manipulation techniques at nanometre resolution in liquid, air and vacuum. Atomic force microscopy (AFM) is a tool that combines these requirem

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-15

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

  5. Synergizing superresolution optical fluctuation imaging with single molecule localization microscopy

    CERN Document Server

    Schidorsky, Shachar; Razvag, Yair; Golan, Yonatan; Weiss, Shimon; Sherman, Eilon

    2016-01-01

    Single molecule localization microscopy (SMLM) techniques enable imaging biological samples well beyond the diffraction limit of light, but they vary significantly in their spatial and temporal resolutions. High-order statistical analysis of temporal fluctuations as in superresolution optical fluctuation imaging (SOFI) also enable imaging beyond diffraction limit, but usually at a lower resolution as compared to SMLM. Since the same data format is acquired for both methods, their algorithms can be applied to the same data set, and thus may be combined synergistically to improve overall imaging performance. Here, we find that SOFI converges much faster than SMLM, provides additive information to SMLM, and can efficiently reject background. We then show how SOFI-assisted SMLM imaging can improve SMLM image reconstruction by rejecting common sources of background, especially under low signal-to-background conditions. The performance of our approach was evaluated using a realistic simulation of fluorescence imagi...

  6. Quantification of photoacoustic microscopy images for ovarian cancer detection

    Science.gov (United States)

    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.

  7. Imaging rat esophagus using combination of reflectance confocal and multiphoton microscopy

    Science.gov (United States)

    Zhuo, S. M.; Chen, J. X.; Jiang, X. S.; Lu, K. C.; Xie, S. S.

    2008-08-01

    We combine reflectance confocal microscopy (RCM) with multiphoton microscopy (MPM) to image rat esophagus. The two imaging modalities allow detection of layered-resolved complementary information from esophagus. In the keratinizing layer, the keratinocytes boundaries can be characterized by RCM, while the keratinocytes cytoplasm (keratin) can be further imaged by multiphoton autofluorescence signal. In the epithelium, the epithelial cellular boundaries and nucleus can be detected by RCM, and MPM can be used for imaging epithelial cell cytoplasm and monitoring metabolic state of epithelium. In the stroma, multiphoton autofluorescence signal is used to image elastin and second harmonic generation signal is utilized to detect collagen, while RCM is used to determine the optical property of stroma. Overall, these results suggest that the combination of RCM and MPM has potential to provide more important and comprehensive information for early diagnosis of esophageal cancer.

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

    Energy Technology Data Exchange (ETDEWEB)

    Stead, A.D.; Ford, T.W. [Univ. of London, Surrey (United Kingdom); Judge, J. [Unilever plc, Sharnbrook (United Kingdom)] [and others

    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.

  9. Divided-aperture differential confocal fast-imaging microscopy

    Science.gov (United States)

    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.

  10. Quantitative second-harmonic generation microscopy in collagen

    Science.gov (United States)

    Stoller, Patrick; Celliers, Peter M.; Reiser, Karen M.; Rubenchik, Alexander M.

    2003-09-01

    The second-harmonic signal in collagen, even in highly organized samples such as rat tail tendon fascicles, varies significantly with position. Previous studies suggest that this variability may be due to the parallel and antiparallel orientation of neighboring collagen fibrils. We applied high-resolution second-harmonic generation microscopy to confirm this hypothesis. Studies in which the focal spot diameter was varied from ~1 to ~6 μm strongly suggest that regions in which collagen fibrils have the same orientation in rat tail tendon are likely to be less than ~1 μm in diameter. These measurements required accurate determination of the focal spot size achieved by use of different microscope objectives; we developed a technique that uses second-harmonic generation in a quartz reference to measure the focal spot diameter directly. We also used the quartz reference to determine a lower limit (dXXX > 0.4 pm/V) for the magnitude of the second-order nonlinear susceptibility in collagen.

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

    Science.gov (United States)

    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.

  12. A femtosecond Raman generator for long wavelength two-photon and third harmonic generation imaging

    Science.gov (United States)

    Trägârdh, J.; Schniete, J.; Parsons, M.; McConnell, G.

    2016-12-01

    We demonstrate a femtosecond single pass Raman generator based on an YVO4 crystal pumped by a high energy fiber laser at a wavelength of 1064 nm and a repetition rate of 1 MHz. The Raman generator shifts the pump wavelength to 1175 nm, in a broadband spectrum, making it suitable for multi-photon microscopy. We use the Raman generator for third harmonic generation imaging of live plant specimens as well as for two-photon fluorescence imaging of red fluorescent protein expressing HeLa cells. We demonstrate that the photo-damage to a live specimen is low.

  13. Low energy electron point source microscopy: beyond imaging.

    Science.gov (United States)

    Beyer, André; Gölzhäuser, Armin

    2010-09-01

    Low energy electron point source (LEEPS) microscopy has the capability to record in-line holograms at very high magnifications with a fairly simple set-up. After the holograms are numerically reconstructed, structural features with the size of about 2 nm can be resolved. The achievement of an even higher resolution has been predicted. However, a number of obstacles are known to impede the realization of this goal, for example the presence of electric fields around the imaged object, electrostatic charging or radiation induced processes. This topical review gives an overview of the achievements as well as the difficulties in the efforts to shift the resolution limit of LEEPS microscopy towards the atomic level. A special emphasis is laid on the high sensitivity of low energy electrons to electrical fields, which limits the structural determination of the imaged objects. On the other hand, the investigation of the electrical field around objects of known structure is very useful for other tasks and LEEPS microscopy can be extended beyond the task of imaging. The determination of the electrical resistance of individual nanowires can be achieved by a proper analysis of the corresponding LEEPS micrographs. This conductivity imaging may be a very useful application for LEEPS microscopes.

  14. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Science.gov (United States)

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094

  15. Second harmonic generation microscopy differentiates collagen type I and type III in COPD

    Science.gov (United States)

    Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

    2012-03-01

    The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

  16. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Directory of Open Access Journals (Sweden)

    Nohra E. Beltran

    2013-01-01

    Full Text Available The gastric mucosa ischemic tissular damage plays an important role in critical care patients’ outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine. The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10% for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (. Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

  17. Second harmonic generation microscopy is a novel technique for differential diagnosis of breast fibroepithelial lesions.

    Science.gov (United States)

    Tan, Wai Jin; Yan, Jie; Xu, Shuoyu; Thike, Aye Aye; Bay, Boon Huat; Yu, Hanry; Tan, Min-Han; Tan, Puay Hoon

    2015-12-01

    Breast fibroepithelial lesions, including fibroadenomas and phyllodes tumours, are commonly encountered in clinical practice. As histological differences between these two related entities may be subtle, resulting in a challenging differential diagnosis, pathological techniques to assist the differential diagnosis of these two entities are of high interest. An accurate diagnosis at biopsy is important given corresponding implications for clinical decision-making including surgical extent and monitoring. Second harmonic generation (SHG) microscopy is a recently developed optical imaging technique capable of robust, powerful and unbiased label-free direct detection of collagen fibril structure in tissue without the use of antibodies. We constructed tissue microarrays emulating limited materials on biopsy to investigate quantitative collagen signal in fibroepithelial lesions using SHG microscopy. Archived formalin-fixed paraffin-embedded materials of 47 fibroepithelial lesions (14 fibroadenomas and 33 phyllodes tumours) were evaluated. Higher collagen signal on SHG microscopy was observed in fibroadenomas than phyllodes tumours on SHG imaging (pmicroscopy for fibroadenoma classification was 71.4% and 84.4%, respectively. To corroborate these findings, we performed immunohistochemistry on tissue array sections using collagen I and III primary antibodies. Both collagen I and III immunohistochemical expressions were also significantly higher in fibroadenomas than in phyllodes tumours (pmicroscopy is a novel imaging approach that can aid the differential diagnosis of fibroepithelial lesions.

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

    Science.gov (United States)

    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.

  19. Volume scanning electron microscopy for imaging biological ultrastructure.

    Science.gov (United States)

    Titze, Benjamin; Genoud, Christel

    2016-11-01

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

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

    CERN Document Server

    Kurvits, Jonathan A; Zia, Rashid

    2015-01-01

    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, which 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 ide...

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

    Science.gov (United States)

    Jones, Chris G

    2012-01-01

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

  2. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1997-01-01

    Transmission Electron Microscopy presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray micronanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fourth edition includes discussion of recent progress, especially in the area of Schottky emission guns, convergent-beam electron diffraction, electron tomography, holography and the high resolution of crystal lattices.

  3. Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy

    Science.gov (United States)

    Lin, Sung-Jan; Hsiao, Chih-Yuan; Sun, Yen; Lo, Wen; Lin, Wei-Chou; Jan, Gwo-Jen; Jee, Shiou-Hwa; Dong, Chen-Yuan

    2005-03-01

    The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25°-60°C. We find that the SHG signal decreases rapidly starting at 45°C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57°C and worsens with increasing temperature. At 57°C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60°C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.

  4. Hybrid Imaging for Extended Depth of Field Microscopy

    Science.gov (United States)

    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.

  5. General Purpose Segmentation for Microorganisms in Microscopy Images

    DEFF Research Database (Denmark)

    Jensen, Sebastian H. Nesgaard; Moeslund, Thomas B.; Rankl, Christian

    2014-01-01

    In this paper, we propose an approach for achieving generalized segmentation of microorganisms in mi- croscopy images. It employs a pixel-wise classification strategy based on local features. Multilayer percep- trons are utilized for classification of the local features and is trained for each...... specific segmentation problem using supervised learning. This approach was tested on five different segmentation problems in bright field, differential interference contrast, fluorescence and laser confocal scanning microscopy. In all instance good results were achieved with the segmentation quality...

  6. In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy

    Science.gov (United States)

    Tanaka, Ryosuke; Fukushima, Shu-ichiro; Sasaki, Kunihiko; Tanaka, Yuji; Murota, Hiroyuki; Matsumoto, Takeshi; Araki, Tsutomu; Yasui, Takeshi

    2013-06-01

    Optical assessment of skin burns is possible with second-harmonic-generation (SHG) microscopy due to its high sensitivity to thermal denaturation of collagen molecules. In contrast to previous studies that were performed using excised tissue specimens ex vivo, in vivo observation of dermal collagen fibers in living rat burn models with SHG microscopy is demonstrated. Changes in signal vanishing patterns in the SHG images are confirmed to be dependent on the burn degree. Comparison of the SHG images with Masson's trichrome-stained images indicated that the observed patterns were caused by the coexistence of molten and fibrous structures of dermal collagen fibers. Furthermore, a quantitative parameter for burn assessment based on the depth profile of the mean SHG intensity across the entire SHG image is proposed. These results and discussions imply a potential of SHG microscopy as a minimally invasive, highly quantitative tool for skin burn assessment.

  7. Confocal supercritical angle fluorescence microscopy for cell membrane imaging

    CERN Document Server

    Sivankutty, Siddharth; Mayet, Céline; Dupuis, Guillaume; Fort, Emmanuel; Lévêque-Fort, Sandrine

    2013-01-01

    We demonstrate sub-wavelength sectioning on biological samples with a conventional confocal microscope. This optical sectioning is achieved by the phenomenon of supercritical angle fuorescence, wherein only a fluorophore next to the interface of a refractive index discontinuity can emit propagating components of radiation into the so-called forbidden angles. The simplicity of this technique allows it to be integrated with a high numerical aperture confocal scanning microscope by only a simple modi?cation on the detection channel. Confocal-SAF microscopy would be a powerful tool to achieve high resolution surface imaging, especially for membrane imaging in biological samples

  8. Neuron Segmentation in Electron Microscopy Images Using Partial Differential Equations.

    Science.gov (United States)

    Jones, Cory; Sayedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2013-01-01

    In connectomics, neuroscientists seek to identify the synaptic connections between neurons. Segmentation of cell membranes using supervised learning algorithms on electron microscopy images of brain tissue is often done to assist in this effort. Here we present a partial differential equation with a novel growth term to improve the results of a supervised learning algorithm. We also introduce a new method for representing the resulting image that allows for a more dynamic thresholding to further improve the result. Using these two processes we are able to close small to medium sized gaps in the cell membrane detection and improve the Rand error by as much as 9% over the initial supervised segmentation.

  9. Nanoscale optical properties of metal nanoparticles probed by Second Harmonic Generation microscopy.

    Science.gov (United States)

    Shen, Hong; Nguyen, Ngoc; Gachet, David; Maillard, Vincent; Toury, Timothée; Brasselet, Sophie

    2013-05-20

    We report spatial and vectorial imaging of local fields' confinement properties in metal nanoparticles with branched shapes, using Second Harmonic Generation (SHG) microscopy. Taking advantage of the coherent nature of this nonlinear process, the technique provides a direct evidence of the coupling between the excitation polarization and both localization and polarization specificities of local fields at the sub-diffraction scale. These combined features, which are governed by the nanoparticles' symmetry, are not accessible using other contrasts such as linear optical techniques or two-photon luminescence.

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

    Science.gov (United States)

    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.

  11. Imaging carious dental tissues with multiphoton fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Lin, Po-Yen; Lyu, Hong-Chou; Hsu, Chin-Ying Stephen; Chang, Chia-Seng; Kao, Fu-Jen

    2011-01-01

    In this study, multiphoton excitation was utilized to image normal and carious dental tissues noninvasively. Unique structures in dental tissues were identified using the available multimodality (second harmonic, autofluorescence, and fluorescence lifetime analysis) without labeling. The collagen in dentin exhibits a strong second harmonic response. Both dentin and enamel emit strong autofluorescence that reveals in detail morphological features (such as dentinal tubules and enamel rods) and, despite their very similar spectral profiles, can be differentiated by lifetime analysis. Specifically, the carious dental tissue exhibits a greatly reduced autofluorescence lifetime, which result is consistent with the degree of demineralization, determined by micro-computed tomography. Our findings suggest that two-photon excited fluorescence lifetime imaging may be a promising tool for diagnosing and monitoring dental caries. PMID:21326645

  12. Canning plasmonic microscopy by image reconstruction from the Fourier space

    CERN Document Server

    Mollet, O; Drezet, A

    2014-01-01

    We demonstrate a simple scheme for high-resolution imaging of nanoplasmonic structures that basically removes most of the resolution limiting allowed light usually transmitted to the far field. This is achieved by implementing a Fourier lens in a near-field scanning optical microscope (NSOM) operating in the leakage-radiation microscopy (LRM) mode. The method consists of reconstructing optical images solely from the plasmonic `forbidden' light collected in the Fourier space. It is demonstrated by using a point-like nanodiamond-based tip that illuminates a thin gold film patterned with a sub-wavelength annular slit. The reconstructed image of the slit shows a spatial resolution enhanced by a factor $\\simeq 4$ compared to NSOM images acquired directly in the real space.

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

    Science.gov (United States)

    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

  14. In vivo optical virtual biopsy of human oral mucosa with harmonic generation microscopy

    Science.gov (United States)

    Tsai, Ming-Rung; Chen, Szu-Yu; Shieh, Dar-Bin; Lou, Pei-Jen; Sun, Chi-Kuang

    2011-01-01

    Recent clinical studies on human skin indicated that in vivo multi-harmonic generation microscopy (HGM) can achieve sub-micron resolution for histopathological analysis with a high penetration depth and leave no energy or photodamages in the interacted tissues. It is thus highly desired to apply HGM for in vivo mucosa histopathological diagnosis. In this paper, the first in vivo optical virtual biopsy of human oral mucosa by using epi-HGM is demonstrated. We modified an upright microscope to rotate the angle of objective for in vivo observation. Our clinical study reveals the capability of HGM to in vivo image cell distributions in human oral mucosa, including epithelium and lamina propria with a high penetration depth greater than 280 μm and a high spatial resolution better than 500 nm. We also found that the third-harmonic-generation (THG) contrast on nucleus depends strongly on its thicknesses, in agreement with a numerical simulation. Besides, 4% acetic acid was found to be able to enhance the THG contrast of nucleus in oral mucosa, while such enhancement was found to decay due to the metabolic clearance of the contrast enhancer by the oral mucosa. Our clinical study indicated that, the combined epi-THG and epi-second-harmonic-generation (SHG) microscopy is a promising imaging tool for in vivo noninvasive optical virtual biopsy and disease diagnosis in human mucosa. PMID:21833368

  15. Virtual biopsy of rat tympanic membrane using higher harmonic generation microscopy

    Science.gov (United States)

    Lee, Wen-Jeng; Lee, Chia-Fone; Chen, Szu-Yu; Chen, Yuh-Shyang; Sun, Chi-Kuang

    2010-07-01

    Multiharmonic optical microscopy has been widely applied in biomedical research due to its unique capability to perform noninvasive studies of biomaterials. In this study, virtual biopsy based on back-propagating multiple optical harmonics, combining second and third harmonics, is applied in unfixed rat tympanic membrane. We show that third harmonic generation can provide morphologic information on the epithelial layers of rat tympanic membrane as well as radial collagen fibers in middle fibrous layers, and that second harmonic generation can provide information on both radial and circular collagen fibers in middle fibrous layers. Through third harmonic generation, the capillary and red blood cells in the middle fibrous layer are also noted. Additionally, the 3-D relationship to adjacent bony structures and spatial variations in thickness and curvature are obtained. Our study demonstrates the feasibility of using a noninvasive optical imaging system for comprehensive evaluation of the tympanic membrane.

  16. Second harmonic generation polarization microscopy with tightly focused linearly and radially polarized beams

    Science.gov (United States)

    Yew, E. Y. S.; Sheppard, C. J. R.

    2007-07-01

    Second harmonic generation microscopy was conducted on rat-tail tendons with linearly and radially polarized beams. Transverse and axial field components were generated in the focal region through tight focusing of linearly and radially polarized. It was found that the generated SHG signals could not be qualitatively explained with a scalar approximation to the electric field at the focus. Only by accounting for the interactions of the axial and transverse components of the electric field interacting through the nonlinear susceptibility χ(2) tensor could the SHG images be explained. For the case of collagen we find that the SHG signal varies as a function of the analyzer angle with a cos2 or sin2 dependency for linearly polarized beams. For tightly focused radially polarized beams we find that the output SHG is radially polarized after collimation and is independent of the analyzer angle.

  17. Combined third-harmonic generation and four-wave mixing microscopy of tissues and embryos.

    Science.gov (United States)

    Mahou, Pierre; Olivier, Nicolas; Labroille, Guillaume; Duloquin, Louise; Sintes, Jean-Marc; Peyriéras, Nadine; Legouis, Renaud; Débarre, Delphine; Beaurepaire, Emmanuel

    2011-10-01

    Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ((3)). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ((3))(-3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ((3))(-2ω(1) + ω(2);ω(1), -ω(2), ω(1)). We show that FWM images provide χ((3)) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.

  18. High resolution surface plasmon microscopy for cell imaging

    Science.gov (United States)

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

    2010-04-01

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

  19. Registration and 3D visualization of large microscopy images

    Science.gov (United States)

    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.

  20. Coherent imaging with incoherent light in digital holographic microscopy

    Science.gov (United States)

    Chmelik, Radim

    2012-01-01

    Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

  1. Acoustic and photoacoustic microscopy imaging of single leukocytes

    Science.gov (United States)

    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.

  2. Label-free determination of hemodynamic parameters in the microcirculaton with third harmonic generation microscopy.

    Directory of Open Access Journals (Sweden)

    Steffen Dietzel

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

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

    CERN Document Server

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

    2016-01-01

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

  4. Compressive Fluorescence Microscopy for Biological and Hyperspectral Imaging

    CERN Document Server

    Studer, Vincent; Chahid, Makhlad; Moussavi, Hamed; Candes, Emmanuel; Dahan, Maxime

    2012-01-01

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

  5. Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

    OpenAIRE

    Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; GAO, BRUCE Z.

    2014-01-01

    Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na ...

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

    Science.gov (United States)

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

    2013-06-01

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

  7. Directional bilateral filters for smoothing fluorescence microscopy images

    Directory of Open Access Journals (Sweden)

    Manasij Venkatesh

    2015-08-01

    Full Text Available 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.

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

    Science.gov (United States)

    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. High resolution magnetic imaging: MicroSQUID Force Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hasselbach, K; Ladam, C; Dolocan, V O; Hykel, D; Crozes, T [Institut Neel, CNRS et Universite Joseph Fourier, BP 166, F-38042 Grenoble Cedex 9 (France); Schuster, K [Institut de RadioAstronomie Millimetrique 300 rue de la Piscine, Domaine Universitaire F-38406 Saint Martin d' Heres (France); Mailly, D [Laboratoire de Photonique et de Nanostructures, CNRS, Site Alcatel de Marcoussis Route de Nozay F-91460 Marcoussis (France)], E-mail: klaus.hasselbach@grenoble.cnrs.fr

    2008-02-01

    Magnetic imaging at the micrometer scale with high sensitivity is a challenge difficult to be met. Magnetic force microscopy has a very high spatial resolution but is limited in magnetic resolution. Hall probe microscopy is very powerful but sensor fabrication at the one micron scale is difficult and effects due to discreteness of charge appear in the form of significant 1/f noise. SQUID microscopy is very powerful, having high magnetic resolution, but spatial resolution is usually of the order of 10 {mu}m. The difficulties lay mostly in an efficient way to couple flux to the sensor. The only way to improve spatial resolution is to place the probe close to the very edge of the support, thus maximising coupling and spatial resolution. If there has been found a way to bring close the tip, there must be also found a reliable a way to maintain distance during scanning. We want to present recent improvements on scanning microsquid microscopy: Namely the improved fabrication of microSQUID tips using silicon micro machining and the precise positioning of the micrometer diameter microSQUID loop by electron beam lithography. The microSQUID is a microbridge DC SQUID, with two opposite microbridges. The constrictions are patterned by high-resolution e-beam lithography and have a width of 20 nm and a length of about 100 nm. The distance control during scanning is obtained by integrating the microSQUID sensor with a piezoelectric tuning fork acting as a force sensor allowing to control height and even topographic imaging. The detector is placed in a custom built near field microscope and the sample temperature can be varied between 0.1 Kelvin and 10 K. The microscope is used to study magnetic flux structures in unconventional superconductors and will be used to observe thermal domains in superconducting detectors in the voltage state.

  10. Confocal Imaging of Biological Tissues Using Second Harmonic Generation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, B-M.; Stoller, P.; Reiser, K.; Eichler, J.; Yan, M.; Rubenchik, A.; Da Silva, L.

    2000-03-06

    A confocal microscopy imaging system was devised to selectively detect Second harmonic signals generated by biological tissues. Several types of biological tissues were examined using this imaging system, including human teeth, bovine blood vessels, and chicken skin. All these tissues generated strong second harmonic signals. There is considerable evidence that the source of these signals in tissue is collagen. Collagen, the predominant component of most tissues, is known to have second order nonlinear susceptibility. This technique may have diagnostic usefulness in pathophysiological conditions characterized by changes in collagen structure including malignant transformation of nevi, progression of diabetic complications, and abnormalities in wound healing.

  11. Scanning electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1985-01-01

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

  12. Next-generation biomarkers based on 100-parameter functional super-resolution microscopy TIS.

    Science.gov (United States)

    Schubert, Walter; Gieseler, Anne; Krusche, Andreas; Serocka, Peter; Hillert, Reyk

    2012-06-15

    Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the power of combinatorial molecular discrimination (PCMD) is at least 2(100) per subcellular data point. It provides the dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies controlling protein network topology and function in the one cell or tissue section. Here we review the technology and findings showing that functional protein networks of the cell surface in different cancers encompass the same hierarchical and spatial coding principle, but express cancer-specific toponome codes within that scheme (referred to as TIS codes). Findings suggest that TIS codes, extracted from large-scale toponome data, have the potential to be next-generation biomarkers because of their cell type and disease specificity. This is functionally substantiated by the observation that blocking toponome-specific lead proteins results in disassembly of molecular networks and loss of function.

  13. Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

    Science.gov (United States)

    Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

    2014-05-01

    Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

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

    Science.gov (United States)

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

    2014-11-01

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

  15. Polarization second harmonic generation microscopy provides quantitative enhanced molecular specificity for tissue diagnostics.

    Science.gov (United States)

    Kumar, Rajesh; Grønhaug, Kirsten M; Romijn, Elisabeth I; Finnøy, Andreas; Davies, Catharina L; Drogset, Jon O; Lilledahl, Magnus B

    2015-09-01

    Due to specific structural organization at the molecular level, several biomolecules (e.g., collagen, myosin etc.) which are strong generators of second harmonic generation (SHG) signals, exhibit unique responses depending on the polarization of the excitation light. By using the polarization second harmonic generation (p-SHG) technique, the values of the second order susceptibility components can be used to differentiate the types of molecule, which cannot be done by the use of a standard SHG intensity image. In this report we discuss how to implement p-SHG on a commercial multiphoton microscope and overcome potential artifacts in susceptibility (χ) image. Furthermore we explore the potential of p-SHG microscopy by applying the technique to different types of tissue in order to determine corresponding reference values of the ratio of second-order χ tensor elements. These values may be used as a bio-marker to detect any structural alterations in pathological tissue for diagnostic purposes. The SHG intensity image (red) in (a) shows the distribution of collagen fibers in ovary tissue but cannot determine the type of collagen fiber. However, the histogram distribution (b) for the values of the χ tensor element ratio can be used to quantitatively identify the types of collagen fibers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Automated cardiac sarcomere analysis from second harmonic generation images

    Science.gov (United States)

    Garcia-Canadilla, Patricia; Gonzalez-Tendero, Anna; Iruretagoyena, Igor; Crispi, Fatima; Torre, Iratxe; Amat-Roldan, Ivan; Bijnens, Bart H.; Gratacos, Eduard

    2014-05-01

    Automatic quantification of cardiac muscle properties in tissue sections might provide important information related to different types of diseases. Second harmonic generation (SHG) imaging provides a stain-free microscopy approach to image cardiac fibers that, combined with our methodology of the automated measurement of the ultrastructure of muscle fibers, computes a reliable set of quantitative image features (sarcomere length, A-band length, thick-thin interaction length, and fiber orientation). We evaluated the performance of our methodology in computer-generated muscle fibers modeling some artifacts that are present during the image acquisition. Then, we also evaluated it by comparing it to manual measurements in SHG images from cardiac tissue of fetal and adult rabbits. The results showed a good performance of our methodology at high signal-to-noise ratio of 20 dB. We conclude that our automated measurements enable reliable characterization of cardiac fiber tissues to systematically study cardiac tissue in a wide range of conditions.

  17. Enhanced CellClassifier: a multi-class classification tool for microscopy images

    Directory of Open Access Journals (Sweden)

    Horvath Peter

    2010-01-01

    Full Text Available Abstract Background Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories. Results We have developed a tool, Enhanced CellClassifier, which circumvents this obstacle. Enhanced CellClassifier starts from images analyzed by CellProfiler, and allows multi-class classification using a Support Vector Machine algorithm. Training of objects can be done by clicking directly "on the microscopy image" in several intuitive training modes. Many routine tasks like out-of focus exclusion and well summary are also supported. Classification results can be integrated with other object measurements including inter-object relationships. This makes a detailed interpretation of the image possible, allowing the differentiation of many complex phenotypes. For the generation of the output, image, well and plate data are dynamically extracted and summarized. The output can be generated as graphs, Excel-files, images with projections of the final analysis and exported as variables. Conclusion Here we describe Enhanced CellClassifier which allows multiple class classification, elucidating complex phenotypes. Our tool is designed for the biologist who wants both, simple and flexible analysis of images without requiring programming skills. This should facilitate the implementation of automated high-content screening.

  18. Mueller matrix signature in advanced fluorescence microscopy imaging

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-03-01

    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.

  20. New generation handheld hyperspectral imager

    Science.gov (United States)

    Wu, Huawen (Owen); Li, Hui; Tang, Shengjun

    2016-10-01

    A miniaturized hyper-spectral imager is enabled with image sensor integrated with dispersing elements in a very compact form factor, removing the need for expensive, moving, bulky and complex optics that have been used in conventional hyper-spectral imagers for decades. The result is a handheld spectral imager that can be installed on miniature UAV drones or conveyor belts in production lines. Eventually, small handhelds can be adapted for use in outpatient medical clinics for point-of-care diagnostics and other in-field applications.

  1. Fluorescence lifetime imaging microscopy of nanodiamonds in vivo

    Science.gov (United States)

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

    2013-03-01

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

  2. Image Restoration Phase-Filtering Lateral Superresolution Confocal Microscopy

    Institute of Scientific and Technical Information of China (English)

    ZHAO Wei-Qian; QIU Li-Rong; CHEN Shan-Shan; FENG Zheng-De

    2006-01-01

    @@ Image restoration phase-filtering lateral superresolution confocal microscopy, a new approach, is proposed to achieve lateral superresolution using a confocal microscope. This approach uses a lateral superresolution pupil filter to preliminarily improve its lateral resolution and uses a single-image superresolution restoration technique based on a maximum likelihood estimate to further improve its lateral resolution. The new approach has the advantages of a low cost and the remarkable superresolution effect without excessive system complexity. Experiments indicate that the proposed approach can improve the lateral resolution of a confocal microscope from 0.3μm to less than 0.1 μm when λ = 632.8 nm and NA =0.85.

  3. Dynamic force microscopy for imaging of viruses under physiological conditions

    Directory of Open Access Journals (Sweden)

    Kienberger Ferry

    2004-01-01

    Full Text Available Dynamic force microscopy (DFM allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2 weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized.

  4. Characterization of tissue-engineered posterior corneas using second- and third-harmonic generation microscopy.

    Directory of Open Access Journals (Sweden)

    Louis Jay

    Full Text Available Three-dimensional tissues, such as the cornea, are now being engineered as substitutes for the rehabilitation of vision in patients with blinding corneal diseases. Engineering of tissues for translational purposes requires a non-invasive monitoring to control the quality of the resulting biomaterial. Unfortunately, most current methods still imply invasive steps, such as fixation and staining, to clearly observe the tissue-engineered cornea, a transparent tissue with weak natural contrast. Second- and third-harmonic generation imaging are well known to provide high-contrast, high spatial resolution images of such tissues, by taking advantage of the endogenous contrast agents of the tissue itself. In this article, we imaged tissue-engineered corneal substitutes using both harmonic microscopy and classic histopathology techniques. We demonstrate that second- and third-harmonic imaging can non-invasively provide important information regarding the quality and the integrity of these partial-thickness posterior corneal substitutes (observation of collagen network, fibroblasts and endothelial cells. These two nonlinear imaging modalities offer the new opportunity of monitoring the engineered corneas during the entire process of production.

  5. Characterization of tissue-engineered posterior corneas using second- and third-harmonic generation microscopy.

    Science.gov (United States)

    Jay, Louis; Bourget, Jean-Michel; Goyer, Benjamin; Singh, Kanwarpal; Brunette, Isabelle; Ozaki, Tsuneyuki; Proulx, Stéphanie

    2015-01-01

    Three-dimensional tissues, such as the cornea, are now being engineered as substitutes for the rehabilitation of vision in patients with blinding corneal diseases. Engineering of tissues for translational purposes requires a non-invasive monitoring to control the quality of the resulting biomaterial. Unfortunately, most current methods still imply invasive steps, such as fixation and staining, to clearly observe the tissue-engineered cornea, a transparent tissue with weak natural contrast. Second- and third-harmonic generation imaging are well known to provide high-contrast, high spatial resolution images of such tissues, by taking advantage of the endogenous contrast agents of the tissue itself. In this article, we imaged tissue-engineered corneal substitutes using both harmonic microscopy and classic histopathology techniques. We demonstrate that second- and third-harmonic imaging can non-invasively provide important information regarding the quality and the integrity of these partial-thickness posterior corneal substitutes (observation of collagen network, fibroblasts and endothelial cells). These two nonlinear imaging modalities offer the new opportunity of monitoring the engineered corneas during the entire process of production.

  6. Real-time in vivo imaging collagen in lymphedematous skin using multiphoton microscopy.

    Science.gov (United States)

    Wu, Xiufeng; Zhuo, Shuangmu; Chen, Jianxin; Liu, Ningfei

    2011-01-01

    Changes of dermal collagen are characteristic for chronic lymphedema. To evaluate these changes, a real-time imaging based on two-photon excited fluorescence and second-harmonic generation was developed for investigating collagen of lymphedematous mouse and rat tail skin in vivo. Our findings showed that the technique could image the morphological changes and distribution of collagen in lymphedematous mouse and rat tail skin in vivo. More importantly, it may allow visualization of dynamic collagen alteration during the progression of lymphedema. Our findings demonstrated that multiphoton microscopy may have potential in a clinical setting as an in vivo diagnostic and monitoring system for therapy in lymphology.

  7. Photon flux requirements for EUV reticle imaging microscopy in the 22 and 16 nm nodes

    Energy Technology Data Exchange (ETDEWEB)

    Wintz, D.; Goldberg, K. A.; Mochi, I.; Huh, S.

    2010-03-12

    EUV-wavelength actinic microscopy yields detailed information about EUV mask patterns, architectures, defects, and the performance of defect repair strategies, without the complications of photoresist imaging. The measured aerial image intensity profiles provide valuable feedback to improve mask and lithography system modeling methods. In order to understand the photon-flux-dependent pattern measurement limits of EUV mask-imaging microscopy, we have investigated the effects of shot noise on aerial image linewidth measurements for lines in the 22 and 16-nm generations. Using a simple model of image formation near the resolution limit, we probe the influence of photon shot noise on the measured, apparent line roughness. With this methodology, we arrive at general flux density requirements independent of the specific EUV microscope configurations. Analytical and statistical analysis of aerial image simulations in the 22 and 16-nm generations reveal the trade-offs between photon energy density (controllable with exposure time), effective pixel dimension on the CCO (controlled by the microscope's magnification ratio), and image log slope (ILS). We find that shot-noise-induced linewidth roughness (LWR) varies imersely with the square root of the photon energy density, and is proportional to the imaging magnification ratio. While high magnification is necessary for adequate spatial resolution, for a given flux density, higher magnification ratios have diminishing benefits. With practical imaging parameters, we find that in order to achieve an LWR (3{sigma}) value of 5% of linewidth for dense, 88-nm mask features with 80% aerial image contrast and 13.5-nm effective pixel width (1000x magnification ratio), a peak photon flux of approximately 1400 photons per pixel per exposure is required.

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

    Science.gov (United States)

    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.

  9. Imaging domains in transmission electron microscopy (invited) (abstract)

    Science.gov (United States)

    Mishra, R. K.

    1987-04-01

    Magnetic domain walls and domains inside thin electron transparent specimens of ferromagnetic materials can be imaged using the Fresnel and Focault techniques in a transmission electron microscope. Combined with the diffraction, microstructural and microchemical capabilities of modern microscopes, Lorentz microscopy offers one of the most powerful tools to study structure-property relationships in magnetic materials. In addition, using this technique, it is possible to deduce the local magnetization distribution around inhomogeneities and complex Bloch and Néel walls. Lorentz images can be used to quantitatively measure domain wall thickness and estimate domain wall energy. With modified sample holders and pole pieces, one can study in situ domain wall motion and the interaction of domains with microstructural features such as second phases, grain boundaries, structural defects, etc. All these will be illustrated with examples of Lorentz images from soft and hard magnets with special emphasis on the Nd-Fe-B hard magnets. Finally, the limitations of the Lorentz imaging technique utilizing the deflected electron intensities will be outlined and a new technique which utilizes the phase changes in the electron beam as it passes through the material in a scanning transmission microscope will be reviewed.

  10. 3D imaging of neutron tracks using confocal microscopy

    Science.gov (United States)

    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

  11. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  12. Enhanced simulator software for image validation and interpretation for multimodal localization super-resolution fluorescence microscopy

    Science.gov (United States)

    Erdélyi, Miklós; Sinkó, József; Gajdos, Tamás.; Novák, Tibor

    2017-02-01

    Optical super-resolution techniques such as single molecule localization have become one of the most dynamically developed areas in optical microscopy. These techniques routinely provide images of fixed cells or tissues with sub-diffraction spatial resolution, and can even be applied for live cell imaging under appropriate circumstances. Localization techniques are based on the precise fitting of the point spread functions (PSF) to the measured images of stochastically excited, identical fluorescent molecules. These techniques require controlling the rate between the on, off and the bleached states, keeping the number of active fluorescent molecules at an optimum value, so their diffraction limited images can be detected separately both spatially and temporally. Because of the numerous (and sometimes unknown) parameters, the imaging system can only be handled stochastically. For example, the rotation of the dye molecules obscures the polarization dependent PSF shape, and only an averaged distribution - typically estimated by a Gaussian function - is observed. TestSTORM software was developed to generate image stacks for traditional localization microscopes, where localization meant the precise determination of the spatial position of the molecules. However, additional optical properties (polarization, spectra, etc.) of the emitted photons can be used for further monitoring the chemical and physical properties (viscosity, pH, etc.) of the local environment. The image stack generating program was upgraded by several new features, such as: multicolour, polarization dependent PSF, built-in 3D visualization, structured background. These features make the program an ideal tool for optimizing the imaging and sample preparation conditions.

  13. Identification by force modulation microscopy of nanoparticles generated in vacuum arcs Identification by force modulation microscopy of nanoparticles generated in vacuum arcs

    Directory of Open Access Journals (Sweden)

    M. Arroyave Franco

    2006-06-01

    Full Text Available An alternative method based on force modulation microscopy (FMM for identification of nanoparticles produced in the plasma generated by the cathode spots of vacuum arcs is presented. FMM technique is enabled for the detection of variations in the mechanical properties of a surface with high sensitiveness. Titanium nitride (TiN coatings deposited on oriented silicon by pulsed vacuum arc process have been analyzed. AFM (Atomic Force Microscopy and FMM images were simultaneously obtained, and in all cases it was possible to identify nanoparticle presence. Further X-ray Diffraction spectra of sample coating were taken. Existence of contaminant particles of 47 nanometers in diameter was reported.En este trabajo se presenta un método alternativo basado en microscopia de modulación de fuerza (FMM, para la identificación de nanogotas producidas en el plasma generado por los spots catódicos de los arcos en vacío. La técnica FMM esta habilitada para la detección de variaciones en las propiedades mecánicas de una superficie, con alta sensibilidad. Se han analizado recubrimientos de nitruro de titanio (TiN depositados sobre Silicio orientado por el proceso de arco en vacío pulsado. Se han obtenido simultáneamente imágenes de microscopia de fuerza atómica (AFM y de microscopia FMM mediante las cuales se ha podido identificar la presencia de nanogotas. Adicionalmente se han tomado espectros de difracción de rayos X (XRD de las muestras recubiertas. Se ha reportado la existencia de partículas contaminantes de 47 nanómetros de diámetro sobre los recubrimientos.

  14. Global error minimization in image mosaicing using graph connectivity and its applications in microscopy

    Directory of Open Access Journals (Sweden)

    Parmeshwar Khurd

    2011-01-01

    Full Text Available 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.

  15. Objective for EUV microscopy, EUV lithography, and x-ray imaging

    Science.gov (United States)

    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.

  16. Improved sampling and analysis of images in corneal confocal microscopy.

    Science.gov (United States)

    Schaldemose, E L; Fontain, F I; Karlsson, P; Nyengaard, J R

    2017-05-26

    Corneal confocal microscopy (CCM) is a noninvasive clinical method to analyse and quantify corneal nerve fibres in vivo. Although the CCM technique is in constant progress, there are methodological limitations in terms of sampling of images and objectivity of the nerve quantification. The aim of this study was to present a randomized sampling method of the CCM images and to develop an adjusted area-dependent image analysis. Furthermore, a manual nerve fibre analysis method was compared to a fully automated method. 23 idiopathic small-fibre neuropathy patients were investigated using CCM. Corneal nerve fibre length density (CNFL) and corneal nerve fibre branch density (CNBD) were determined in both a manual and automatic manner. Differences in CNFL and CNBD between (1) the randomized and the most common sampling method, (2) the adjusted and the unadjusted area and (3) the manual and automated quantification method were investigated. The CNFL values were significantly lower when using the randomized sampling method compared to the most common method (p = 0.01). There was not a statistical significant difference in the CNBD values between the randomized and the most common sampling method (p = 0.85). CNFL and CNBD values were increased when using the adjusted area compared to the standard area. Additionally, the study found a significant increase in the CNFL and CNBD values when using the manual method compared to the automatic method (p ≤ 0.001). The study demonstrated a significant difference in the CNFL values between the randomized and common sampling method indicating the importance of clear guidelines for the image sampling. The increase in CNFL and CNBD values when using the adjusted cornea area is not surprising. The observed increases in both CNFL and CNBD values when using the manual method of nerve quantification compared to the automatic method are consistent with earlier findings. This study underlines the importance of improving the analysis of the

  17. Quantitative differentiation of normal and scarred tissues using second-harmonic generation microscopy.

    Science.gov (United States)

    Yildirim, Murat; Quinn, Kyle P; Kobler, James B; Zeitels, Steven M; Georgakoudi, Irene; Ben-Yakar, Adela

    2016-11-01

    The aim of this study was to differentiate normal and scarred hamster cheek pouch samples by applying a quantitative image analysis technique for determining collagen fiber direction and density in second-harmonic generation microscopy images. This paper presents a collagen tissue analysis of scarred cheek pouches of four adult male Golden Syrian hamsters as an animal model for vocal fold scarring. One cheek pouch was scarred using an electrocautery unit and the other cheek was used as a control for each hamster. A home-built upright microscope and a compact ultrafast fiber laser were used to acquire depth resolved epi-collected second-harmonic generation images of collagen fibers. To quantify the average fiber direction and fiber density in each image, we applied two-dimensional Fourier analysis and intensity thresholding at five different locations for each control and scarred tissue sample, respectively. The resultant depth-resolved average fiber direction variance for scarred hamster cheek pouches (0.61 ± 0.03) was significantly lower (p tissue (0.73 ± 0.04), indicating increased fiber alignment within the scar. Depth-resolved average voxel density measurements indicated scarred tissues contained greater (p image analysis of both fiber alignment and density from depth-resolved second-harmonic generation images in epi-detection mode enabled the quantification of the increased collagen fiber deposition and alignment typically observed in fibrosis. The epi-detection geometry is the only viable method for in vivo imaging as well as imaging thick turbid tissues. These quantitative endpoints, clearly differentiating between control and scarred hamster cheek pouches, provide an objective means to characterize the extent of vocal fold scarring in vivo in preclinical and clinical research. In particular, this non-invasive method offers advantages for monitoring scar treatments in live animals and following the effects of scarring-related treatments such as

  18. Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

    Directory of Open Access Journals (Sweden)

    Sean C Warren

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

  19. Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy.

    Science.gov (United States)

    Bushby, Andrew J; P'ng, Kenneth M Y; Young, Robert D; Pinali, Christian; Knupp, Carlo; Quantock, Andrew J

    2011-06-01

    In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2000-09-01

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

  3. Imaging Photon Lattice States by Scanning Defect Microscopy

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Tada Yoshitaka

    2006-11-01

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

  5. Super-resolved multimodal multiphoton microscopy with spatial frequency-modulated imaging

    CERN Document Server

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

    2015-01-01

    Super-resolved 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 super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any contrast mechanism, and with single-pixel detection. We show multimodal super-resolved 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 has the potential to not only pro...

  6. In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy

    Science.gov (United States)

    Tsai, M.-R.; Chen, S.-Y.; Shieh, D.-B.; Lou, P.-J.; Sun, C.-K.

    2010-02-01

    Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for noninvasive in vivo diseases diagnosis.

  7. Analysis of second-harmonic generation microscopy under refractive index mismatch

    Institute of Scientific and Technical Information of China (English)

    Wang Xiang-Hui; Lin Lie; Zhang Yang

    2007-01-01

    On the basis of the vector diffraction theory and Green's function method, this paper investigates the effects of refractive index mismatch on second-harmonic generation (SHG) microscopy. The polarization distribution and SHG intensity are calculated as functions of the sample radius and probe depth. The numerical results show that refractive index mismatch can result in peak intensity degradation, increase secondary lobes and extension of secondharmonic polarization distribution. Because of the attenuation of polarization intensity, the detected SHG intensity significantly decreases with increasing probe depth, which can limit the imaging depth of SHG microcopy inside thick samples. Forward SHG intensity decays slowly than backward SHG, due to the combination of extension secondharmonic polarization distribution and strong dependency of forward SHG on sample radius.

  8. Directional and singular surface plasmon generation in chiral and achiral nanostructures demonstrated by Leakage Radiation Microscopy

    CERN Document Server

    Jiang, Quanbo; Berthel, Martin; Huant, Serge; Bellessa, Joel; Genet, Cyriaque; Drezet, Aurélien

    2016-01-01

    In this paper, we describe the implementation of leakage radiation microscopy (LRM) to probe the chirality of plasmonic nanostructures. We demonstrate experimentally spin-driven directional coupling as well as vortex generation of surface plasmon polaritons (SPPs) by nanostructures built with T-shaped and $\\Lambda$- shaped apertures. Using this far-field method, quantitative inspections, including directivity and extinction ratio measurements, are achieved via polarization analysis in both image and Fourier planes. To support our experimental findings, we develop an analytical model based on a multidipolar representation of $\\Lambda$- and T-shaped aperture plasmonic coupler allowing a theoretical explanation of both directionality and singular SPP formation. Furthermore, the roles of symmetry breaking and phases are emphasized in this work. This quantitative characterization of spin-orbit interactions paves the way for developing new directional couplers for subwavelength routing.

  9. Single-wavelength reflected confocal and multiphoton microscopy for tissue imaging

    Science.gov (United States)

    Chen, Wei-Liang; Chou, Chen-Kuan; Lin, Ming-Gu; Chen, Yang-Fang; Jee, Shiou-Hwa; Tan, Hsin-Yuan; Tsai, Tsung-Hua; Kim, Ki-Hean; Kim, Daekeun; So, Peter T. C.; Lin, Sung-Jan; Dong, Chen-Yuan

    2009-09-01

    Both reflected confocal and multiphoton microscopy can have clinical diagnostic applications. The successful combination of both modalities in tissue imaging enables unique image contrast to be achieved, especially if a single laser excitation wavelength is used. We apply this approach for skin and corneal imaging using the 780-nm output of a femtosecond, titanium-sapphire laser. We find that the near-IR, reflected confocal (RC) signal is useful in characterizing refractive index varying boundaries in bovine cornea and porcine skin, while the multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) intensities can be used to image cytoplasm and connective tissues (collagen), respectively. In addition, quantitative analysis shows that we are able to detect MAF from greater imaging depths than with the near-IR RC signal. Furthermore, by performing RC imaging at 488, 543, and 633 nm, we find that a longer wavelength leads to better image contrast for deeper imaging of the bovine cornea and porcine skin tissue. Finally, by varying power of the 780-nm source, we find that comparable RC image quality was achieved in the 2.7 to 10.7-mW range.

  10. Optical Imaging and Microscopy Techniques and Advanced Systems

    CERN Document Server

    Török, Peter

    2007-01-01

    This text on contemporary optical systems is intended for optical researchers and engineers, graduate students and optical microscopists in the biological and biomedical sciences. This second edition contains two completely new chapters. In addition most of the chapters from the first edition have been revised and updated. The book consists of three parts: The first discusses high-aperture optical systems, which form the backbone of optical microscopes. An example is a chapter new in the second edition on the emerging field of high numerical aperture diffractive lenses which seems to have particular promise in improving the correction of lenses. In this part particular attention is paid to optical data storage. The second part is on the use of non-linear optical techniques, including nonlinear optical excitation (total internal reflection fluorescence, second and third harmonic generation and two photon microscopy) and non-linear spectroscopy (CARS). The final part of the book presents miscellaneous technique...

  11. Fiber-based 1150-nm femtosecond laser source for the minimally invasive harmonic generation microscopy

    Science.gov (United States)

    Huang, Jing-Yu; Guo, Lun-Zhang; Wang, Jing-Zun; Li, Tse-Chung; Lee, Hsin-Jung; Chiu, Po-Kai; Peng, Lung-Han; Liu, Tzu-Ming

    2017-03-01

    Harmonic generation microscopy (HGM) has become one unique tool of optical virtual biopsy for the diagnosis of cancer and the in vivo cytometry of leukocytes. Without labeling, HGM can reveal the submicron features of tissues and cells in vivo. For deep imaging depth and minimal invasiveness, people commonly adopt 1100- to 1300-nm femtosecond laser sources. However, those lasers are typically based on bulky oscillators whose performances are sensitive to environmental conditions. We demonstrate a fiber-based 1150-nm femtosecond laser source, with 6.5-nJ pulse energy, 86-fs pulse width, and 11.25-MHz pulse repetition rate. It was obtained by a bismuth borate or magnesium-doped periodically poled lithium niobate (MgO:PPLN) mediated frequency doubling of the 2300-nm solitons, generated from an excitation of 1550-nm femtosecond pulses on a large mode area photonic crystal fiber. Combined with a home-built laser scanned microscope and a tailor-made frame grabber, we achieve a pulse-per-pixel HGM imaging in vivo at a 30-Hz frame rate. This integrated solution has the potential to be developed as a stable HGM system for routine clinical use.

  12. Imaging arterial cells, atherosclerosis, and restenosis by multimodal nonlinear optical microscopy

    Science.gov (United States)

    Wang, Han-Wei; Simianu, Vlad; Locker, Matthew J.; Sturek, Michael; Cheng, Ji-Xin

    2008-02-01

    By integrating sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on a coherent anti-Stokes Raman scattering (CARS) microscope platform, multimodal nonlinear optical (NLO) imaging of arteries and atherosclerotic lesions was demonstrated. CARS signals arising from CH II-rich membranes allowed visualization of endothelial cells and smooth muscle cells in a carotid artery. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are rich in CH II bonds in their cross-linking residues. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. The system is capable of identifying different atherosclerotic lesion stages with sub-cellular resolution. The stages of atherosclerosis, such as macrophage infiltration, lipid-laden foam cell accumulation, extracellular lipid distribution, fibrous tissue deposition, plaque establishment, and formation of other complicated lesions could be viewed by our multimodal CARS microscope. Collagen percentages in the region adjacent to coronary artery stents were resolved. High correlation between NLO and histology imaging evidenced the validity of the NLO imaging. The capability of imaging significant components of an arterial wall and distinctive stages of atherosclerosis in a label-free manner suggests the potential application of multimodal nonlinear optical microscopy to monitor the onset and progression of arterial diseases.

  13. A workflow for the automatic segmentation of organelles in electron microscopy image stacks.

    Science.gov (United States)

    Perez, Alex J; Seyedhosseini, Mojtaba; Deerinck, Thomas J; Bushong, Eric A; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H

    2014-01-01

    Electron microscopy (EM) facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson's and Alzheimer's diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM). Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime.

  14. Transmission electron microscopy physics of image formation and microanalysis

    CERN Document Server

    Reimer, Ludwig

    1984-01-01

    The aim of this book is to outline the physics of image formation, electron­ specimen interactions and image interpretation in transmission electron mic­ roscopy. The book evolved from lectures delivered at the University of Munster and is a revised version of the first part of my earlier book Elek­ tronenmikroskopische Untersuchungs- und Priiparationsmethoden, omitting the part which describes specimen-preparation methods. In the introductory chapter, the different types of electron microscope are compared, the various electron-specimen interactions and their applications are summarized and the most important aspects of high-resolution, analytical and high-voltage electron microscopy are discussed. The optics of electron lenses is discussed in Chapter 2 in order to bring out electron-lens properties that are important for an understanding of the function of an electron microscope. In Chapter 3, the wave optics of elec­ trons and the phase shifts by electrostatic and magnetic fields are introduced; Fresne...

  15. Observation of tendon repair in animal model using second-harmonic-generation microscopy

    Science.gov (United States)

    Hase, Eiji; Minamikawa, Takeo; Sato, Katsuya; Takahashi, Mitsuhiko; Yasui, Takashi

    2016-03-01

    Tendon rupture is a trauma difficult to recover the condition before injury. In previous researches, tensile test and staining method have been widely used to elucidate the mechanism of the repair process from the viewpoints of the mechanical property and the histological findings. However, since both methods are destructive and invasive, it is difficult to obtain both of them for the same sample. If both the mechanical property and the histological findings can be obtained from the same sample, one may obtain new findings regarding mechanisms of tendon repairing process. In this paper, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, deep penetration, and without additional staining. Since SHG light intensity sensitively reflects the structural maturity of collagen molecule and its aggregates, it will be a good indicator for the repairing degree of the ruptured tendon. From comparison of SHG images between the 4-weeks-repaired tendon and the sound tendon in the animal model, we confirmed that SHG light intensity of the repaired tendon was significantly lower than that of the sound tendon, indicating that the collagen structure in the repaired tendon is still immature. Furthermore, we performed both SHG imaging and the tensile test for the same sample, and confirmed a correlation between them. This result shows a potential of SHG light for an indicator of the histological and mechanical recovery of the ruptured tendon.

  16. Generating Stereoscopic Television Images With One Camera

    Science.gov (United States)

    Coan, Paul P.

    1996-01-01

    Straightforward technique for generating stereoscopic television images involves use of single television camera translated laterally between left- and right-eye positions. Camera acquires one of images (left- or right-eye image), and video signal from image delayed while camera translated to position where it acquires other image. Length of delay chosen so both images displayed simultaneously or as nearly simultaneously as necessary to obtain stereoscopic effect. Technique amenable to zooming in on small areas within broad scenes. Potential applications include three-dimensional viewing of geological features and meteorological events from spacecraft and aircraft, inspection of workpieces moving along conveyor belts, and aiding ground and water search-and-rescue operations. Also used to generate and display imagery for public education and general information, and possible for medical purposes.

  17. NI-78LABEL-FREE MULTIPHOTON MICROSCOPY: A NOVEL TOOL FOR THE IMAGING OF BRAIN TUMORS

    Science.gov (United States)

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

    2014-01-01

    Changes in tissue composition caused by brain tumor growth involve a series of complex biochemical alterations which can be imaged on unstained native tissue using multiphoton microscopy: We used coherent anti-Stokes Raman scattering (CARS) imaging that resonantly excites the symmetric stretching vibration of CH2 groups at 2850 cm−1 and visualizes lipid content in combination with imaging of endogenous two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to discern different types of tumors from normal tissue in unstained, native brain samples. Experimental brain tumors were induced in nude mice NMRI nu/nu (n = 25) by stereotactic implantation of glioblastoma (U87), melanoma (A375) and breast cancer (MCF-7) cell lines. Label-free multiphoton microscopy of brain cryosections provided exhaustive information of the tumor morphochemistry. The tumor border was defined with cellular resolution by a strong reduction of CARS signal intensity to 61% (glioblastoma), 71% (melanoma) and 68% (breast cancer). This reduction of lipid content within the tumor was confirmed by Raman spectroscopy. Micrometastases infiltrating normal tissue (size 50 - 200 µm) were identified in glioblastoma and melanoma. Additionally, multiphoton microscopy proved a reduction of CARS signal intensity in all human glioblastoma samples analyzed (to 72%, n = 6). Additionally, relevant SHG and TPEF signals were detected in human primary and secondary brain tumor samples and enabled to image variations in tumor associated vasculature, fibrosis, necrosis and nuclear size and density. All primary or secondary brain tumors investigated were characterized by a lower intensity of the CARS signal, therefore offering a simple tool for objective tumor detection and delineation. The combination of techniques allows retrieving a quantity of information on native unstained tissue which is comparable to H&E staining. Therefore, label-free multiphoton microscopy has the potential to become a

  18. Label-free imaging of rat spinal cords based on multiphoton microscopy

    Science.gov (United States)

    Liao, Chenxi; Wang, Zhenyu; Zhou, Linquan; Zhu, Xiaoqin; Liu, Wenge; Chen, Jianxin

    2016-10-01

    As an integral part of the central nervous system, the spinal cord is a communication cable between the body and the brain. It mainly contains neurons, glial cells, nerve fibers and fiber tracts. The recent development of the optical imaging technique allows high-resolution imaging of biological tissues with the great potential for non-invasively looking inside the body. In this work, we evaluate the imaging capacity of multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) for the cells and extracellular matrix in the spinal cord at molecular level. Rat spinal cord tissues were sectioned and imaged by MPM to demonstrate that MPM is able to show the microstructure including white matter, gray matter, ventral horns, dorsal horns, and axons based on the distinct intrinsic sources in each region of spinal cord. In the high-resolution and high-contrast MPM images, the cell profile can be clearly identified as dark shadows caused by nuclei and encircled by cytoplasm. The nerve fibers in white matter region emitted both SHG and TPEF signals. The multiphoton microscopic imaging technique proves to be a fast and effective tool for label-free imaging spinal cord tissues, based on endogenous signals in biological tissue. It has the potential to extend this optical technique to clinical study, where the rapid and damage-free imaging is needed.

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

    Science.gov (United States)

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

    2011-02-01

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

  20. Neural imaging in songbirds using fiber optic fluorescence microscopy

    Science.gov (United States)

    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.

  1. SISGR: Room Temperature Single-Molecule Detection and Imaging by Stimulated Emission Microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xiaoliang Sunney [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology

    2017-03-13

    specificity than absorption and fluorescence. Current sensitivity limit of SRS microscopy has not yet reached single molecule detection. We proposed to capitalize on our state-of-the-art SRS microscopy and develop near-resonance enhanced SRS for single molecule detection of carotenoids and heme proteins. The specific aims we pursued are: (1) building the next SRS generation microscope that utilizes near resonance enhancement to allow detection and imaging of single molecules with undetectable fluorescence, such as -carotene. (2) using near-resonance SRS as a contrast mechanism to study dye-sensitize semiconductor interface, elucidating the heterogeneous electron ejection kinetics with high spatial and temporal resolution. (3) studying the binding and unbinding of oxygen in single hemoglobin molecules in order to gain molecular level understanding of the long-standing issue of cooperativity. The new methods developed in the fund period of this grant have advanced the detection sensitivity in many aspects. Near-resonance SRS improved the signal by using shorter wavelengths for SRS microscopy. Frequency modulation and multi-color SRS target the reduction of background to improve the chemical specificity of SRS while maintaining the high imaging speed. Time-domain coherent Raman scattering microscopy targets to reduce the noise floor of coherent Raman microscopy. These methods have already demonstrated first-of-a-kind new applications in biology and medical research. However, we are still one order of magnitude away from single molecule limit. It is important to continue to improve the laser specification and develop new imaging methods to finally achieve label-free single molecule microscopy.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-07

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

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

    Science.gov (United States)

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

    2011-09-07

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

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

    Science.gov (United States)

    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.

  5. Snow crystal imaging using scanning electron microscopy: III. Glacier ice, snow and biota

    Science.gov (United States)

    Rango, A.; Wergin, W.P.; Erbe, E.F.; Josberger, E.G.

    2000-01-01

    Low-temperature scanning electron microscopy (SEM) was used to observe metamorphosed snow, glacial firn, and glacial ice obtained from South Cascade Glacier in Washington State, USA. Biotic samples consisting of algae (Chlamydomonas nivalis) and ice worms (a species of oligochaetes) were also collected and imaged. In the field, the snow and biological samples were mounted on copper plates, cooled in liquid nitrogen, and stored in dry shipping containers which maintain a temperature of -196??C. The firn and glacier ice samples were obtained by extracting horizontal ice cores, 8 mm in diameter, at different levels from larger standard glaciological (vertical) ice cores 7.5 cm in diameter. These samples were cooled in liquid nitrogen and placed in cryotubes, were stored in the same dry shipping container, and sent to the SEM facility. In the laboratory, the samples were sputter coated with platinum and imaged by a low-temperature SEM. To image the firn and glacier ice samples, the cores were fractured in liquid nitrogen, attached to a specimen holder, and then imaged. While light microscope images of snow and ice are difficult to interpret because of internal reflection and refraction, the SEM images provide a clear and unique view of the surface of the samples because they are generated from electrons emitted or reflected only from the surface of the sample. In addition, the SEM has a great depth of field with a wide range of magnifying capabilities. The resulting images clearly show the individual grains of the seasonal snowpack and the bonding between the snow grains. Images of firn show individual ice crystals, the bonding between the crystals, and connected air spaces. Images of glacier ice show a crystal structure on a scale of 1-2 mm which is considerably smaller than the expected crystal size. Microscopic air bubbles, less than 15 ??m in diameter, clearly marked the boundaries between these crystal-like features. The life forms associated with the glacier were

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

    Directory of Open Access Journals (Sweden)

    Cotarla Ion

    2008-01-01

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

  7. Second Harmonic Generation in Scanning Probe Microscopy for Edge Localization

    Institute of Scientific and Technical Information of China (English)

    HU Xiao-Gen; LI Yu-He; LIN Hao-Shan; WANG Dong-Sheng; QI Xin

    2011-01-01

    We present an approach of second harmonic generation for edge localization of nano-scale defects measurement,based on the impact of the oscillating tip on the sample that induces higher harmonics of the excitation frequency.The harmonic signals of tip motion are measured by the heterodyne interferornetry. The edge amplitude ratio for the edge characterization can be calculated by a mechanics model and the threshold of edge localization is experimentally determined by second harmonic profiles. This approach has been successfully utilized to measure the pitch of a standard sample. The results show that the second harmonic is sensitive to locating the edge of nano-scale defects with high accuracy.%@@ We present an approach of second harmonic generation for edge localization of nano-scale defects measurement,based on the impact of the oscillating tip on the sample that induces higher harmonics of the excitation frequency.The harmonic signals of tip motion are measured by the heterodyne interferometry.The edge amplitude ratio for the edge characterization can be calculated by a mechanics model and the threshold of edge localization is experimentally determined by second harmonic profiles.This approach has been successfully utilized to measure the pitch of a standard sample.The results show that the second harmonic is sensitive to locating the edge of nano-scale defects with high accuracy.

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

    Directory of Open Access Journals (Sweden)

    Pia C. Lansåker

    2014-10-01

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

  9. Near-infrared microscopy imaging for quantitative analysis of active component in counterfeit imidacloprid

    Science.gov (United States)

    Huang, Yue; Cao, Jinli; Ye, Shengfeng; Duan, Jia; Wu, Lijun; Li, Qianqian; Min, Shungeng

    2012-01-01

    Near-infrared (NIR) imaging systems simultaneously record spectral and spatial information. Near-infrared imaging was applied to the identification of imidacloprid in both artificially mixed samples and commercial formulation in this study. The distributions of technical imidacloprid and additive in the heterogeneous counterfeit were obtained by the relationship imaging (RI) mode. Furthermore a series of samples which consisted of different contents of uniformly distributed imidacloprid were prepared and three data cubes were generated at each content. Extracted spectra from those images were imported to establish the partial least squares model. The model's results were: R2 99.21%, RMSEC 0.0306, RMSECV 0.0183, RMSECV/mean value 0.0348 and RSEP 0.0784. The prediction relative error of commercial formulation is 0.0680, indicating the predicted value was correlated to the real content. Lastly the chemical value reconstruction image of imidacloprid formulation products was calculated by MATLAB program. NIR microscopy imaging manifests herein its potential in qualitatively identifying the active component in counterfeit pesticide and quantifying the active component in scanned image.

  10. Chromatic confocal microscopy for multi-depth imaging of epithelial tissue.

    Science.gov (United States)

    Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E; Maitland, Kristen C

    2013-05-01

    We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue.

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

    Science.gov (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-22

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

  12. Robust image alignment for cryogenic transmission electron microscopy.

    Science.gov (United States)

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

    2017-03-01

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Enhancing resolution and contrast in second-harmonic generation microscopy using an advanced maximum likelihood estimation restoration method

    Science.gov (United States)

    Sivaguru, Mayandi; Kabir, Mohammad M.; Gartia, Manas Ranjan; Biggs, David S. C.; Sivaguru, Barghav S.; Sivaguru, Vignesh A.; Berent, Zachary T.; Wagoner Johnson, Amy J.; Fried, Glenn A.; Liu, Gang Logan; Sadayappan, Sakthivel; Toussaint, Kimani C.

    2017-02-01

    Second-harmonic generation (SHG) microscopy is a label-free imaging technique to study collagenous materials in extracellular matrix environment with high resolution and contrast. However, like many other microscopy techniques, the actual spatial resolution achievable by SHG microscopy is reduced by out-of-focus blur and optical aberrations that degrade particularly the amplitude of the detectable higher spatial frequencies. Being a two-photon scattering process, it is challenging to define a point spread function (PSF) for the SHG imaging modality. As a result, in comparison with other two-photon imaging systems like two-photon fluorescence, it is difficult to apply any PSF-engineering techniques to enhance the experimental spatial resolution closer to the diffraction limit. Here, we present a method to improve the spatial resolution in SHG microscopy using an advanced maximum likelihood estimation (AdvMLE) algorithm to recover the otherwise degraded higher spatial frequencies in an SHG image. Through adaptation and iteration, the AdvMLE algorithm calculates an improved PSF for an SHG image and enhances the spatial resolution by decreasing the full-width-at-halfmaximum (FWHM) by 20%. Similar results are consistently observed for biological tissues with varying SHG sources, such as gold nanoparticles and collagen in porcine feet tendons. By obtaining an experimental transverse spatial resolution of 400 nm, we show that the AdvMLE algorithm brings the practical spatial resolution closer to the theoretical diffraction limit. Our approach is suitable for adaptation in micro-nano CT and MRI imaging, which has the potential to impact diagnosis and treatment of human diseases.

  14. Generating Virtual Images from Oblique Frames

    Directory of Open Access Journals (Sweden)

    Carlos R. T. Caldeira

    2013-04-01

    Full Text Available Image acquisition systems based on multi-head arrangement of digital cameras are attractive alternatives enabling a larger imaging area when compared to a single frame camera. The calibration of this kind of system can be performed in several steps or by using simultaneous bundle adjustment with relative orientation stability constraints. The paper will address the details of the steps of the proposed approach for system calibration, image rectification, registration and fusion. Experiments with terrestrial and aerial images acquired with two Fuji FinePix S3Pro cameras were performed. The experiments focused on the assessment of the results of self-calibrating bundle adjustment with and without relative orientation constraints and the effects to the registration and fusion when generating virtual images. The experiments have shown that the images can be accurately rectified and registered with the proposed approach, achieving residuals smaller than one pixel.

  15. Multiphoton microscopy and image guided light activated therapy using nanomaterials (Conference Presentation)

    Science.gov (United States)

    Prasad, Paras N.

    2017-02-01

    This talk will focus on design and applications of nanomaterials exhibiting strong multiphoton upconversion for multiphoton microscopy as well as for image-guided and light activated therapy .1-3 Such processes can occur by truly nonlinear optical interactions proceeding through virtual intermediate states or by stepwise coupled linear excitations through real intermediate states. Multiphoton processes in biocompatible multifunctional nanoparticles allow for 3D deep tissue imaging. In addition, they can produce in-situ photon conversion of deep tissue penetrating near IR light into a needed shorter wavelength light for photo-activated therapy at a targeted site, thus overcoming the limited penetration of UV or visible light into biological media. We are using near IR emitters such as silicon quantum dots which also exhibit strong multiphoton excitation for multiphoton microscopy. Another approach involves nonlinear nanocrystals such as ZnO which can produce four wave mixing, sum frequency generation as well as second harmonic generation to convert a deep tissue penetrating Near IR light at the targeted biological site to a desired shorter wavelength light suitable for bio imaging or activation of a therapy. We have utilized this approach to activate a photosensitizer for photodynamic therapy. Yet another type of upconversion materials is rare-earth ion doped optical nanotransformers which transform a Near IR (NIR) light from an external source by sequential single photon absorption, in situ and on demand, to a needed wavelength. Applications of these nanotransformers in multiphoton photoacoustic imaging will also be presented. An exciting direction pursued by us using these multiphoton nanoparticles, is functional imaging of brain. Simultaneously, they can effect optogenetics for regioselective stimulation of neurons for providing an effective intervention/augmentation strategy to enhance the cognitive state and lead to a foundation for futuristic vision of super

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

    Science.gov (United States)

    Duan, Yubo; Chen, Nanguang

    2015-11-15

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

  17. Imaging Biological Systems using Dielectric Near-Field Microscopy

    Science.gov (United States)

    Brown, Keith; Issadore, David; Hunt, Tom; Westervelt, Robert

    2007-03-01

    We have developed a dielectric spectrometer for use on biological systems. The spectrum of dielectric response to RF electric fields is analogous to color as an optical response. Measurement of the dielectric spectrum from ˜ 10 kHz to ˜ 3 GHz will reveal information about the structure and conditions of protein solutions, protein crystals and biological tissues. We designed and built a system to test biological samples in a microfluidic chamber mounted on a circuit board. The apparatus measures the RF dielectric spectrum directly, or by analyzing the pulse response in the time domain. We have constructed several versions of the hardware for sensitive capacitive measurements, including two types of capacitive bridges, and a transmission line, incorporating precision electronics and local generation of pulses. A goal is to scale the system down and implement many dielectric spectrometers as an array of pixels on a CMOS chip for dielectric near-field microscopy of biological samples. This work made possible by NSEC NSF grant PHY-0117795 and the NCI MIT-Harvard CCNE.

  18. Learning generative models of natural images.

    Science.gov (United States)

    Wu, Jiann-Ming; Lin, Zheng-Han

    2002-04-01

    This work proposes an unsupervised learning process for analysis of natural images. The derivation is based on a generative model, a stochastic coin-flip process directly operating on many disjoint multivariate Gaussian distributions. Following the maximal likelihood principle and using the Potts encoding, the goodness-of-fit of the generative model to tremendous patches randomly sampled from natural images is quantitatively expressed by an objective function subject to a set of constraints. By further combination of the objective function and the minimal wiring criterion, we achieve a mixed integer and linear programming. A hybrid of the mean field annealing and the gradient descent method is applied to the mathematical framework and produces three sets of interactive dynamics for the learning process. Numerical simulations show that the learning process is effective for extraction of orientation, localization and bandpass features and the generative model can make an ensemble of a sparse code for natural images.

  19. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

    OpenAIRE

    Supatto, Willy; McMahon, Amy; Fraser, Scott E.; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and progr...

  20. Timing and Operating Mode Design for Time-Gated Fluorescence Lifetime Imaging Microscopy

    OpenAIRE

    Chao Liu; Xinwei Wang; Yan Zhou; Yuliang Liu

    2013-01-01

    Steady-state fluorence imaging and time-resolved fluorescence imaging are two important areas in fluorescence imaging research. Fluorescence lifetime imaging is an absolute measurement method which is independent of excitation laser intensity, fluorophore concentration, and photobleaching compared to fluorescence intensity imaging techniques. Time-gated fluorescence lifetime imaging microscopy (FLIM) can provide high resolution and high imaging frame during mature FLIM methods. An abstract ti...

  1. Effect of molecular organization on the image histograms of polarization SHG microscopy.

    Science.gov (United States)

    Psilodimitrakopoulos, Sotiris; Amat-Roldan, Ivan; Loza-Alvarez, Pablo; Artigas, David

    2012-10-01

    Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.

  2. Efficient parallel Levenberg-Marquardt model fitting towards real-time automated parametric imaging microscopy.

    Science.gov (United States)

    Zhu, Xiang; Zhang, Dianwen

    2013-01-01

    We present a fast, accurate and robust parallel Levenberg-Marquardt minimization optimizer, GPU-LMFit, which is implemented on graphics processing unit for high performance scalable parallel model fitting processing. GPU-LMFit can provide a dramatic speed-up in massive model fitting analyses to enable real-time automated pixel-wise parametric imaging microscopy. We demonstrate the performance of GPU-LMFit for the applications in superresolution localization microscopy and fluorescence lifetime imaging microscopy.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    de Siqueira, Alexandre Fioravante; Cabrera, Flavio Camargo [UNESP; Pagamisse, Aylton; Job,Aldo Eloizo

    2016-01-01

    Electronic microscopy has been used for morphology evaluation of different materials structures. However, microscopy results may be affected by several factors. Image processing methods can be used to correct and improve the quality of these results. In this article, we propose an algorithm based on starlets to perform the segmentation of scanning electron microscopy images. An application is presented in order to locate gold nanoparticles in natural rubber membranes. In this application, our...

  5. A fast image registration approach of neural activities in light-sheet fluorescence microscopy images

    Science.gov (United States)

    Meng, Hui; Hui, Hui; Hu, Chaoen; Yang, Xin; Tian, Jie

    2017-03-01

    The ability of fast and single-neuron resolution imaging of neural activities enables light-sheet fluorescence microscopy (LSFM) as a powerful imaging technique in functional neural connection applications. The state-of-art LSFM imaging system can record the neuronal activities of entire brain for small animal, such as zebrafish or C. elegans at single-neuron resolution. However, the stimulated and spontaneous movements in animal brain result in inconsistent neuron positions during recording process. It is time consuming to register the acquired large-scale images with conventional method. In this work, we address the problem of fast registration of neural positions in stacks of LSFM images. This is necessary to register brain structures and activities. To achieve fast registration of neural activities, we present a rigid registration architecture by implementation of Graphics Processing Unit (GPU). In this approach, the image stacks were preprocessed on GPU by mean stretching to reduce the computation effort. The present image was registered to the previous image stack that considered as reference. A fast Fourier transform (FFT) algorithm was used for calculating the shift of the image stack. The calculations for image registration were performed in different threads while the preparation functionality was refactored and called only once by the master thread. We implemented our registration algorithm on NVIDIA Quadro K4200 GPU under Compute Unified Device Architecture (CUDA) programming environment. The experimental results showed that the registration computation can speed-up to 550ms for a full high-resolution brain image. Our approach also has potential to be used for other dynamic image registrations in biomedical applications.

  6. An epifluorescence microscopy method for generalized polarization imaging

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard; Helix Nielsen, Claus

    2011-01-01

    Generalized polarization (GP) microscopy represents an excellent tool to study lipid–lipid and lipid–protein interactions in situ and in vitro. Here, we present an efficient and cost effective method to perform GP microscopy using a standard light-emitting diode (LED) epifluorescence microscope...

  7. Isotropic image in structured illumination microscopy patterned with a spatial light modulator.

    Science.gov (United States)

    Chang, Bo-Jui; Chou, Li-Jun; Chang, Yun-Ching; Chiang, Su-Yu

    2009-08-17

    We developed a structured illumination microscopy (SIM) system that uses a spatial light modulator (SLM) to generate interference illumination patterns at four orientations - 0 degrees, 45 degrees, 90 degrees, and 135 degrees, to reconstruct a high-resolution image. The use of a SLM for pattern alterations is rapid and precise, without mechanical calibration; moreover, our design of SLM patterns allows generating the four illumination patterns of high contrast and nearly equivalent periods to achieve a near isotropic enhancement in lateral resolution. We compare the conventional image of 100-nm beads with those reconstructed from two (0 degrees +90 degrees or 45 degrees +135 degrees) and four (0 degrees +45 degrees +90 degrees +135 degrees) pattern orientations to show the differences in resolution and image, with the support of simulations. The reconstructed images of 200-nm beads at various depths and fine structures of actin filaments near the edge of a HeLa cell are presented to demonstrate the intensity distributions in the axial direction and the prospective application to biological systems.

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

    Science.gov (United States)

    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. Groundtruth Generation and Document Image Degradation

    Science.gov (United States)

    2005-05-01

    the author in [32] uses this model to design a linear filtering scheme instead of generating synthetic images. The necessity and the work...TrueType specifications”, https://www.microsoft.com/ typography /specs/default.htm [28] Esko Ukkonen, “Algorithm for Approximate String Matching

  10. Research of second harmonic generation images based on texture analysis

    Science.gov (United States)

    Liu, Yao; Li, Yan; Gong, Haiming; Zhu, Xiaoqin; Huang, Zufang; Chen, Guannan

    2014-09-01

    Texture analysis plays a crucial role in identifying objects or regions of interest in an image. It has been applied to a variety of medical image processing, ranging from the detection of disease and the segmentation of specific anatomical structures, to differentiation between healthy and pathological tissues. Second harmonic generation (SHG) microscopy as a potential noninvasive tool for imaging biological tissues has been widely used in medicine, with reduced phototoxicity and photobleaching. In this paper, we clarified the principles of texture analysis including statistical, transform, structural and model-based methods and gave examples of its applications, reviewing studies of the technique. Moreover, we tried to apply texture analysis to the SHG images for the differentiation of human skin scar tissues. Texture analysis method based on local binary pattern (LBP) and wavelet transform was used to extract texture features of SHG images from collagen in normal and abnormal scars, and then the scar SHG images were classified into normal or abnormal ones. Compared with other texture analysis methods with respect to the receiver operating characteristic analysis, LBP combined with wavelet transform was demonstrated to achieve higher accuracy. It can provide a new way for clinical diagnosis of scar types. At last, future development of texture analysis in SHG images were discussed.

  11. A Deep Generative Deconvolutional Image Model

    Energy Technology Data Exchange (ETDEWEB)

    Pu, Yunchen; Yuan, Xin; Stevens, Andrew J.; Li, Chunyuan; Carin, Lawrence

    2016-05-09

    A deep generative model is developed for representation and analysis of images, based on a hierarchical convolutional dictionary-learning framework. Stochastic unpooling is employed to link consecutive layers in the model, yielding top-down image generation. A Bayesian support vector machine is linked to the top-layer features, yielding max-margin discrimination. Deep deconvolutional inference is employed when testing, to infer the latent features, and the top-layer features are connected with the max-margin classifier for discrimination tasks. The model is efficiently trained using a Monte Carlo expectation-maximization (MCEM) algorithm; the algorithm is implemented on graphical processor units (GPU) to enable large-scale learning, and fast testing. Excellent results are obtained on several benchmark datasets, including ImageNet, demonstrating that the proposed model achieves results that are highly competitive with similarly sized convolutional neural networks.

  12. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    Directory of Open Access Journals (Sweden)

    Alex Joseph Perez

    2014-11-01

    Full Text Available Electron microscopy (EM facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson’s and Alzheimer’s diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM. Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime.

  13. Generating passive NIR images from active LIDAR

    Science.gov (United States)

    Hagstrom, Shea; Broadwater, Joshua

    2016-05-01

    Many modern LIDAR platforms contain an integrated RGB camera for capturing contextual imagery. However, these RGB cameras do not collect a near-infrared (NIR) color channel, omitting information useful for many analytical purposes. This raises the question of whether LIDAR data, collected in the NIR, can be used as a substitute for an actual NIR image in this situation. Generating a LIDAR-based NIR image is potentially useful in situations where another source of NIR, such as satellite imagery, is not available. LIDAR is an active sensing system that operates very differently from a passive system, and thus requires additional processing and calibration to approximate the output of a passive instrument. We examine methods of approximating passive NIR images from LIDAR for real-world datasets, and assess differences with true NIR images.

  14. B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Shilpa Dilipkumar

    2015-03-01

    Full Text Available An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy. A comparative study of the proposed technique with the state-of-art maximum likelihood (ML and maximum-a-posteriori (MAP with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

  15. A computational approach to real-time image processing for serial time-encoded amplified microscopy

    Science.gov (United States)

    Oikawa, Minoru; Hiyama, Daisuke; Hirayama, Ryuji; Hasegawa, Satoki; Endo, Yutaka; Sugie, Takahisa; Tsumura, Norimichi; Kuroshima, Mai; Maki, Masanori; Okada, Genki; Lei, Cheng; Ozeki, Yasuyuki; Goda, Keisuke; Shimobaba, Tomoyoshi

    2016-03-01

    High-speed imaging is an indispensable technique, particularly for identifying or analyzing fast-moving objects. The serial time-encoded amplified microscopy (STEAM) technique was proposed to enable us to capture images with a frame rate 1,000 times faster than using conventional methods such as CCD (charge-coupled device) cameras. The application of this high-speed STEAM imaging technique to a real-time system, such as flow cytometry for a cell-sorting system, requires successively processing a large number of captured images with high throughput in real time. We are now developing a high-speed flow cytometer system including a STEAM camera. In this paper, we describe our approach to processing these large amounts of image data in real time. We use an analog-to-digital converter that has up to 7.0G samples/s and 8-bit resolution for capturing the output voltage signal that involves grayscale images from the STEAM camera. Therefore the direct data output from the STEAM camera generates 7.0G byte/s continuously. We provided a field-programmable gate array (FPGA) device as a digital signal pre-processor for image reconstruction and finding objects in a microfluidic channel with high data rates in real time. We also utilized graphics processing unit (GPU) devices for accelerating the calculation speed of identification of the reconstructed images. We built our prototype system, which including a STEAM camera, a FPGA device and a GPU device, and evaluated its performance in real-time identification of small particles (beads), as virtual biological cells, owing through a microfluidic channel.

  16. Analysis of human aorta using fluorescence lifetime imaging microscopy (FLIM)

    Science.gov (United States)

    Vieira-Damiani, Gislaine; Adur, J.; Ferro, D. P.; Adam, R. L.; Pelegati, V.; Thomáz, A.; Cesar, C. L.; Metze, K.

    2012-03-01

    The use of photonics has improved our understanding of biologic phenomena. For the study of the normal and pathologic architecture of the aorta the use of Two-Photon Excited Fluorescence (TPEF) and Second Harmonic Generation showed interesting details of morphologic changes of the elastin-collagen architecture during aging or development of hypertension in previous studies. In this investigation we tried to apply fluorescence lifetime imaging (FLIM) for the morphologic analysis of human aortas. The aim of our study was to use FLIM in non-stained formalin-fixed and paraffin-embedded samples of the aorta ascendants in hypertensive and normotensive patients of various ages, examining two different topographical regions. The FLIM-spectra of collagen and elastic fibers were clearly distinguishable, thus permitting an exact analysis of unstained material on the microscopic level. Moreover the FLIM spectrum of elastic fibers revealed variations between individual cases, which indicate modifications on a molecular level and might be related to FLIM age or diseases states and reflect modifications on a molecular level.

  17. Adult Human Neurogenesis: from Microscopy to Magnetic Resonance Imaging

    Directory of Open Access Journals (Sweden)

    Amanda eSierra

    2011-04-01

    Full Text Available Neural stem cells reside in well-defined areas of the adult human brain and are capable of gene-rating 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.

  18. Next-generation endomyocardial biopsy: the potential of confocal and super-resolution microscopy.

    Science.gov (United States)

    Crossman, David J; Ruygrok, Peter N; Hou, Yu Feng; Soeller, Christian

    2015-03-01

    Confocal laser scanning microscopy and super-resolution microscopy provide high-contrast and high-resolution fluorescent imaging, which has great potential to increase the diagnostic yield of endomyocardial biopsy (EMB). EMB is currently the gold standard for identification of cardiac allograft rejection, myocarditis, and infiltrative and storage diseases. However, standard analysis is dominated by low-contrast bright-field light and electron microscopy (EM); this lack of contrast makes quantification of pathological features difficult. For example, assessment of cardiac allograft rejection relies on subjective grading of H&E histology, which may lead to diagnostic variability between pathologists. This issue could be solved by utilising the high contrast provided by fluorescence methods such as confocal to quantitatively assess the degree of lymphocytic infiltrate. For infiltrative diseases such as amyloidosis, the nanometre resolution provided by EM can be diagnostic in identifying disease-causing fibrils. The recent advent of super-resolution imaging, particularly direct stochastic optical reconstruction microscopy (dSTORM), provides high-contrast imaging at resolution approaching that of EM. Moreover, dSTORM utilises conventional fluorescence dyes allowing for the same structures to be routinely imaged at the cellular scale and then at the nanoscale. The key benefit of these technologies is that the high contrast facilitates quantitative digital analysis and thereby provides a means to robustly assess critical pathological features. Ultimately, this technology has the ability to provide greater accuracy and precision to EMB assessment, which could result in better outcomes for patients.

  19. Imaging ectopic fat deposition in Caenorhabditis elegans muscles using nonlinear microscopy.

    Science.gov (United States)

    Mari, Meropi; Filippidis, George; Palikaras, Konstantinos; Petanidou, Barbara; Fotakis, Costas; Tavernarakis, Nektarios

    2015-06-01

    The elucidation of the molecular mechanisms that lead to the development of metabolic syndrome, a complex of pathological conditions including type-2 diabetes, hypertension, and cardiovascular diseases, is an important issue with high biological significance and requires accurate methods capable of monitoring lipid storage distribution and dynamics in vivo. In this study, the nonlinear phenomena of second and third harmonic generation (SHG, THG) have been employed simultaneously as label-free, nondestructive diagnostic techniques, for the monitoring and the complementary three-dimensional (3D) imaging and analysis of the muscular areas and the lipid content localization. THG microscopy was used as a quantitative tool in order to record the accumulation of lipids in nonadipose tissues in the pharyngeal muscles of 18 Caenorhabditis elegans (C. elegans) specimens, while the SHG imaging provided the detailed anatomical information about the structure of the muscles. The ectopic accumulation of fat on the pharyngeal muscles increases in wild-type (N2) C. elegans between 1 and 9 days of adulthood. This suggests a correlation of ectopic fat accumulation with the process of aging. Our results can contribute to the unraveling of the link between the deposition of ectopic fat and aging, but mainly to the validation of SHG and THG microscopy modalities as new, noninvasive tools to localize and quantify selectively lipid formation and distribution.

  20. Dental caries imaging using hyperspectral stimulated Raman scattering microscopy

    Science.gov (United States)

    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.

  1. Super-resolution deep imaging with hollow Bessel beam STED microscopy

    CERN Document Server

    Yu, Wentao; Dong, Dashan; Yang, Xusan; Xiao, Yunfeng; Gong, Qihuang; Xi, Peng; Shi, Kebin

    2015-01-01

    Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method beating the diffraction barrier for improved lateral spatial resolution in cellular imaging, lithography, etc. Due to specimen-induced aberrations and scattering distortion, it has been a great challenge for STED to maintain consistent lateral resolution deeply inside the specimens. Here we report on a deep imaging STED microscopy by using Gaussian beam for excitation and hollow Bessel beam for depletion (GB-STED). The proposed scheme shows the improved imaging depth up to ~155{\\mu}m in solid agarose sample, ~115{\\mu}m in PDMS and ~100{\\mu}m in phantom of gray matter in brain tissue with consistent super resolution, while the standard STED microscopy shown a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB-STED, making it a promising tool for deep 3D imaging optical nanoscopy and laser fabrication.

  2. Accuracy in Robot Generated Image Data Sets

    DEFF Research Database (Denmark)

    Aanæs, Henrik; Dahl, Anders Bjorholm

    2015-01-01

    In this paper we present a practical innovation concerning how to achieve high accuracy of camera positioning, when using a 6 axis industrial robots to generate high quality data sets for computer vision. This innovation is based on the realization that to a very large extent the robots positioning...... error is deterministic, and can as such be calibrated away. We have successfully used this innovation in our efforts for creating data sets for computer vision. Since the use of this innovation has a significant effect on the data set quality, we here present it in some detail, to better aid others...... in using robots for image data set generation....

  3. Multiphoton microscopy for imaging infectious keratitis: demonstration of the pattern of microbial spread in an experimental model

    Science.gov (United States)

    Sun, Yen; Lo, Wen; Wu, Ruei-Jhih; 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 application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for imaging and monitoring the disease progress of infectious keratitis in an experimental model, and to investigate the possible correlation of tissue architecture with spreading patterns of pathogens in an experimental model. Porcine eyes are to be obtained from slaughter house and processed and placed in organ culture system. Fungal infections by common pathogens of infectious keratitis are to be induced in porcine cornea buttons. Multiphoton fluorescence and SHG microscopy will be used for imaging and for monitoring the progression and extension of tissue destruction and possibly the pattern of pathogen spreading. We found that SHG imaging is useful in identifying alterations to collagen architecture while autofluorescence microscopy can be used to visualize the fungi and cells within the stroma. In summary, multiphoton fluorescence and second harmonic generation microscopy can non-invasively demonstrate and monitor tissue destruction associated with infectious keratitis. The pattern of pathogen spreading and its correlation with the tissue architecture can also be shown, which can be useful for future studies of the tissue-microbial interactions for infectious keratitis.

  4. Nonlinear photoacoustic microscopy via a loss modulation technique: from detection to imaging.

    Science.gov (United States)

    Lai, Yu-Hung; Lee, Szu-Yu; Chang, Chieh-Feng; Cheng, Yu-Hsiang; Sun, Chi-Kuang

    2014-01-13

    In order to achieve high-resolution deep-tissue imaging, multi-photon fluorescence microscopy and photoacoustic tomography had been proposed in the past two decades. However, combining the advantages of these two imaging systems to achieve optical-spatial resolution with an ultrasonic-penetration depth is still a field with challenges. In this paper, we investigate the detection of the two-photon photoacoustic ultrasound, and first demonstrate background-free two-photon photoacoustic imaging in a phantom sample. To generate the background-free two-photon photoacoustic signals, we used a high-repetition rate femtosecond laser to induce narrowband excitation. Combining a loss modulation technique, we successfully created a beating on the light intensity, which not only provides pure sinusoidal modulation, but also ensures the spectrum sensitivity and frequency selectivity. By using the lock-in detection, the power dependency experiment validates our methodology to frequency-select the source of the nonlinearity. This ensures our capability of measuring the background-free two-photon photoacoustic waves by detecting the 2nd order beating signal directly. Furthermore, by mixing the nanoparticles and fluorescence dyes as contrast agents, the two-photon photoacoustic signal was found to be enhanced and detected. In the end, we demonstrate subsurface two-photon photoacoustic bio-imaging based on the optical scanning mechanism inside phantom samples.

  5. Compound Cellular Imaging of Laser Scanning Confocal Microscopy by Using Gold Nanoparticles and Dyes

    Directory of Open Access Journals (Sweden)

    Jiunn-Woei Liaw

    2008-04-01

    Full Text Available Combining the scattered light of gold nanoparticles (GNPs and the fluorescence of dye molecules, a compound cellular imaging of laser scanning confocal microscopy (LSCM is obtained. The human breast cancer cell line (MDA-MB-435S, BCRC 60429 is used for experiment. These cells are incubated with a glucose medium containing GNPs for 26 hours, and then are stained by Prodium Iodide (PI for their nuclei. By using a single laser to illuminate these cells and adjusting the ranges of two bandpass filters for the detection, the scattered light from the GNPs and the fluorescence of PI can be induced simultaneously, but be detected separately without crosstalk. Furthermore, a compound cellular image can be obtained by merging the two images of the expressions of GNP and PI together. From the TEM images of these cells, it is observed that GNPs are aggregated in the vesicles of the cytoplasm due to the cell’s endocytosis. The aggregation of GNPs makes the surface plasmon resonance band of GNPs broadened, so that strong scattered light from GNPs can be generated by the illumination of different-wavelength lasers (458, 488, 514, 561, and 633 nm.

  6. Post-processing for statistical image analysis in light microscopy.

    Science.gov (United States)

    Cardullo, Richard A; Hinchcliffe, Edward H

    2013-01-01

    Image processing of images serves a number of important functions including noise reduction, contrast enhancement, and feature extraction. Whatever the final goal, an understanding of the nature of image acquisition and digitization and subsequent mathematical manipulations of that digitized image is essential. Here we discuss the basic mathematical and statistical processes that are routinely used by microscopists to routinely produce high quality digital images and to extract key features of interest using a variety of extraction and thresholding tools. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Second harmonic generation microscopy reveals hidden polar organization in fluoride doped MIL-53(Fe)

    NARCIS (Netherlands)

    Markey, K.; Putzeys, T.; Horcajada, P.; Devic, T.; Guillou, N.; Wübbenhorst, M.; Van Cleuvenbergen, S.; Verbiest, T.; De Vos, D.E.; Van der Veen, M.A.

    2016-01-01

    Polar metal–organic frameworks have potential applications as functional non-linear optical, piezoelectric, pyroelectric and ferroelectric materials. Using second harmonic generation microscopy we found that fluoride doping of the microporous iron(III) terephthalate MOF MIL-53(Fe) induces a polar or

  8. Second-Harmonic Generation Scanning Microscopy on Domains in Al Surfaces

    DEFF Research Database (Denmark)

    Pedersen, Kjeld; Bozhevolnyi, Sergey I.

    1999-01-01

    Scanning optical second-harmonic generation microscopy has been used to investigate domains in the surface of polycrystaline Al. Strong contrast among the crystalline grains is obtained due to variations in their crystallographic orientations and thus also nonlinear response. The origin of the co...

  9. Second-Harmonic Generation Scanning Microscopy on Domains in Al Surfaces

    DEFF Research Database (Denmark)

    Pedersen, Kjeld; Bozhevolnyi, Sergey I.

    1999-01-01

    Scanning optical second-harmonic generation microscopy has been used to investigate domains in the surface of polycrystaline Al. Strong contrast among the crystalline grains is obtained due to variations in their crystallographic orientations and thus also nonlinear response. The origin of the co...

  10. Automatic detection of cell divisions (mitosis) in live-imaging microscopy images using Convolutional Neural Networks.

    Science.gov (United States)

    Shkolyar, Anat; Gefen, Amit; Benayahu, Dafna; Greenspan, Hayit

    2015-08-01

    We propose a semi-automated pipeline for the detection of possible cell divisions in live-imaging microscopy and the classification of these mitosis candidates using a Convolutional Neural Network (CNN). We use time-lapse images of NIH3T3 scratch assay cultures, extract patches around bright candidate regions that then undergo segmentation and binarization, followed by a classification of the binary patches into either containing or not containing cell division. The classification is performed by training a Convolutional Neural Network on a specially constructed database. We show strong results of AUC = 0.91 and F-score = 0.89, competitive with state-of-the-art methods in this field.

  11. Nanoscale Imaging Technology for THz Frequency Transmission Microscopy

    Science.gov (United States)

    2014-12-16

    Microscopy, Analytical Chemistry , (06 2013): 0. doi: 10.1021/ac4010088 Yung Yu Wang, Peter J. Burke. Polyelectrolyte multilayer electrostatic gating of...C. G. Densmore, S. K. Doorn, P. J. Burke. Effect of source, surfactant , and deposition process on electronic properties of nanotube arrays, (09

  12. Laser differential fitting confocal microscopy with high imaging efficiency.

    Science.gov (United States)

    Sheng, Zhong; Wang, Yun; Zhao, Weiqian; Qiu, Lirong; Sun, Yingbin

    2016-09-01

    Based on the optical arrangement of a bipolar differential confocal microscopy (BDCM), laser differential fitting confocal microscopy (DFCM) is proposed in this paper using the feature of BDCM that a zero-crossing point (ZCP) of the axial response curve precisely corresponds to the focus of the system objective. A linear segment of the DFCM axial response around the ZCP is used to fit a straight line. Focus can be determined by solving the equations of the fitting lines, and then, the sample surface could be measured and reconstructed with a high resolution. Compared with the curve-fitting peak detection, which is an algorithm for focus detection widely used in conventional confocal microscopy, the line-fitting zero solution method used in DFCM has several advantages, such as high precision and sensitivity. Most importantly, precise focus detection can be realized using less data, i.e., DFCM has a high measurement efficiency. Furthermore, DFCM can effectively eliminate common-mode noise in a confocal microscopy system and has good noise suppression and disturbance resistance capability.

  13. Infrared multiphoton microscopy: subcellular-resolved deep tissue imaging.

    NARCIS (Netherlands)

    Andresen, V.; Alexander, S.; Heupel, W.M.; Hirschberg, M.; Hoffman, R.M.; Friedl, P.H.A.

    2009-01-01

    Multiphoton microscopy (MPM) is the method of choice for investigating cells and cellular functions in deep tissue sections and organs. Here we present the setup and applications of infrared-(IR-)MPM using excitation wavelengths above 1080 nm. IR-MPM enables the use of red fluorophores and

  14. Modeling of Image Formation in Cryo-Electron Microscopy

    NARCIS (Netherlands)

    Vulovic, M.

    2013-01-01

    Knowledge of the structure of biological specimens is crucial for understanding life. Cryo-electron microscopy (cryo-EM) permits structural studies of biological specimen at their near-native state. The research performed in this thesis represents one of two subprojects of the FOM industrial partner

  15. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  16. Nonlinear Image Restoration in Confocal Microscopy : Stability under Noise

    NARCIS (Netherlands)

    Roerdink, J.B.T.M.

    1995-01-01

    In this paper we study the noise stability of iterative algorithms developed for attenuation correction in Fluorescence Confocal Microscopy using FT methods. In each iteration the convolution of the previous estimate is computed. It turns out that the estimators are robust to noise perturbation.

  17. SIMS ion microscopy as a novel, practical tool for subcellular chemical imaging in cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, S

    2003-01-15

    The development of cryogenic sample preparations, subcellular image quantification schemes, and correlative confocal laser scanning microscopy and ion microscopy have made dynamic SIMS a versatile tool in biology and medicine. For example, ion microscopy can provide much needed, novel information on calcium influx and intracellular calcium stores at organelle resolution in normal and transformed cells in order to better understand the altered calcium signaling in malignant cells. 3-D SIMS imaging of cells revealed dynamic gradients of calcium in cells undergoing mitosis and cytokinesis. Studies of subcellular localization of anticancer drugs is another area of research where ion microscopy can provide novel observations in many types of cancers. Ion microscopy is already an essential tool in boron neutron capture therapy (BNCT) of brain cancer as it can be used to quantitatively image the subcellular location of boron in cells and tissues. This information is critically needed for testing the efficacy of boronated agents and for calculations of radiation dosimetry.

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

    Science.gov (United States)

    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.

  19. Improving spatial resolution of confocal Raman microscopy by super-resolution image restoration.

    Science.gov (United States)

    Cui, Han; Zhao, Weiqian; Wang, Yun; Fan, Ying; Qiu, Lirong; Zhu, Ke

    2016-05-16

    A new super-resolution image restoration confocal Raman microscopy method (SRIR-RAMAN) is proposed for improving the spatial resolution of confocal Raman microscopy. This method can recover the lost high spatial frequency of the confocal Raman microscopy by using Poisson-MAP super-resolution imaging restoration, thereby improving the spatial resolution of confocal Raman microscopy and realizing its super-resolution imaging. Simulation analyses and experimental results indicate that the spatial resolution of SRIR-RAMAN can be improved by 65% to achieve 200 nm with the same confocal Raman microscopy system. This method can provide a new tool for high spatial resolution micro-probe structure detection in physical chemistry, materials science, biomedical science and other areas.

  20. Hair Image Generation Using Connected Texels

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiaopeng; CHEN Yanyun; WU Enhua

    2001-01-01

    Generation of photo-realistic images of human hair is a challenging topic in computer graphics. The difficulty in solving the problem in this aspect comes mainly from the extremely large number of hairs and the high complexity of the hair shapes. Regarding to the modeling and rendering of hair-type objects,Kajiya proposed a so-called texel model for producing furry surfaces. However,Kajiya's model could be only used for the generation of short hairs. In this paper,a concise and practical approach is presented to solve the problem of rendering long hairs, and in particular the method of rendering the smooth segmental texels for the generation of long hairs is addressed.

  1. Adenovirus Structure as Revealed by X-Ray Crystallography, Electron Microscopy, and Difference Imaging

    Science.gov (United States)

    Stewart, Phoebe L.; Burnett, Roger M.

    1993-03-01

    The three-dimensional structure of human type 2 adenovirus was studied by combining X-ray crystallography and electron microscopy in a novel way. The 2.9 Å crystal structure of the major capsid protein, hexon, was positioned into a three-dimensional image reconstruction of the intact virus that was derived from cryo-electron micrographs. A three-dimensional difference map was generated by subtracting 240 copies of the crystallographic hexon from the density of the intact virus. This map revealed several minor structural proteins acting as “cement” to stabilize the assembly. The current state of structural knowledge concerning the location of the polypeptide components and the viral DNA is presented.

  2. Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

    Science.gov (United States)

    Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

    2016-01-01

    Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

  3. Microstructure and properties of laser clad coatings studied by orientation imaging microscopy

    NARCIS (Netherlands)

    Ocelik, V.; Furar, I.; De Hosson, J. Th. M.

    2010-01-01

    In this work orientation imaging microscopy (OIM), based on electron backscatter diffraction in scanning electron microscopy, was employed to examine in detail the relationship between laser cladding processing parameters and he properties and the microstructure of single and overlapping laser track

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

    Science.gov (United States)

    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…

  5. Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticles and Cells

    CERN Document Server

    Fairbairn, N; Carter, R; Fernandes, R; Kanaras, A G; Elliott, T J; Somekh, M G; Pitter, M C; Muskens, O L

    2012-01-01

    Spatial modulation microscopy is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the spatial modulation microscopy technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticles.

  6. High resolution 3D imaging of synchrotron generated microbeams

    Energy Technology Data Exchange (ETDEWEB)

    Gagliardi, Frank M., E-mail: frank.gagliardi@wbrc.org.au [Alfred Health Radiation Oncology, The Alfred, Melbourne, Victoria 3004, Australia and School of Medical Sciences, RMIT University, Bundoora, Victoria 3083 (Australia); Cornelius, Iwan [Imaging and Medical Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia and Centre for Medical Radiation Physics, University of Wollongong, Wollongong, New South Wales 2500 (Australia); Blencowe, Anton [Division of Health Sciences, School of Pharmacy and Medical Sciences, The University of South Australia, Adelaide, South Australia 5000, Australia and Division of Information Technology, Engineering and the Environment, Mawson Institute, University of South Australia, Mawson Lakes, South Australia 5095 (Australia); Franich, Rick D. [School of Applied Sciences and Health Innovations Research Institute, RMIT University, Melbourne, Victoria 3000 (Australia); Geso, Moshi [School of Medical Sciences, RMIT University, Bundoora, Victoria 3083 (Australia)

    2015-12-15

    Purpose: Microbeam radiation therapy (MRT) techniques are under investigation at synchrotrons worldwide. Favourable outcomes from animal and cell culture studies have proven the efficacy of MRT. The aim of MRT researchers currently is to progress to human clinical trials in the near future. The purpose of this study was to demonstrate the high resolution and 3D imaging of synchrotron generated microbeams in PRESAGE® dosimeters using laser fluorescence confocal microscopy. Methods: Water equivalent PRESAGE® dosimeters were fabricated and irradiated with microbeams on the Imaging and Medical Beamline at the Australian Synchrotron. Microbeam arrays comprised of microbeams 25–50 μm wide with 200 or 400 μm peak-to-peak spacing were delivered as single, cross-fire, multidirectional, and interspersed arrays. Imaging of the dosimeters was performed using a NIKON A1 laser fluorescence confocal microscope. Results: The spatial fractionation of the MRT beams was clearly visible in 2D and up to 9 mm in depth. Individual microbeams were easily resolved with the full width at half maximum of microbeams measured on images with resolutions of as low as 0.09 μm/pixel. Profiles obtained demonstrated the change of the peak-to-valley dose ratio for interspersed MRT microbeam arrays and subtle variations in the sample positioning by the sample stage goniometer were measured. Conclusions: Laser fluorescence confocal microscopy of MRT irradiated PRESAGE® dosimeters has been validated in this study as a high resolution imaging tool for the independent spatial and geometrical verification of MRT beam delivery.

  7. Deep Imaging in Scattering Media with Single Photon Selective Plane Illumination Microscopy (SPIM)

    CERN Document Server

    Pediredla, Adithya Kumar; Avants, Ben; Ye, Fan; Nagayama, Shin; Chen, Ziying; Kemere, Caleb; Robinson, Jacob; Veeraraghavan, Ashok

    2016-01-01

    In most biological tissues, light scattering due to small differences in refractive index limits the depth of optical imaging systems. Two-photon microscopy (2PM), which significantly reduces the scattering of the excitation light, has emerged as the most common method to image deep within scattering biological tissue. This technique, however, requires high-power pulsed lasers that are both expensive and difficult to integrate into compact portable systems. In this paper, using a combination of theoretical and experimental techniques, we show that Selective Plane Illumination Microscopy (SPIM) can image nearly as deep as 2PM without the need for a high-powered pulsed laser. Compared to other single photon imaging techniques like epifluorescence and confocal microscopy, SPIM can image more than twice as deep in scattering media (approximately 10 times the mean scattering length). These results suggest that SPIM has the potential to provide deep imaging in scattering media in situations where 2PM systems would ...

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

    Science.gov (United States)

    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.

  9. Voltammetric scanning electrochemical cell microscopy: dynamic imaging of hydrazine electro-oxidation on platinum electrodes

    NARCIS (Netherlands)

    Chen, C.-H.; Jacobse, L.; McKelvey, K.; Lai, S.C.S.; Koper, M.T.M.; Unwin, P.R.

    2015-01-01

    Voltammetric scanning electrochemical cell microscopy (SECCM) incorporates cyclic voltammetry measurements in the SECCM imaging protocol, by recording electrochemical currents in a wide potential window at each pixel in a map. This provides much more information compared to traditional fixed potenti

  10. Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

    Science.gov (United States)

    Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

    2017-03-01

    Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

  11. Tomographic diffractive microscopy with agile illuminations for imaging targets in a noisy background.

    Science.gov (United States)

    Zhang, T; Godavarthi, C; Chaumet, P C; Maire, G; Giovannini, H; Talneau, A; Prada, C; Sentenac, A; Belkebir, K

    2015-02-15

    Tomographic diffractive microscopy is a marker-free optical digital imaging technique in which three-dimensional samples are reconstructed from a set of holograms recorded under different angles of incidence. We show experimentally that, by processing the holograms with singular value decomposition, it is possible to image objects in a noisy background that are invisible with classical wide-field microscopy and conventional tomographic reconstruction procedure. The targets can be further characterized with a selective quantitative inversion.

  12. Nanoscale Subsurface Imaging of Nanocomposites via Resonant Difference-Frequency Atomic Force Ultrasonic Microscopy

    Science.gov (United States)

    Cantrell, Sean A.; Cantrell, John H.; Lillehei, Peter T.

    2007-01-01

    A scanning probe microscope methodology, called resonant difference-frequency atomic force ultrasonic microscopy (RDF-AFUM), has been developed. The method employs an ultrasonic wave launched from the bottom of a sample while the cantilever of an atomic force microscope engages the sample top surface. The cantilever is driven at a frequency differing from the ultrasonic frequency by one of the contact resonance frequencies of the cantilever. The nonlinear mixing of the oscillating cantilever and the ultrasonic wave at the sample surface generates difference-frequency oscillations at the cantilever contact resonance. The resonance-enhanced difference-frequency signals are used to create amplitude and phase-generated images of nanoscale near-surface and subsurface features. RDF-AFUM phase images of LaRC-CP2 polyimide polymer containing embedded nanostructures are presented. A RDF-AFUM micrograph of a 12.7 micrometer thick film of LaRC-CP2 containing a monolayer of gold nanoparticles embedded 7 micrometers below the specimen surface reveals the occurrence of contiguous amorphous and crystalline phases within the bulk of the polymer and a preferential growth of the crystalline phase in the vicinity of the gold nanoparticles. A RDF-AFUM micrograph of LaRC-CP2 film containing randomly dispersed carbon nanotubes reveals the growth of an interphase region at certain nanotube-polymer interfaces.

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

    Science.gov (United States)

    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.

  14. Electron Microscopy and Image Analysis for Selected Materials

    Science.gov (United States)

    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.

  15. In vivo imaging of spinal cord in contusion injury model mice by multi-photon microscopy

    Science.gov (United States)

    Oshima, Y.; Horiuchi, H.; Ogata, T.; Hikita, A.; Miura, H.; Imamura, T.

    2014-03-01

    Fluorescent imaging technique is a promising method and has been developed for in vivo applications in cellular biology. In particular, nonlinear optical imaging technique, multi-photon microscopy has make it possible to analyze deep portion of tissues in living animals such as axons of spinal code. Traumatic spinal cord injuries (SCIs) are usually caused by contusion damages. Therefore, observation of spinal cord tissue after the contusion injury is necessary for understanding cellular dynamics in response to traumatic SCI and development of the treatment for traumatic SCI. Our goal is elucidation of mechanism for degeneration of axons after contusion injuries by establishing SCI model and chronic observation of injured axons in the living animals. Firstly we generated and observed acute SCI model by contusion injury. By using a multi-photon microscope, axons in dorsal cord were visualized approximately 140 micron in depth from the surface. Immediately after injury, minimal morphological change of spinal cord was observed. At 3 days after injury, spinal cord was swelling and the axons seem to be fragmented. At 7 days after injury, increased degradation of axons could be observed, although the image was blurred due to accumulation of the connective tissue. In the present study, we successfully observed axon degeneration after the contusion SCI in a living animal in vivo. Our final goal is to understand molecular mechanisms and cellular dynamics in response to traumatic SCIs in acute and chronic stage.

  16. Gradient light interference microscopy (GLIM) for imaging thick specimens (Conference Presentation)

    Science.gov (United States)

    Nguyen, Tan H.; Kandel, Mikhail E.; Popescu, Gabriel

    2016-03-01

    Compared to the Phase Contrast, Differential Interference Contrast (DIC) has been known to give higher depth sectioning as well as a halo-free images when investigating transparent specimens. Thanks to relying on generating two slightly shifted replicas with a small amount of shift, within the coherence area, DIC is able to operate with very low coherence light. More importantly, the method is able to work with very large numerical aperture of the illumination, which offer comparable sectioning capability to bright field microscopy. However, DIC is still a qualitative method, which limits potential applications of the technique. In this paper, we introduce a method that extends the capability of DIC by combining it with a phase shifting module to extract the phase gradient information. A theoretical model of the image formation is developed and the possibility of integrating the gradient function is analyzed.. Our method is benchmarked on imaging embryos during their 7-day development, HeLa cells during mitosis, and control samples.

  17. Quantitative Imaging of Microwave Electric Fields through Near-Field Scanning Microwave Microscopy

    Science.gov (United States)

    Dutta, S. K.; Vlahacos, C. P.; Steinhauer, D. E.; Thanawalla, A.; Feenstra, B. J.; Wellstood, F. C.; Anlage, Steven M.; Newman, H. S.

    1998-03-01

    The ability to non-destructively image electric field patterns generated by operating microwave devices (e.g. filters, antennas, circulators, etc.) would greatly aid in the design and testing of these structures. Such detailed information can be used to reconcile discrepancies between simulated behavior and experimental data (such as scattering parameters). The near-field scanning microwave microscope we present uses a coaxial probe to provide a simple, broadband method of imaging electric fields.(S. M. Anlage, et al.) IEEE Trans. Appl. Supercond. 7, 3686 (1997).^,(See http://www.csr.umd.edu/research/hifreq/micr_microscopy.html) The signal that is measured is related to the incident electric flux normal to the face of the center conductor of the probe, allowing different components of the field to be measured by orienting the probe appropriately. By using a simple model of the system, we can also convert raw data to absolute electric field. Detailed images of standing waves on copper microstrip will be shown and compared to theory.

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

    Science.gov (United States)

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

    2014-03-01

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

  19. Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering.

    Science.gov (United States)

    Dumas, D; Gaborit, N; Grossin, L; Riquelme, B; Gigant-Huselstein, C; De Isla, N; Gillet, P; Netter, P; Stoltz, J F

    2004-01-01

    Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).

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

    Science.gov (United States)

    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.

  1. Magni: A Python Package for Compressive Sampling and Reconstruction of Atomic Force Microscopy Images

    DEFF Research Database (Denmark)

    Oxvig, Christian Schou; Pedersen, Patrick Steffen; Arildsen, Thomas

    2014-01-01

    provides researchers in compressed sensing with a selection of algorithms for reconstructing undersampled general images, and offers a consistent and rigorous way to efficiently evaluate the researchers own developed reconstruction algorithms in terms of phase transitions. The package also serves......Magni is an open source Python package that embraces compressed sensing and Atomic Force Microscopy (AFM) imaging techniques. It provides AFM-specific functionality for undersampling and reconstructing images from AFM equipment and thereby accelerating the acquisition of AFM images. Magni also...

  2. In situ visualization of dermal collagen dynamics during skin burn healing using second-harmonic-generation microscopy

    Science.gov (United States)

    Yasui, Takeshi; Hase, Eiji; Tanaka, Ryosuke; Fukushima, Shu-ichiro; Araki, Tsutomu

    2015-06-01

    Burn healing is a process to repair thermally damaged tissues. Although burn healing has many aspects, it is common for dynamics of collagen fiber, such as decomposition, production, or growth, to be closely related with burn healing. If such healing process can be visualized from the viewpoint of the collagen dynamics, one may obtain new findings regarding biological repairing mechanisms in the healing process. To this end, second-harmonic-generation (SHG) light will be an effective optical probe because of high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional (3D) sectioning, minimal invasiveness, deep penetration, the absence of interference from background light, and in situ measurement without additional staining. Furthermore, since SHG light arises from a non-centrosymmetric triple helix of three polypeptide chains in the collagen molecule, its intensity decreases and finally disappears when thermal denaturation caused by the skin burn changes the structure of this molecule to a centrosymmetric random coil. Therefore, optical assessment of skin burn has been investigated by SHG microscopy. In this paper, we applied SHG microscopy for in situ imaging of the healing process in animal skin burn and successfully visualized the decomposition, production, and growth of renewal collagen fibers as a series of time-lapse images in the same subject.

  3. Automatic detection of NIL defects using microscopy and image processing

    KAUST Repository

    Pietroy, David

    2013-12-01

    Nanoimprint Lithography (NIL) is a promising technology for low cost and large scale nanostructure fabrication. This technique is based on a contact molding-demolding process, that can produce number of defects such as incomplete filling, negative patterns, sticking. In this paper, microscopic imaging combined to a specific processing algorithm is used to detect numerically defects in printed patterns. Results obtained for 1D and 2D imprinted gratings with different microscopic image magnifications are presented. Results are independent on the device which captures the image (optical, confocal or electron microscope). The use of numerical images allows the possibility to automate the detection and to compute a statistical analysis of defects. This method provides a fast analysis of printed gratings and could be used to monitor the production of such structures. © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    Carvalho, Lara; Heisenberg, Carl-Philipp

    2009-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    唐志列; 杨初平; 裴红津; 梁瑞生; 刘颂豪

    2002-01-01

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

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

    Science.gov (United States)

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

    2013-07-01

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

  8. Electron Microscopy Imaging of Single-Wall Carbon Nanotube Networks in Polymers

    Science.gov (United States)

    Jesse, Stephen; Guillorn, Michael; Ivanov, Ilia; Puretzky, Alex; Howe, Jane; Britt, Phillip; Geohegan, David

    2004-03-01

    Scanning electron microscopy (SEM) imaging techniques have been applied to study the electrical transport properties of conducting networks of single-walled carbon nanotubes (SWNTs) in insulating polymers. Two SEM techniques were used. One approach uses specimen current (SC) measurements to visualize current flow within the SWNT network. Another and novel approach is highly sensitive to electrical potential within the networks and occurs as a result of the large electric fields generated in the vicinity of the nanotube bundles. High-resolution transmission electron microscopy was used to characterize the SWNT bundles in the PMMA. These techniques permit a direct experimental approach to characterize and understand potential distribution and current flow through percolation networks formed by nanotube bundles in polymers, or more generally, nanorods or nanowires in various matrices. This research was sponsored by NASA-Langley Research Center and the Laboratory-Directed Research and Development Program at ORNL, and the U.S. Department of Energy under contract DE-AC05-00OR22725 with the Oak Ridge National Laboratory, managed by UT-Battelle, LLC.

  9. Whole slide imaging of unstained tissue using lensfree microscopy

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. High resolution imaging using scanning ion conductance microscopy with improved distance feedback control

    Institute of Scientific and Technical Information of China (English)

    Chao Li; Nicholas Johnson; Victor Ostanin; Andrew Shevchuk; Liming Ying; Yuri Korchev; David Klenerman

    2008-01-01

    Microscopy is an essential technique for observation on living cells. There is currently great interest in applying scanning probe microscopy to image-living biological cells in their natural environment at the nanometer scale. Scanning ion conductance microscopy is a new form of scanning probe microscopy, which enables non-contact high-resolution imaging of living biological cells. Based on a scanned nanopipette in physiological buffer, the distance feedback control uses the ion current to control the distance between the pipette tip and the sample surface. However, this feedback control has difficulties over slopes on convoluted cell surfaces, which limits its resolution. In this study, we present an improved form of feedback control that removes the contribution of up to the third-order slope from the ion current signal, hence providing a more accurate signal for controlling the distance. We show that this allows faster and lower noise topographic high-resolution imaging.

  12. A custom CMOS imager for multi-beam laser scanning microscopy and an improvement of scanning speed

    Science.gov (United States)

    Seo, Min-Woong; Kagawa, Keiichiro; Yasutomi, Keita; Kawahito, Shoji

    2013-02-01

    Multi-beam laser scanning confocal microscopy with a 256 × 256-pixel custom CMOS imager performing focal-plane pinhole effect, in which any rotating disk is not required, is demonstrated. A specimen is illuminated by 32 × 32 diffraction limited light spots whose wavelength and pitch are 532nm and 8.4 μm, respectively. The spot array is generated by a microlens array, which is scanned by two-dimensional piezo actuator according to the scanning of the image sensor. The frame rate of the prototype is 0.17 Hz, which is limited by the actuator. The confocal effect has been confirmed by comparing the axial resolution in the confocal imaging mode with that of the normal imaging mode. The axial resolution in the confocal mode measured by the full width at half maximum (FWHM) for a planar mirror was 8.9 μm, which is showed that the confocality has been achieved with the proposed CMOS image sensor. The focal-plane pinhole effect in the confocal microscopy with the proposed CMOS imager has been demonstrated at low frame rate. An improvement of the scanning speed and a CMOS imager with photo-sensitivity modulation pixels suitable for high-speed scanning are also discussed.

  13. A minimal optical trapping and imaging microscopy system.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.

  14. Transillumination spatially modulated illumination microscopy for human chromosome imaging

    Science.gov (United States)

    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.

  15. Quantitative imaging of complex samples by spiral phase contrast microscopy.

    Science.gov (United States)

    Bernet, Stefan; Jesacher, Alexander; Fürhapter, Severin; Maurer, Christian; Ritsch-Marte, Monika

    2006-05-01

    Recently a spatial spiral phase filter in a Fourier plane of a microscopic imaging setup has been demonstrated to produce edge enhancement and relief-like shadow formation of amplitude and phase samples. Here we demonstrate that a sequence of at least 3 spatially filtered images, which are recorded with different rotational orientations of the spiral phase plate, can be used to obtain a quantitative reconstruction of both, amplitude and phase information of a complex microscopic sample, i.e. an object consisting of mixed absorptive and refractive components. The method is demonstrated using a calibrated phase sample, and an epithelial cheek cell.

  16. Resonant second-harmonic-generation circular-dichroism microscopy reveals molecular chirality in native biological tissues

    CERN Document Server

    Chen, Mei-Yu; Kan, Che-Wei; Lin, Yen-Yin; Ye, Cin-Wei; Wu, Meng-Jer; Liu, Hsiang-Lin; Chu, Shi-Wei

    2016-01-01

    Conventional linear optical activity effects are widely used for studying chiral materials. However, poor contrast and artifacts due to sample anisotropy limit the applicability of these methods. Here we demonstrate that nonlinear second-harmonic-generation circular dichroism spectral microscopy can overcome these limits. In intact collagenous tissues, clear spectral resonance is observed with sub-micrometer spatial resolution. By performing gradual protein denaturation studies, we show that the resonant responses are dominantly due to the molecular chirality.

  17. Multimodal optical setup for nonlinear and fluorescence lifetime imaging microscopies: improvement on a commercial confocal inverted microscope

    Science.gov (United States)

    Pelegati, V. B.; Adur, J.; de Thomaz, A. A.; Almeida, D. B.; Baratti, M. O.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    In this work we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus FV300) to include nonlinear optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). The NLO microscopies included two-photon fluorescence (TPFE), Second Harmonic Generation (SHG) and Third Harmonic Generation (THG). The whole system, including FLIM, used only one laser source composed of an 80 MHz femtosecond laser. The commercial Ti:sapphire lasers can be tuned up to 690-1040 nm bringing the THG signal to the 350 nm region where most microscope optics do not work. However, the third harmonic is only generated at the sample, meaning that we only have to take care of the collection optics. To do that we used a remote photomultiplier to acquire the THG signal at the 310-350 nm wavelength window. After performing the tests to guarantee that we are observing actually SHG/THG signals we than used this system to acquire multimodal images of several biological samples, from epithelial cancer to vegetables. The ability to see the collagen network together with the cell nuclei proved to be important for cancer tissues diagnosis. Moreover, FLIM provides information about the cell metabolism, also very important for cancer cell processes.

  18. Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy.

    Directory of Open Access Journals (Sweden)

    Kazuo Yamagata

    Full Text Available 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.

  19. Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daldrup-Link, Heike E. [Department of Radiology, UCSF Medical Center, University of California in San Francisco, 513 Parnassus Ave, CA 94143, San Francisco (United States); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Institute of Pathology, Technical University, Munich (Germany); Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Department of Radiology, Technical University, Munich (Germany); Pichler, Bernd [Department of Biomedical Engineering, University of California Davis, Davis (United States); Heinzmann, Ulrich [National Research Center for Environment and Health, Technical University, Munich (Germany); Oostendorp, Robert A.J. [3. Clinic of Internal Medicine, Laboratory of Stem Cell Physiology, Technical University, Munich (Germany)

    2004-09-01

    The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1 x 10{sup 6}-3 x 10{sup 8} labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10{sup 6} cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells

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

    Science.gov (United States)

    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

  1. Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

    Science.gov (United States)

    Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

    2014-01-01

    Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis

    NARCIS (Netherlands)

    Ritsma, L.; Steller, E.J.; Beerling, E.; Loomans, C.J.; Zomer, A.; Gerlach, C.; Vrisekoop, N.; Seinstra, D.; van Gurp, L.; Schafer, R.; Raats, D.A.; de Graaff, A.; Schumacher, T.N.; de Koning, E.; Rinkes, I.H.; Kranenburg, O.; van Rheenen, J.

    2012-01-01

    Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We

  3. Combination of a spinning disc confocal unit with frequency-domain fluorescence lifetime imaging microscopy.

    NARCIS (Netherlands)

    van Munster, E.B.; Goedhart, J.; Kremers, G.J.; Manders, E.M.M.; Gadella, Th.W.J.

    2007-01-01

    BACKGROUND: Wide-field frequency-domain fluorescence lifetime imaging microscopy (FLIM) is an established technique to determine fluorescence lifetimes. Disadvantage of wide-field imaging is that measurements are compromised by out-of-focus blur. Conventional scanning confocal typically means long

  4. Cellular features of psoriatic skin: imaging and quantification using in vivo reflectance confocal microscopy

    NARCIS (Netherlands)

    Wolberink, E.A.W.; Erp, P.E.J. van; Teussink, M.M.; Kerkhof, P.C.M. van de; Gerritsen, M.J.P.

    2011-01-01

    BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250

  5. Submolecular Resolution Imaging of molecules by Atomic Force Microscopy:The influence of the Electrostatic Force

    NARCIS (Netherlands)

    van der Lit, J.; Cicco, F.; Hapala, P.; Jelinek, P.; Swart, Ingmar

    2016-01-01

    The forces governing the contrast in submolecular resolution imaging of molecules with atomic force microscopy (AFM) have recently become a topic of intense debate. Here, we show that the electrostatic force is essential to understand the contrast in atomically resolved AFM images of polar molecules

  6. Application of quantitative second-harmonic generation microscopy to dynamic conditions.

    Science.gov (United States)

    Kabir, Mohammad M; Inavalli, V V G Krishna; Lau, Tung-Yuen; Toussaint, Kimani C

    2013-01-01

    We present a quantitative second-harmonic generation (SHG) imaging technique that quantifies the 2D spatial organization of collagen fiber samples under dynamic conditions, as an image is acquired. The technique is demonstrated for both a well-aligned tendon sample and a randomly aligned, sparsely distributed collagen scaffold sample. For a fixed signal-to-noise ratio, we confirm the applicability of this method for various window sizes (pixel areas) as well as with using a gridded overlay map that allows for correlations of fiber orientations within a given image. This work has direct impact to in vivo biological studies by incorporating simultaneous SHG image acquisition and analysis.

  7. The Next Generation of HLA Image Products

    Science.gov (United States)

    Gaffney, N. I.; Casertano, S.; Ferguson, B.

    2012-09-01

    We present the re-engineered pipeline based on existing and improved algorithms with the aim of improving processing quality, cross-instrument portability, data flow management, and software maintenance. The Hubble Legacy Archive (HLA) is a project to add value to the Hubble Space Telescope data archive by producing and delivering science-ready drizzled data products and source lists derived from these products. Initially, ACS, NICMOS, and WFCP2 data were combined using instrument-specific pipelines based on scripts developed to process the ACS GOODS data and a separate set of scripts to generate source extractor and DAOPhot source lists. The new pipeline, initially designed for WFC3 data, isolates instrument-specific processing and is easily extendable to other instruments and to generating wide-area mosaics. Significant improvements have been made in image combination using improved alignment, source detection, and background equalization routines. It integrates improved alignment procedures, better noise model, and source list generation within a single code base. Wherever practical, PyRAF based routines have been replaced with non-IRAF based python libraries (e.g. NumPy and PyFITS). The data formats have been modified to handle better and more consistent propagation of information from individual exposures to the combined products. A new exposure layer stores the effective exposure time for each pixel in the sky which is key in properly interpreting combined images from diverse data that were not initially planned to be mosaiced. We worked to improve the validity of the metadata within our FITS headers for these products relative to standard IRAF/PyRAF processing. Any keywords that pertain to individual exposures have been removed from the primary and extension headers and placed in a table extension for more direct and efficient perusal. This mechanism also allows for more detailed information on the processing of individual images to be stored and propagated

  8. Three-dimensional second-harmonic generation imaging of fibrillar collagen in biological tissues.

    Science.gov (United States)

    Xie, Jiansong; Ferbas, John; Juan, Gloria

    2012-07-01

    Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues. Some technical considerations are also addressed.

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

    Science.gov (United States)

    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.

  10. Imaging of dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy.

    Science.gov (United States)

    Wang, Xiaohua; Teng, Yan; Wang, Qinli; Li, Xiaojuan; Sheng, Xianyong; Zheng, Maozhong; Samaj, Jozef; Baluska, Frantisek; Lin, Jinxing

    2006-08-01

    Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

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

    Science.gov (United States)

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

    2010-02-01

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

  12. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    Directory of Open Access Journals (Sweden)

    Emilio J Gualda

    2014-08-01

    Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  13. Automated analysis of heterogeneous carbon nanostructures by high-resolution electron microscopy and on-line image processing

    Energy Technology Data Exchange (ETDEWEB)

    Toth, P., E-mail: toth.pal@uni-miskolc.hu [Department of Chemical Engineering, University of Utah, 50 S. Central Campus Drive, Salt Lake City, UT 84112-9203 (United States); Farrer, J.K. [Department of Physics and Astronomy, Brigham Young University, N283 ESC, Provo, UT 84602 (United States); Palotas, A.B. [Department of Combustion Technology and Thermal Energy, University of Miskolc, H3515, Miskolc-Egyetemvaros (Hungary); Lighty, J.S.; Eddings, E.G. [Department of Chemical Engineering, University of Utah, 50 S. Central Campus Drive, Salt Lake City, UT 84112-9203 (United States)

    2013-06-15

    High-resolution electron microscopy is an efficient tool for characterizing heterogeneous nanostructures; however, currently the analysis is a laborious and time-consuming manual process. In order to be able to accurately and robustly quantify heterostructures, one must obtain a statistically high number of micrographs showing images of the appropriate sub-structures. The second step of analysis is usually the application of digital image processing techniques in order to extract meaningful structural descriptors from the acquired images. In this paper it will be shown that by applying on-line image processing and basic machine vision algorithms, it is possible to fully automate the image acquisition step; therefore, the number of acquired images in a given time can be increased drastically without the need for additional human labor. The proposed automation technique works by computing fields of structural descriptors in situ and thus outputs sets of the desired structural descriptors in real-time. The merits of the method are demonstrated by using combustion-generated black carbon samples. - Highlights: ► The HRTEM analysis of heterogeneous nanostructures is a tedious manual process. ► Automatic HRTEM image acquisition and analysis can improve data quantity and quality. ► We propose a method based on on-line image analysis for the automation of HRTEM image acquisition. ► The proposed method is demonstrated using HRTEM images of soot particles.

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

    Science.gov (United States)

    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.

  15. Imaging of magnetic and electric fields by electron microscopy.

    Science.gov (United States)

    Zweck, Josef

    2016-10-12

    Nanostructured materials become more and more a part of our daily life, partly as self-assembled particles or artificially patterned. These nanostructures often possess intrinsic magnetic and/or electric fields which determine (at least partially) their physical properties. Therefore it is important to be able to measure these fields reliably on a nanometre scale. A rather common instrument for the investigation of these fields is the transmission electron microscope as it offers high spatial resolution. The use of an electron microscope to image electric and magnetic fields on a micron down to sub-nanometre scale is treated in detail for transmission electron microscopes (TEM) and scanning transmission electron microscopes (STEM). The formation of contrast is described for the most common imaging modes, the specific advantages and disadvantages of each technique are discussed and examples are given. In addition, the experimental requirements for the use of the techniques described are listed and explained.

  16. Simulation study of secondary electron images in scanning ion microscopy

    CERN Document Server

    Ohya, K

    2003-01-01

    The target atomic number, Z sub 2 , dependence of secondary electron yield is simulated by applying a Monte Carlo code for 17 species of metals bombarded by Ga ions and electrons in order to study the contrast difference between scanning ion microscopes (SIM) and scanning electron microscopes (SEM). In addition to the remarkable reversal of the Z sub 2 dependence between the Ga ion and electron bombardment, a fine structure, which is correlated to the density of the conduction band electrons in the metal, is calculated for both. The brightness changes of the secondary electron images in SIM and SEM are simulated using Au and Al surfaces adjacent to each other. The results indicate that the image contrast in SIM is much more sensitive to the material species and is clearer than that for SEM. The origin of the difference between SIM and SEM comes from the difference in the lateral distribution of secondary electrons excited within the escape depth.

  17. Imaging of magnetic and electric fields by electron microscopy

    Science.gov (United States)

    Zweck, Josef

    2016-10-01

    Nanostructured materials become more and more a part of our daily life, partly as self-assembled particles or artificially patterned. These nanostructures often possess intrinsic magnetic and/or electric fields which determine (at least partially) their physical properties. Therefore it is important to be able to measure these fields reliably on a nanometre scale. A rather common instrument for the investigation of these fields is the transmission electron microscope as it offers high spatial resolution. The use of an electron microscope to image electric and magnetic fields on a micron down to sub-nanometre scale is treated in detail for transmission electron microscopes (TEM) and scanning transmission electron microscopes (STEM). The formation of contrast is described for the most common imaging modes, the specific advantages and disadvantages of each technique are discussed and examples are given. In addition, the experimental requirements for the use of the techniques described are listed and explained.

  18. Tomographic imaging and scanning thermal microscopy: thermal impedance tomography

    OpenAIRE

    2002-01-01

    The application of tomographic imaging techniques developed for medical applications to the data provided by the scanning thermal microscope will give access to true three-dimensional information on the thermal properties of materials on a mm length scale. In principle, the technique involves calculating and inverting a sensitivity matrix for a uniform isotropic material, collecting ordered data at several modulation frequencies, and multiplying the inverse of the matrix with the data vector....

  19. Imaging by Electrochemical Scanning Tunneling Microscopy and Deconvolution Resolving More Details of Surfaces Nanomorphology

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov

    to crystallographic-surface structures. Within the wide range of new technologies, those images surface features, the electrochemical scanning tunneling microscope (ESTM) provides means of atomic resolution where the tip participates actively in the process of imaging. Two metallic surfaces influence ions trapped...... of the characteristic details of the images. A large proportion of the observed noise may be explained by the scanning actions of the feedback circuitry while a minor fraction of the image details may be explained by surface drift phenomena. As opposed to the method of deconvolution, conventional methods of filtering......Upon imaging, electrochemical scanning tunneling microscopy (ESTM), scanning electrochemical micro-scopy (SECM) and in situ STM resolve information on electronic structures and on surface topography. At very high resolution, imaging processing is required, as to obtain information that relates...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Picosecond anti-Stokes generation in a photonic-crystal fiber for interferometric CARS microscopy.

    Science.gov (United States)

    Andresen, Esben Ravn; Keiding, Søren Rud; Potma, Eric Olaf

    2006-08-07

    We generate tunable picosecond anti-Stokes pulses by four-wave mixing of two picosecond pump and Stokes pulse trains in a photonic-crystal fiber. The visible, spectrally narrow anti-Stokes pulses with shifts over 150 nm are generated without generating other spectral features. As a demonstration, we employ the generated anti-Stokes pulses as reference pulses in an interferometric coherent anti-Stokes Raman scattering imaging experiment showing that interpulse coherence among the pump, Stokes and anti-Stokes beams is retained.

  3. Slide-free histology via MUSE: UV surface excitation microscopy for imaging unsectioned tissue (Conference Presentation)

    Science.gov (United States)

    Levenson, Richard M.; Harmany, Zachary; Demos, Stavros G.; Fereidouni, Farzad

    2016-03-01

    Widely used methods for preparing and viewing tissue specimens at microscopic resolution have not changed for over a century. They provide high-quality images but can involve time-frames of hours or even weeks, depending on logistics. There is increasing interest in slide-free methods for rapid tissue analysis that can both decrease turn-around times and reduce costs. One new approach is MUSE (microscopy with UV surface excitation), which exploits the shallow penetration of UV light to excite fluorescent signals from only the most superficial tissue elements. The method is non-destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin embedding, or thin sectioning. It requires no lasers, confocal, multiphoton or optical coherence tomography optics. MUSE generates diagnostic-quality histological images that can be rendered to resemble conventional hematoxylin- and eosin-stained samples, with enhanced topographical information, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in research facilities, hospital-based and stand-alone clinical settings, in local or regional pathology labs, as well as in low-resource environments.

  4. Imaging of normal and pathologic joint synovium using nonlinear optical microscopy as a potential diagnostic tool

    Science.gov (United States)

    Tiwari, Nivedan; Chabra, Sanjay; Mehdi, Sheherbano; Sweet, Paula; Krasieva, Tatiana B.; Pool, Roy; Andrews, Brian; Peavy, George M.

    2010-09-01

    An estimated 1.3 million people in the United States suffer from rheumatoid arthritis (RA). RA causes profound changes in the synovial membrane of joints, and without early diagnosis and intervention, progresses to permanent alterations in joint structure and function. The purpose of this study is to determine if nonlinear optical microscopy (NLOM) can utilize the natural intrinsic fluorescence properties of tissue to generate images that would allow visualization of the structural and cellular composition of fresh, unfixed normal and pathologic synovial tissue. NLOM is performed on rabbit knee joint synovial samples using 730- and 800-nm excitation wavelengths. Less than 30 mW of excitation power delivered with a 40×, 0.8-NA water immersion objective is sufficient for the visualization of synovial structures to a maximum depth of 70 μm without tissue damage. NLOM imaging of normal and pathologic synovial tissue reveals the cellular structure, synoviocytes, adipocytes, collagen, vascular structures, and differential characteristics of inflammatory infiltrates without requiring tissue processing or staining. Further study to evaluate the ability of NLOM to assess the characteristics of pathologic synovial tissue and its potential role for the management of disease is warranted.

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

    Science.gov (United States)

    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. ©RSNA, 2015.

  6. Third Harmonic Generation microscopy as a diagnostic tool for the investigation of microglia BV-2 and breast cancer cells activation

    Science.gov (United States)

    Gavgiotaki, E.; Filippidis, G.; Psilodimitrakopoulos, S.; Markomanolaki, H.; Kalognomou, M.; Agelaki, S.; Georgoulias, V.; Athanassakis, I.

    2015-07-01

    Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field. Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the activation state of different cell types by using the THG imaging microscopy as a diagnostic tool. BV-2 microglia cell line was used as a representative biological model enabling the study of resting and activated state of the cells linked to various pathological conditions. Third Harmonic Generation (THG) and Two Photon Excitation Fluorescence (TPEF) measurements were simultaneously collected from stained breast cancer cells, by employing a single homemade experimental apparatus and it was shown that high THG signals mostly arise from lipid bodies. Continuously, BV-2 microglia cells were examined with or without activation by lipopolysaccharide (LPS) in order to discriminate between control and activated cells based on the quantification of THG signals. Statistically quantification was accomplished in both mean area and mean intensity values of THG. The values for mean total area and mean THG intensity values have been increased in activated versus the non-activated cells. Similar studies of quantification are underway in breast cancer cells for the exact discrimination on different cell lines. Furthermore, laser polarization dependence of SHG and THG signal in unstained biological samples is investigated.

  7. Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy.

    Science.gov (United States)

    Ranjit, Suman; Dvornikov, Alexander; Dobrinskikh, Evgenia; Wang, Xiaoxin; Luo, Yuhuan; Levi, Moshe; Gratton, Enrico

    2017-07-01

    The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet.

  8. Image simulations of kinked vortices for transmission electron microscopy

    DEFF Research Database (Denmark)

    Beleggia, Marco; Pozzi, G.; Tonomura, A.

    2010-01-01

    We present an improved model of kinked vortices in high-Tc superconductors suitable for the interpretation of Fresnel or holographic observations carried out with a transmission electron microscope. A kinked vortex is composed of two displaced half-vortices, perpendicular to the film plane...... observations of high-Tc superconducting films, where the Fresnel contrast associated with some vortices showed a dumbbell like appearance. Here, we show that under suitable conditions the JV segment may reveal itself in Fresnel imaging or holographic phase mapping in a transmission electron microscope....

  9. Vibrational imaging based on stimulated Raman scattering microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Nandakumar, P; Kovalev, A; Volkmer, A [3. Physikalisches Institut, Universitaet Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart (Germany)], E-mail: a.volkmer@physik.uni-stuttgart.de

    2009-03-15

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  10. Vibrational imaging based on stimulated Raman scattering microscopy

    Science.gov (United States)

    Nandakumar, P.; Kovalev, A.; Volkmer, A.

    2009-03-01

    A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76 MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes.

  11. Multimodal imaging of vocal fold scarring in a rabbit model by multiphoton microscopy

    Science.gov (United States)

    Kazarine, Alexei; Bouhabel, Sarah; Douillette, Annie H.; Kost, Karen; Li-Jessen, Nicole Y. K.; Mongeau, Luc; Wiseman, Paul W.

    2017-02-01

    Vocal fold scarring as a result of injury or disease can lead to voice disorders which can significantly affect the quality of life. During the scarring process, the normally elastic tissue of the vocal fold lamina propria is replaced by a much stiffer collagen-based fibrotic tissue, which impacts the fold's ability to vibrate. Surgical removal of this tissue is often ineffective and can result in further scarring. Injectable biomaterials, a form of tissue engineering, have been proposed as a potential solution to reduce existing scars or prevent scarring altogether. In order to properly evaluate the effectiveness of these new materials, multiphoton microscopy emerges as an effective tool due to its intrinsic multiple label free contrast mechanisms that highlight extracellular matrix elements. In this study, we evaluate the spatial distribution of collagen and elastin fibers in a rabbit model using second harmonic generation (SHG), third harmonic generation (THG) and two photon autofluorescence (TPAF) applied to unlabeled tissue sections. In comparison to traditional methods that rely on histological staining or immunohistochemistry, SHG, THG and TPAF provide a more reliable detection of these native proteins. The evaluation of collagen levels allows us to follow the extent of scarring, while the presence of elastin fibers is thought to be indicative of the level of healing of the injured fold. Using these imaging modalities, we characterize the outcome of injectable biomaterial treatments in order to direct future treatments for tissue engineering.

  12. Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

    Directory of Open Access Journals (Sweden)

    Jun Miyazaki

    2017-04-01

    Full Text Available Photothermal microscopy is useful to visualize the distribution of non-fluorescence chromoproteins in biological specimens. Here, we developed a high sensitivity and high resolution photothermal microscopy with low-cost and compact laser diodes as light sources. A new detection scheme for improving signal to noise ratio more than 4-fold is presented. It is demonstrated that spatial resolution in photothermal microscopy is up to nearly twice as high as that in the conventional widefield microscopy. Furthermore, we demonstrated the ability for distinguishing or identifying biological molecules with simultaneous muti-wavelength imaging. Simultaneous photothermal and fluorescence imaging of mouse brain tissue was conducted to visualize both neurons expressing yellow fluorescent protein and endogenous non-fluorescent chromophores.

  13. Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

    CERN Document Server

    Rupprecht, Peter; Groessl, Florian; Haubensak, Wulf E; Vaziri, Alipasha

    2015-01-01

    A number of questions in systems 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 organism. However, the broader applicability of light-sculpting microscopy 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 par...

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

    Science.gov (United States)

    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.

  15. Organization of astaxanthin within oil bodies of Haematococcus pluvialis studied with polarization-dependent harmonic generation microscopy.

    Science.gov (United States)

    Tokarz, Danielle; Cisek, Richard; El-Ansari, Omar; Espie, George S; Fekl, Ulrich; Barzda, Virginijus

    2014-01-01

    Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Efficient Parallel Levenberg-Marquardt Model Fitting towards Real-Time Automated Parametric Imaging Microscopy

    OpenAIRE

    Xiang Zhu; Dianwen Zhang

    2013-01-01

    We present a fast, accurate and robust parallel Levenberg-Marquardt minimization optimizer, GPU-LMFit, which is implemented on graphics processing unit for high performance scalable parallel model fitting processing. GPU-LMFit can provide a dramatic speed-up in massive model fitting analyses to enable real-time automated pixel-wise parametric imaging microscopy. We demonstrate the performance of GPU-LMFit for the applications in superresolution localization microscopy and fluorescence lifetim...

  19. Deep focus; a digital image processing technique to produce improved focal depth in light microscopy:

    OpenAIRE

    Goldsmith, Noel T.

    2000-01-01

    In light microscopy, the spatial transverse resolution is a function of the wavelength and numerical aperture. The depth resolution is another function of these parameters. The factors that enable the detection of fine detail, make the sharp focusing of more than a thin slice of the depth in an object impossible. When the examination of fracture surfaces is attempted using light reflection microscopy, the roughness will often restrict the in-focus parts of an image to a small portion of the f...

  20. Using Second Harmonic Generation Microscopy to Study the Three-Dimensional Structure of Collagen and its Degradation Mechanism

    Science.gov (United States)

    Mega, Yair

    Collagen is one of the most abundant proteins found in the human body. Its crystalline structure possesses no centrosymmetry, allowing it to emit second-harmonic waves. Second harmonic generation (SHG) microscopy utilizes the latter quality to produce high-resolution images of collagen rich tissues and therefore become a key research tool in the biomedical field. We developed a new model, intended to be used together with second harmonic generation (SHG) microscopy, to thoroughly investigate collagen-based tissues. We use our SHG model to reveal information in real time from enzymatic biochemical processes. We also present a novel method used to measure quantitatively the direction of the fibers within the tissue, from SHG images. Using this method, we were able to reconstruct an angular map of the orientation of collagen fibers from multiple sections across the entire area of a human cornea. The structure we obtained demonstrates the criss-crossing structure of the human cornea, previously suggested in the literature. In addition, we also report work on a unique step-wise three-photon fluorescence excitation discovered in melanin. This unique fluorescence mechanism was exploited to discriminate melanin on a small-size, low-cost and low laser power setup which was used as a prototype for a handheld device. The latter study is a part of a larger on-going effort in our group to explore new diagnosis methods to be used for early skin cancer screening. Finally, this work demonstrates a spectroscopy-based method to correct for blood vessel thickness effect. The method analyzes spectral shift from a molecular imaging agent and correlate the shifts to the length of the optical path in blood. The correction method described in this work is intended to be implemented on a guided catheter near infrared fluorescence (NIRF) intra-vascular imaging system. In this imaging system, this study's results will used to correct for the radial distance between the imaging tip of the

  1. Label-free 3D visualization of cellular and tissue structures in intact muscle with second and third harmonic generation microscopy.

    Directory of Open Access Journals (Sweden)

    Markus Rehberg

    Full Text Available Second and Third Harmonic Generation (SHG and THG microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy.

  2. Imaging of membrane proteins using antenna-based optical microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hoeppener, Christiane; Novotny, Lukas [Institute of Optics and Department of Biomedical Engineering, University of Rochester, Rochester, NY 14627 (United States)], E-mail: novotny@optics.rochester.edu

    2008-09-24

    The localization and identification of individual proteins is of key importance for the understanding of biological processes on the molecular scale. Here, we demonstrate near-field fluorescence imaging of single proteins in their native cell membrane. Incident laser radiation is localized and enhanced with an optical antenna in the form of a spherical gold particle attached to a pointed dielectric tip. Individual proteins can be identified with a diffraction-unlimited spatial resolution of {approx}50 nm. Besides determining the concentration and distribution of specific membrane proteins, this approach makes it possible to study the colocalization of different membrane proteins. Moreover, it enables a simultaneous recording of the membrane topology. Protein distributions can be correlated with the local membrane topology, thereby providing important information on the chemical and structural organization of cellular membranes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Jesse, Stephen [ORNL; Vasudevan, Dr. Rama [Oak Ridge National Laboratory (ORNL); Collins, Liam [University College, Dublin; Strelcov, Evgheni [ORNL; Okatan, Mahmut B [ORNL; Belianinov, Alex [ORNL; Baddorf, Arthur P [ORNL; Proksch, Roger [Asylum Research, Santa Barbara, CA; Kalinin, Sergei V [ORNL

    2014-01-01

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

  4. Photoelectron microscopy in the life sciences: Imaging neuron networks

    Energy Technology Data Exchange (ETDEWEB)

    Mercanti, D. (Istituto di Neurobiologia del CNR, Viale Marx 15, 00100 Roma (Italy)); De Stasio, G. (ISM-CNR, Via E. Fermi 38, 00044 Frascati, Roma (Italy)); Ciotti, M.T. (Istituto di Neurobiologia del CNR, Viale Marx 15, 00100 Roma (Italy)); Capasso, C.; Ng, W.; Ray-Chaudhuri, A.K.; Liang, S.H.; Cole, R.K.; Guo, Z.Y.; Wallace, J. (Department of Physics, University of Wisconsin, Madison, WI (USA) Electrical and Computer Engineering, University of Wisconsin, Madison, WI (USA)); Margaritondo, G. (Institut de Physique Appliquee, Ecole Polytechnique Federale de Lausanne, Ecublens (Switzerland)); Cerrina, F. (Departments of Physics, University of Wisconsin, Madison, WI (USA) Electrical and Computer Engineering, University of Wisconsin, Madison, WI (USA)); Underwood, J.; Perera, R.; Kortright, J. (Center for X-ray Optics, Lawrence Berkeley Laboratory, Berkeley, CA 94720 (USA))

    1991-05-01

    Photoemission techniques like electron spectroscopy for chemical analysis are the leading electronic probes in materials science---but their impact in the life sciences has been minimal. A critical problem is that the lateral resolution in ordinary photoemission does not exceed a few tenths of a millimeter. This space-averaged probe is nearly useless for most of the fundamental problems in biophysics and biochemistry, which deal with microstructures in the submicron range or smaller. This limit is being overcome with photoemission microscopes, such as our scanning instrument MAXIMUM. The first scanning photoelectron micrographs of a cellular system with submicron resolution are presented. Minute details of neuron networks are imaged on MAXIMUM, thereby opening the way to novel applications of photoemission in the life sciences. The details include individual neurons, axons, dendrites, and synapses, and composite large-area scanning micrographs were routinely produced with a lateral resolution of 0.5 {mu}m.

  5. Video Object Tracking in Neural Axons with Fluorescence Microscopy Images

    Directory of Open Access Journals (Sweden)

    Liang Yuan

    2014-01-01

    tracking. In this paper, we describe two automated tracking methods for analyzing neurofilament movement based on two different techniques: constrained particle filtering and tracking-by-detection. First, we introduce the constrained particle filtering approach. In this approach, the orientation and position of a particle are constrained by the axon’s shape such that fewer particles are necessary for tracking neurofilament movement than object tracking techniques based on generic particle filtering. Secondly, a tracking-by-detection approach to neurofilament tracking is presented. For this approach, the axon is decomposed into blocks, and the blocks encompassing the moving neurofilaments are detected by graph labeling using Markov random field. Finally, we compare two tracking methods by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and the experimental results show that both methods demonstrate good performance in comparison with the existing approaches, and the tracking accuracy of the tracing-by-detection approach is slightly better between the two.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    Science.gov (United States)

    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.

  9. High-sensitivity chemical imaging for biomedicine by SRS microscopy (Conference Presentation)

    Science.gov (United States)

    Min, Wei

    2017-02-01

    Innovations in spectroscopy principles and microscopy technology have significantly impacted modern biology and medicine. While most of the contemporary bio-imaging modalities harness electronic transition, nuclear spin or radioactivity, vibrational spectroscopy has not been widely used yet. Here we will discuss an emerging chemical imaging platform, stimulated Raman scattering (SRS) microscopy, which can enhance the otherwise feeble spontaneous Raman eight orders of magnitude by virtue of stimulated emission. When coupled with stable isotopes (e.g., deuterium and 13C) or bioorthogonal chemical moieties (e.g., alkynes), SRS microscopy is well suited for probing in vivo metabolic dynamics of small bio-molecules which cannot be labeled by bulky fluorophores. Physical principle of the underlying optical spectroscopy and exciting biomedical applications such as imaging lipid metabolism, protein synthesis, DNA replication, protein degradation, RNA synthesis, glucose uptake, drug trafficking and tumor metabolism will be presented.

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

    Science.gov (United States)

    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.

  11. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy

    Directory of Open Access Journals (Sweden)

    Segall Jeffrey E

    2005-05-01

    Full Text Available Abstract Background The development of multiphoton laser scanning microscopy has greatly facilitated the imaging of living tissues. However, the use of genetically encoded fluorescent proteins to distinguish different cell types in living animals has not been described at single cell resolution using multiphoton microscopy. Results Here we describe a method for the simultaneous imaging, by multiphoton microscopy, of Green Fluorescent Protein, Cyan Fluorescent Protein and collagen in vivo in living tumors. This novel method enables: 1 the simultaneous visualization of overall cell shape and sub-cellular structures such as the plasma membrane or proteins of interest in cells inside living animals, 2 direct comparison of the behavior of single cells from different cell lines in the same microenvironment in vivo. Conclusion Using this multi-fluor, multiphoton technique, we demonstrate that motility and metastatic differences between carcinoma cells of differing metastatic potential can be imaged in the same animal simultaneously at sub-cellular resolution.

  12. Automated quadrilateral mesh generation for digital image structures

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    With the development of advanced imaging technology, digital images are widely used. This paper proposes an automatic quadrilateral mesh generation algorithm for multi-colour imaged structures. It takes an original arbitrary digital image as an input for automatic quadrilateral mesh generation, this includes removing the noise, extracting and smoothing the boundary geometries between different colours, and automatic all-quad mesh generation with the above boundaries as constraints. An application example is...

  13. Orientation analysis of collagen fibers in healing tendon by using second-harmonic-generation microscopy

    Science.gov (United States)

    Hase, E.; Minamikawa, T.; Sato, K.; Yonekura, D.; Takahashi, M.; Yasui, T.

    2017-02-01

    Tendon rupture is a trauma that is difficult to fully recover from. Therefore, non-destructive and non-invasive evaluation method for the tendon healing is strongly required. In this study, we performed the orientation analysis of collagen fiber in healing tendon by two-dimensional Fourier transform (2D-FT) of SHG image. The extracted 2D-FT power spectra imply the correlation with the degree of the tendon healing. These results indicate that SHG microscopy has a unique potential as a non-destructive and non-invasive indicator of tendon healing.

  14. Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Leitner, Michael [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Stock, Lorenz G. [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Traxler, Lukas [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria); Leclercq, Laurent [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Bonazza, Klaus; Friedbacher, Gernot [Institute of Chemical Technologies and Analytics, Vienna University of Technology, Getreidemarkt 9/164, 1060 Vienna (Austria); Cottet, Hervé [Institut des Biomolécules Max Mousseron (IBMM, UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier), Place Eugène Bataillon, CC 1706, 34095 Montpellier (France); Stutz, Hanno [Division of Chemistry and Bioanalytics, Department of Molecular Biology, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Christian Doppler Laboratory for Innovative Tools for the Characterization of Biosimilars, University Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg (Austria); Ebner, Andreas, E-mail: andreas.ebner@jku.at [Institute of Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020 Linz (Austria)

    2016-08-03

    Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated. - Highlights: • SMIL coating allows generation of homogeneous ultra-flat surfaces. • Molecular electrostatic adhesion forces can be determined in the inner wall of CZE capillary with picoNewton accuracy. • Topographical images and simultaneously acquired adhesion maps yield morphological and chemical information at the nanoscale.

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

    Science.gov (United States)

    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. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

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

    Science.gov (United States)

    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.

  17. Correlation of live-cell imaging with volume scanning electron microscopy.

    Science.gov (United States)

    Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

    2017-01-01

    Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  19. Nanoscale imaging of untreated mammalian cells in a medium with low radiation damage using scanning electron-assisted dielectric microscopy

    Science.gov (United States)

    Okada, Tomoko; Ogura, Toshihiko

    2016-01-01

    Imaging of untreated living cells in a medium at a nanometre-scale resolution under physiological conditions is a significant challenge. Scanning electron microscopy (SEM) is widely used to observe cells in various atmospheric holders or special equipment. However, untreated biological specimens in aqueous solution generally incur heavy radiation damage from the direct electron beam (EB); and these images exhibit very poor contrast. Therefore, a new method for generating high-contrast images of living cells under physiological conditions without radiation damage has been strongly desired. Here, we demonstrate the first nanoscale observation of living cultured mammalian cells using our newly developed scanning-electron assisted dielectric microscopy (SE-ADM) method with a culture dish holder. Using the difference in relative permittivity between water and specimens, our SE-ADM system aids in the visualisation of untreated biological samples in aqueous solution. In addition, specimens incurred only a low level of radiation damage because the tungsten (W)-coated silicon nitride (SiN) film absorbs irradiated electrons. Untreated cells and organelles are clearly visible in high-contrast and high-resolution images without staining and fixation. Furthermore, our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage. PMID:27375121

  20. Nanoscale imaging of untreated mammalian cells in a medium with low radiation damage using scanning electron-assisted dielectric microscopy

    Science.gov (United States)

    Okada, Tomoko; Ogura, Toshihiko

    2016-07-01

    Imaging of untreated living cells in a medium at a nanometre-scale resolution under physiological conditions is a significant challenge. Scanning electron microscopy (SEM) is widely used to observe cells in various atmospheric holders or special equipment. However, untreated biological specimens in aqueous solution generally incur heavy radiation damage from the direct electron beam (EB); and these images exhibit very poor contrast. Therefore, a new method for generating high-contrast images of living cells under physiological conditions without radiation damage has been strongly desired. Here, we demonstrate the first nanoscale observation of living cultured mammalian cells using our newly developed scanning-electron assisted dielectric microscopy (SE-ADM) method with a culture dish holder. Using the difference in relative permittivity between water and specimens, our SE-ADM system aids in the visualisation of untreated biological samples in aqueous solution. In addition, specimens incurred only a low level of radiation damage because the tungsten (W)-coated silicon nitride (SiN) film absorbs irradiated electrons. Untreated cells and organelles are clearly visible in high-contrast and high-resolution images without staining and fixation. Furthermore, our method enables the detection of changes in organelle structures within cells via time-lapse imaging with minimal radiation damage.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  3. Methods of peripheral nerve tissue preparation for second harmonic generation imaging of collagen fibers.

    Science.gov (United States)

    Vijayaraghavan, Surabhi; Huq, Rumana; Hausman, Michael R

    2014-03-15

    Second harmonic generation (SHG) imaging of the peripheral nerve using multi-photon microscopy is a novel technique with little documentation. It affords the significant possibility of non-destructive imaging of internal nerve anatomy. The nature of nerve tissue, especially its size and viscoelastic properties, present special challenges for microscopy. While nerves are under an innate in situ strain, they retract once dissected, thus distorting microscopic structure. The challenge is to preserve the nerve in its natural strain range to obtain images that most truly reveal its structure. This study examined backscattered SHG images of rat median nerve prepared by several different methods to compare image quality and content. Nerve segments were fixed under strained (constant load or length) and unstrained conditions and imaged as whole nerve as well as plastic (methyl methacrylate) and paraffin embedded sections. These were tested for optimal excitation wavelength, quantitative image contrast, and overall quality. Root mean squared (RMS) contrast proved to be a reliable measure of the level of image contrast perceived by eye. We concluded that images obtained from tissue sections (plastic and paraffin) provided the most accurate and revealing SHG images of peripheral nerve structure. Removing the embedding material prior to imaging significantly improved image quality. Optimal excitation wavelengths were consistent regardless of the preparation method. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Hofer, Manuel; Adamsmaier, Stefan [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Zanten, Thomas S. van [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Chtcheglova, Lilia A. [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Manzo, Carlo [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); Duman, Memed [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Mayer, Barbara [Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Ebner, Andreas [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Moertelmaier, Manuel; Kada, Gerald [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria); Garcia-Parajo, Maria F. [IBEC-Institute for Bioengineering of Catalonia and CIBER-Bbn, Baldiri i Reixac 15-21, Barcelona 08028 (Spain); ICREA-Institucio Catalana de Recerca i Estudis Avancats, 08010 Barcelona (Spain); Hinterdorfer, Peter, E-mail: peter.hinterdorfer@jku.at [University of Linz, Institute for Biophysics, Altenbergerstr. 69, 4040 Linz (Austria); Christian Doppler Laboratory for Nanoscopic Methods in Biophysics, Institute for Biophysics, University of Linz, Altenbergerstr. 69, 4040 Linz (Austria); Kienberger, Ferry [Agilent Technologies Austria GmbH, Aubrunnerweg 11, 4040 Linz (Austria)

    2010-05-15

    We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2{sup o}. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

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

    Science.gov (United States)

    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.

  6. Single-exposure quantitative phase imaging in color-coded LED microscopy (Conference Presentation)

    Science.gov (United States)

    Lee, Wonchan; Jung, Daeseong; Joo, Chulmin

    2017-02-01

    Quantitative phase-gradient or phase imaging in LED microscopy has been recently demonstrated. The methods enable measurement of phase distribution of transparent specimens in a simple and cost-effective manner, but require multiple image acquisitions with different source or pupil configurations to improve phase accuracy. Here, we demonstrate a strategy for single-shot quantitative phase imaging in color-coded LED microscopy. We employ a circular LED illumination pattern that is trisected into subregions with equal area, assigned to red, green and blue colors, respectively. Additional color filter is also employed to mitigate the color leakage of light into different color channels of the image sensor. Image acquisition with a color image sensor and subsequent computation based on the weak object transfer function allow for quantitative amplitude and phase measurements of a specimen. We describe computational model and single-shot quantitative phase imaging capability of our method by presenting phase images of calibrated phase sample and dynamics of cells. Phase measurement accuracy is validated with pre-characterized phase plate, and single-shot phase imaging capability is demonstrated with time-lapse imaging of cells acquired at 30 Hz.

  7. A software framework for the analysis of complex microscopy image data.

    Science.gov (United States)

    Chao, Jerry; Ward, E Sally; Ober, Raimund J

    2010-07-01

    Technological advances in both hardware and software have made possible the realization of sophisticated biological imaging experiments using the optical microscope. As a result, modern microscopy experiments are capable of producing complex image datasets. For a given data analysis task, the images in a set are arranged, based on the requirements of the task, by attributes such as the time and focus levels at which they were acquired. Importantly, different tasks performed over the course of an analysis are often facilitated by the use of different arrangements of the images. We present a software framework that supports the use of different logical image arrangements to analyze a physical set of images. This framework, called the Microscopy Image Analysis Tool (MIATool), realizes the logical arrangements using arrays of pointers to the images, thereby removing the need to replicate and manipulate the actual images in their storage medium. In order that they may be tailored to the specific requirements of disparate analysis tasks, these logical arrangements may differ in size and dimensionality, with no restrictions placed on the number of dimensions and the meaning of each dimension. MIATool additionally supports processing flexibility, extensible image processing capabilities, and data storage management.

  8. Performance of a malaria microscopy image analysis slide reading device

    Directory of Open Access Journals (Sweden)

    Prescott William R

    2012-05-01

    Full Text Available Abstract Background Viewing Plasmodium in Romanovsky-stained blood has long been considered the gold standard for diagnosis and a cornerstone in management of the disease. This method however, requires a subjective evaluation by trained, experienced diagnosticians and establishing proficiency of diagnosis is fraught with many challenges. Reported here is an evaluation of a diagnostic system (a “device” consisting of a microscope, a scanner, and a computer algorithm that evaluates scanned images of standard Giemsa-stained slides and reports species and parasitaemia. Methods The device was challenged with two independent tests: a 55 slide, expert slide reading test the composition of which has been published by the World Health Organization (“WHO55” test, and a second test in which slides were made from a sample of consenting subjects participating in a malaria incidence survey conducted in Equatorial Guinea (EGMIS test. These subjects’ blood was tested by malaria RDT as well as having the blood smear diagnosis unequivocally determined by a worldwide panel of a minimum of six reference microscopists. Only slides with unequivocal microscopic diagnoses were used for the device challenge, n = 119. Results On the WHO55 test, the device scored a “Level 4” using the WHO published grading scheme. Broken down by more traditional analysis parameters this result was translated to 89% and 70% sensitivity and specificity, respectively. Species were correctly identified in 61% of the slides and the quantification of parasites fell within acceptable range of the validated parasitaemia in 10% of the cases. On the EGMIS test it scored 100% and 94% sensitivity/specificity, with 64% of the species correct and 45% of the parasitaemia within an acceptable range. A pooled analysis of the 174 slides used for both tests resulted in an overall 92% sensitivity and 90% specificity with 61% species and 19% quantifications correct. Conclusions In its

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

    Science.gov (United States)

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

    2014-11-30

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

  10. Simultaneous whole-animal 3D-imaging of neuronal activity using light field microscopy

    CERN Document Server

    Prevedel, R; Hoffmann, M; Pak, N; Wetzstein, G; Kato, S; Schrödel, T; Raskar, R; Zimmer, M; Boyden, E S; Vaziri, A

    2014-01-01

    3D functional imaging of neuronal activity in entire organisms at single cell level and physiologically relevant time scales faces major obstacles due to trade-offs between the size of the imaged volumes, and spatial and temporal resolution. Here, using light-field microscopy in combination with 3D deconvolution, we demonstrate intrinsically simultaneous volumetric functional imaging of neuronal population activity at single neuron resolution for an entire organism, the nematode Caenorhabditis elegans. The simplicity of our technique and possibility of the integration into epi-fluoresence microscopes makes it an attractive tool for high-speed volumetric calcium imaging.

  11. Laser Doppler holographic microscopy in transmission: application to fish embryo imaging

    CERN Document Server

    Verrier, Nicolas; Gross, Michel

    2014-01-01

    We have extended Laser Doppler holographic microscopy to transmission geometry. The technique is validated with living fish embryos imaged by a modified upright bio-microcope. By varying the frequency of the holographic reference beam, and the combination of frames used to calculate the hologram, multimodal imaging has been performed. Doppler images of the blood vessels for different Doppler shifts, images where the flow direction is coded in RGB colors or movies showing blood cells individual motion have been obtained as well. The ability to select the Fourier space zone that is used to calculate the signal, makes the method quantitative.

  12. X-ray imaging microscopy at 25 keV with Fresnel zone plate optics

    CERN Document Server

    Awaji, M; Takeuchi, A; Takano, H; Kamijo, N; Tamura, S; Yasumoto, M

    2001-01-01

    X-ray imaging microscopy with a sputtered-sliced Fresnel zone plate (SS-FZP) has been developed at an X-ray energy of 25 keV. Objects were imaged in transmission with the SS-FZP as an objective with a magnification of 10.2 times, and detected with a X-ray image sensor. The performance of the imaging microscope has been tested with a gold mesh and a resolution test pattern at an undulator beamline 47XU of SPring-8. The resolution test patterns up to 0.5 mu m line-and-space structures have been resolved.

  13. Magni: A Python Package for Compressive Sampling and Reconstruction of Atomic Force Microscopy Images

    Directory of Open Access Journals (Sweden)

    Christian Schou Oxvig

    2014-10-01

    Full Text Available Magni is an open source Python package that embraces compressed sensing and Atomic Force Microscopy (AFM imaging techniques. It provides AFM-specific functionality for undersampling and reconstructing images from AFM equipment and thereby accelerating the acquisition of AFM images. Magni also provides researchers in compressed sensing with a selection of algorithms for reconstructing undersampled general images, and offers a consistent and rigorous way to efficiently evaluate the researchers own developed reconstruction algorithms in terms of phase transitions. The package also serves as a convenient platform for researchers in compressed sensing aiming at obtaining a high degree of reproducibility of their research.

  14. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    Kecheng Li; Douglas W. Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. Withthe thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration, and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

  15. IMAGING WOOD PULP FIBRE SURFACE LIGNIN BY FLUORESCENCE CONFOCAL LASER SCANNING MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    KechengLi; DouglasW.Reeve

    2004-01-01

    A novel methodology for imaging wood pulp fibre surface lignin by fluorescence confocal laser scanning microscopy was developed. Various imaging modes and imaging conditions were explored for quantitative analysis. Acridine Orange was used for labelling lignin and the orthochromatic labelling condition was developed. With the thusly established methodology, the distribution of lignin across the fibre wall was clearly imaged. It was found that surface lignin concentration is about 2-4 times higher than bulk lignin concentration and that high concentration of lignin was also found on the fibre lumen surfaces and pit borders.

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

    Science.gov (United States)

    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).

  17. Imaging stability in force-feedback high-speed atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Byung I., E-mail: ByungKim@boisestate.edu [Department of Physics, Boise State University, 1910 University Drive Boise, ID 83725-1570, United States of America (United States); Boehm, Ryan D. [Department of Physics, Boise State University, 1910 University Drive Boise, ID 83725-1570, United States of America (United States)

    2013-02-15

    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 0.2 s per frame, suggesting that the force-feedback HSAFM was stable for various imaging speeds in imaging softer adhesive biological samples. - Highlights: ► We investigated the imaging stability of force-feedback HSAFM. ► Stable–unstable imaging transitions rely on applied force and sample hydrophilicity. ► The stable–unstable transitions are found to be independent of imaging rate.

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

    Science.gov (United States)

    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.

  19. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis.

    Science.gov (United States)

    Supatto, Willy; McMahon, Amy; Fraser, Scott E; Stathopoulos, Angelike

    2009-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five-step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multi-photon microscopy, to 3D cell tracking using image processing, registration of tracking data and their quantitative analysis using computational tools. It uses commercially available equipment and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to carry out the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple modifications, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements and to quantify the collective nature of cell migration.

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

    Science.gov (United States)

    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.

  1. Lossless compression of JPEG2000 whole slide images is not required for diagnostic virtual microscopy.

    Science.gov (United States)

    Kalinski, Thomas; Zwönitzer, Ralf; Grabellus, Florian; Sheu, Sien-Yi; Sel, Saadettin; Hofmann, Harald; Roessner, Albert

    2011-12-01

    The use of lossy compression in medical imaging is controversial, although it is inevitable to reduce large data amounts. In contrast with lossy compression, lossless compression does not impair image quality. In addition to our previous studies, we evaluated virtual 3-dimensional microscopy using JPEG2000 whole slide images of gastric biopsy specimens with or without Helicobacter pylori gastritis using lossless compression (1:1) or lossy compression with different compression levels: 5:1, 10:1, and 20:1. The virtual slides were diagnosed in a blinded manner by 3 pathologists using the updated Sydney classification. The results showed no significant differences in the diagnosis of H pylori between the different levels of compression in virtual microscopy. We assume that lossless compression is not required for diagnostic virtual microscopy. The limits of lossy compression in virtual microscopy without a loss of diagnostic quality still need to be determined. Analogous to the processes in radiology, recommendations for the use of lossy compression in diagnostic virtual microscopy have to be worked out by pathology societies.

  2. Three-dimensional microscopy and sectional image reconstruction using optical scanning holography.

    Science.gov (United States)

    Lam, Edmund Y; Zhang, Xin; Vo, Huy; Poon, Ting-Chung; Indebetouw, Guy

    2009-12-01

    Fast acquisition and high axial resolution are two primary requirements for three-dimensional microscopy. However, they are sometimes conflicting: imaging modalities such as confocal imaging can deliver superior resolution at the expense of sequential acquisition at different axial planes, which is a time-consuming process. Optical scanning holography (OSH) promises to deliver a good trade-off between these two goals. With just a single scan, we can capture the entire three-dimensional volume in a digital hologram; the data can then be processed to obtain the individual sections. An accurate modeling of the imaging system is key to devising an appropriate image reconstruction algorithm, especially for real data where random noise and other imaging imperfections must be taken into account. In this paper we demonstrate sectional image reconstruction by applying an inverse imaging sectioning technique to experimental OSH data of biological specimens and visualizing the sections using the OSA Interactive Science Publishing software.

  3. Polarization-resolved second harmonic generation microscopy of chiral G-shaped metamaterials

    Science.gov (United States)

    Mamonov, Evgeniy A.; Maydykovskiy, Anton I.; Kolmychek, Irina A.; Magnitskiy, Sergey A.; Murzina, Tatiana V.

    2017-08-01

    Chiral planar metamaterials are known for their possibility to show strong nonlinear optical effects such as second harmonic generation (SHG) circular dichroism or asymmetric SHG. The underlying mechanisms are commonly discussed in terms of local field effects and formation of localized SHG sources (so called "hotspots") that are sensitive to the shape and size of meta-atoms. Nevertheless, a full characterization of the polarization state of the nonlinear optical radiation from the hotspots has not been performed until now. Here we present the results of the polarization-resolved second harmonic generation microscopy studies of planar chiral G-shaped metamaterials. We demonstrate that the SHG radiation coming from the hotspots that are localized within a single meta-atom is partially polarized; moreover, the SHG polarization state reveals the chirality of the structure. The observed effects are attributed to the induced plasmonic current oscillations at the fundamental frequency along with the local field distribution.

  4. Monitoring of collagen shrinkage by use of second harmonic generation microscopy

    Science.gov (United States)

    Lin, Sung-Jan; Chen, Jau-Shiuh; Lo, Wen; Sun, Yen; Chen, Wei-Liang; Chan, Jung-Yi; Tan, Hsin-Yuan; Lin, Wei-Chou; Hsu, Chih-Jung; Young, Tai-Horng; Jee, Shiou-Hwa; Dong, Chen-Yuan

    2006-02-01

    Thermal treatment induced collagen shrinkage has a great number of applications in medical practice. Clinically, the there is lack of reliable non-invasive methods to quantify the shrinkage. Overt treatment by heat application can lead to devastating results. We investigate the serial changes of collagen shrinkage by thermal treatment of rat tail tendons. The change in length is correlated with the finding in second harmonic generation microscopy and histology. Rat tail tendon shortens progressively during initial thermal treatment. After a certain point in time, the length then remains almost constant despite further thermal treatment. The intensity of second harmonic generation signals also progressively decreases initially and then remains merely detectable upon further thermal treatment. It prompts us to develop a mathematic model to quantify the dependence of collagen shrinkage on changes of SHG intensity. Our results show that SHG intensity can be used to predict the degree of collagen shrinkage during thermal treatment for biomedical applications.

  5. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine.

    Science.gov (United States)

    Evans, Conor L; Xie, X Sunney

    2008-01-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique that is capable of real-time, nonperturbative examination of living cells and organisms based on molecular vibrational spectroscopy. Recent advances in detection schemes, understanding of contrast mechanisms, and developments of laser sources have enabled superb sensitivity and high time resolution. Emerging applications, such as metabolite and drug imaging and tumor identification, raise many exciting new possibilities for biology and medicine.

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

    Science.gov (United States)

    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.

  7. Joint denoising and distortion correction of atomic scale scanning transmission electron microscopy images

    OpenAIRE

    Berkels, Benjamin; Wirth, Benedikt

    2016-01-01

    Nowadays, modern electron microscopes deliver images at atomic scale. The precise atomic structure encodes information about material properties. Thus, an important ingredient in the image analysis is to locate the centers of the atoms shown in micrographs as precisely as possible. Here, we consider scanning transmission electron microscopy (STEM), which acquires data in a rastering pattern, pixel by pixel. Due to this rastering combined with the magnification to atomic scale, movements of th...

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

    Science.gov (United States)

    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 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.

  9. Automated three-dimensional detection and shape classification of dendritic spines from fluorescence microscopy images.

    Directory of Open Access Journals (Sweden)

    Alfredo Rodriguez

    Full Text Available A fundamental challenge in understanding how dendritic spine morphology controls learning and memory has been quantifying three-dimensional (3D spine shapes with sufficient precision to distinguish morphologic types, and sufficient throughput for robust statistical analysis. The necessity to analyze large volumetric data sets accurately, efficiently, and in true 3D has been a major bottleneck in deriving reliable relationships between altered neuronal function and changes in spine morphology. We introduce a novel system for automated detection, shape analysis and classification of dendritic spines from laser scanning microscopy (LSM images that directly addresses these limitations. The system is more accurate, and at least an order of magnitude faster, than existing technologies. By operating fully in 3D the algorithm resolves spines that are undetectable with standard two-dimensional (2D tools. Adaptive local thresholding, voxel clustering and Rayburst Sampling generate a profile of diameter estimates used to classify spines into morphologic types, while minimizing optical smear and quantization artifacts. The technique opens new horizons on the objective evaluation of spine changes with synaptic plasticity, normal development and aging, and with neurodegenerative disorders that impair cognitive function.

  10. Image processing for electron microscopy single-particle analysis using XMIPP.

    Science.gov (United States)

    Scheres, Sjors H W; Núñez-Ramírez, Rafael; Sorzano, Carlos O S; Carazo, José María; Marabini, Roberto

    2008-01-01

    We describe a collection of standardized image processing protocols for electron microscopy single-particle analysis using the XMIPP software package. These protocols allow performing the entire processing workflow starting from digitized micrographs up to the final refinement and evaluation of 3D models. A particular emphasis has been placed on the treatment of structurally heterogeneous data through maximum-likelihood refinements and self-organizing maps as well as the generation of initial 3D models for such data sets through random conical tilt reconstruction methods. All protocols presented have been implemented as stand-alone, executable python scripts, for which a dedicated graphical user interface has been developed. Thereby, they may provide novice users with a convenient tool to quickly obtain useful results with minimum efforts in learning about the details of this comprehensive package. Examples of applications are presented for a negative stain random conical tilt data set on the hexameric helicase G40P and for a structurally heterogeneous data set on 70S Escherichia coli ribosomes embedded in vitrified ice.

  11. Direct imaging of phase objects enables conventional deconvolution in bright field light microscopy.

    Directory of Open Access Journals (Sweden)

    Carmen Noemí Hernández Candia

    Full Text Available In transmitted optical microscopy, absorption structure and phase structure of the specimen determine the three-dimensional intensity distribution of the image. The elementary impulse responses of the bright field microscope therefore consist of separate absorptive and phase components, precluding general application of linear, conventional deconvolution processing methods to improve image contrast and resolution. However, conventional deconvolution can be applied in the case of pure phase (or pure absorptive objects if the corresponding phase (or absorptive impulse responses of the microscope are known. In this work, we present direct measurements of the phase point- and line-spread functions of a high-aperture microscope operating in transmitted bright field. Polystyrene nanoparticles and microtubules (biological polymer filaments serve as the pure phase point and line objects, respectively, that are imaged with high contrast and low noise using standard microscopy plus digital image processing. Our experimental results agree with a proposed model for the response functions, and confirm previous theoretical predictions. Finally, we use the measured phase point-spread function to apply conventional deconvolution on the bright field images of living, unstained bacteria, resulting in improved definition of cell boundaries and sub-cellular features. These developments demonstrate practical application of standard restoration methods to improve imaging of phase objects such as cells in transmitted light microscopy.

  12. Covalently Immobilised Cytochrome C Imaged by In Situ Scanning Tunnelling Microscopy

    DEFF Research Database (Denmark)

    Andersen, Jens Enevold Thaulov; Olesen, Klaus G.; Danilov, Alexey I.

    1997-01-01

    In situ scanning tunnelling microscopy (STM) imaging of cytochrome c (cyt c) on polycrystalline Pt surfaces and on Au(lll) was achieved first by covalent immobilisation of 3-aminopropyltriethoxysilane (3-APTS) brought to react with oxide present on the Pt surfaces. Covalently bound 3-APTS forms a...

  13. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a Scanni

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

    NARCIS (Netherlands)

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

    2016-01-01

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

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

    Science.gov (United States)

    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.

  16. Refractive Index Sensing of Green Fluorescent Proteins in Living Cells Using Fluorescence Lifetime Imaging Microscopy

    NARCIS (Netherlands)

    Manen, van Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; Berg, van den Timo K.; Roos, Dirk; Otto, Cees

    2008-01-01

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91phox, which are both subunits of the phagocyte NADPH oxidase

  17. Batch fabrication of scanning microscopy probes for thermal and magnetic imaging using standard micromachining

    NARCIS (Netherlands)

    Sarajlic, Edin; Vermeer, Rolf; Delalande, M.Y.; Siekman, Martin Herman; Huijink, R.; Fujita, H.; Abelmann, Leon

    2010-01-01

    We present a process for batch fabrication of a novel scanning microscopy probe for thermal and magnetic imaging using standard micromachining and conventional optical contact lithography. The probe features an AFM-type cantilever with a sharp pyramidal tip composed of four freestanding silicon

  18. Atomic scale imaging of hydroxyapatite and brushite in air by force microscopy

    Science.gov (United States)

    Siperko, Lorraine M.; Landis, William J.

    1992-11-01

    A method for obtaining atomic scale images of powder samples by force microscopy has been used to determine surface structures of hydroxyapatite and brushite. From isolated hydroxyapatite crystal clusters, two crystal planes have been identified. The and spacings obtained agree well with published crystallographic values. Groups of brushite platelets yielded atomic spacings which are presumed to be those of the crystal plane.

  19. A Study of the Probe Effect on the Apparent Image of Biological Atomic Force Microscopy

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The probe effect on the apparent image of biological atomic force microscopy was explored in this study, and the potential of AFM in conformational study of gene related biological processes was illustrated by the specific nanostructural information of a new antitumor drug binding to DNA.

  20. Evaluation of Yogurt Microstructure Using Confocal Laser Scanning Microscopy and Image Analysis

    DEFF Research Database (Denmark)

    Skytte, Jacob Lercke; Ghita, Ovidiu; Whelan, Paul F.

    2015-01-01

    The microstructure of protein networks in yogurts defines important physical properties of the yogurt and hereby partly its quality. Imaging this protein network using confocal scanning laser microscopy (CSLM) has shown good results, and CSLM has become a standard measuring technique for fermente...

  1. A MONTE CARLO SIMULATION OF SECONDARY ELECTRON AND BACKSCATTERED ELECTRON IMAGES IN SCANNING ELECTRON MICROSCOPY

    Institute of Scientific and Technical Information of China (English)

    H.M. Li; Z.J. Ding

    2005-01-01

    A new parallel Monte Carlo simulation method of secondary electron (SE) and backscattered electron images (BSE) of scanning electron microscopy (SEM) for a complex geometric structure has been developed. This paper describes briefly the simulation method and the modification to the conventional sampling method for the step length. Example simulation results have been obtained for several artificial structures.

  2. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  5. EVOLUTION OF MICROSTRUCTURE IN LASER CLAD COATINGS STUDIED BY ORIENTATION IMAGING MICROSCOPY

    NARCIS (Netherlands)

    Ocelik, Vaclav; Furar, Ivan; De Hosson, Jeff Th. M.

    2010-01-01

    Laser powder deposition of thick metallic coatings is a surface engineering technique that provides coatings resistant against high loading impact, severe wear and corrosion at high temperatures. In this work Orientation Imaging Microscopy (OIM) based on Electron Back Scatter Diffraction in a Scanni

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

    NARCIS (Netherlands)

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

    2009-01-01

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

  7. Precise Determination of the Crystallographic Orientations in Single ZnS Nanowires by Second-Harmonic Generation Microscopy

    CERN Document Server

    Hongbo, Hu; Hua, Long; Weiwei, Liu; Bing, Wang; Peixiang, Lu

    2015-01-01

    We report on the systematical study of the second-harmonic generation (SHG) in single zinc sulfide nanowires (ZnS NWs). The high quality ZnS NWs with round cross-section were fabricated by chemical vapor deposition method. The transmission electron microscopy images show that the actual growth-axis has a deviation angle of 0o~20o with the preferential growth direction [120], which leads to the various polarization-dependent SHG response patterns in different individual ZnS NWs. The SHG response is quite sensitive to the orientations of c-axis as well as the (100) and (010) crystal-axis of ZnS NWs, thus all the three crystal-axis orientations of ZnS NWs are precisely determined by the SHG method. A high SHG conversion efficiency of 7*10^(-6) is obtained in single ZnS NWs, which shows potential applications in nanoscale ultraviolet light source, nonlinear optical microscopy and nanophotonic devices.

  8. Synchronized and timing-stabilized pulse generation from a gain-switched laser diode for stimulated Raman scattering microscopy

    Science.gov (United States)

    Tokunaga, Kyoya; Fang, Yi-Cheng; Yokoyama, Hiroyuki; Ozeki, Yasuyuki

    2016-03-01

    We present a picosecond laser source based on a gain-switched laser diode (GS-LD) that can be applied to stimulated Raman scattering (SRS) microscopy. A 1.06-μm GS-LD was used to generate 14-ps pulses at a repetition rate of 38 MHz. The GS-LD was driven by 200-ps electrical pulses, which were triggered through a toggle flip-flop (T-FF). As a result, the GS-LD pulses were subharmonically synchronized to Ti:sapphire laser (TSL) pulses at a repetition rate of 76 MHz. We investigated the timing jitter of GS-LD pulses and found it to be less than 2.5 ps. We also show that the trigger delay can be less sensitive to the optical power of TSL pulses by controlling the threshold voltage of the T-FF. As a result, GS-LD pulses sufficiently overlapped with TSL pulses even when we scanned the wavelength of the TSL pulses. We demonstrate the SRS imaging of HeLa cells with GS-LD pulses and TSL pulses, proving that GS-LD is readily applicable to SRS microscopy as a compact and stable pulse source.

  9. Tip-induced deformation of a phospholipid bilayer: Theoretical perspective of sum frequency generation imaging

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, Victor [Bereozovaya 2A, Konstantinovo, Moscow Region 140207 (Russian Federation)

    2014-10-21

    The paper addresses theory of Sum Frequency Generation imaging of an atomic force microscopy tip-induced deformation of a bilayer phospholipid membrane deposited over a pore: known as a nano-drum system. Image modeling employed nonlinearities of the normal modes specific to hydrocarbon terminal methyls, which are distributed about the deformed surfaces of inner and outer leaflets. The deformed profiles are according to the solutions of shape equation for Canham-Helfrich Hamiltonian accounting properties of four membranes, which differ in elasticity and adhesion. The results indicate that in continuous deformed surfaces, the difference in the curvature of the outer and inner leaflets dominates in the imaged nonlinearity. This is different comparing to the results for a perfect bilayer spherical cap system (the subject of previous study), where nonlinear image response is dominated by the mismatch of the inner and outer leaflets’ surface areas (as projected to the image plane) at the edge of perfectly spherical structure. The results of theoretical studies, here, demonstrate that Sum Frequency Generation imaging in continuous and deformed bilayer surfaces are helpful to address curvature locally and anticipate mechanical properties of membrane. The articles discuss applicability and practical limitations of the approach. Combination of Atomic Force Microscopy and Sum Frequency Generation imaging under controlled tip-induced deformation provides a good opportunity to probe and test membranes physical properties with rigor of adopted theory.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  12. Overview of telepathology, virtual microscopy, and whole slide imaging: prospects for the future.

    Science.gov (United States)

    Weinstein, Ronald S; Graham, Anna R; Richter, Lynne C; Barker, Gail P; Krupinski, Elizabeth A; Lopez, Ana Maria; Erps, Kristine A; Bhattacharyya, Achyut K; Yagi, Yukako; Gilbertson, John R

    2009-08-01

    Telepathology, the practice of pathology at a long distance, has advanced continuously since 1986. Today, fourth-generation telepathology systems, so-called virtual slide telepathology systems, are being used for education applications. Both conventional and innovative surgical pathology diagnostic services are being designed and implemented as well. The technology has been commercialized by more than 30 companies in Asia, the United States, and Europe. Early adopters of telepathology have been laboratories with special challenges in providing anatomic pathology services, ranging from the need to provide anatomic pathology services at great distances to the use of the technology to increase efficiency of services between hospitals less than a mile apart. As to what often happens in medicine, early adopters of new technologies are professionals who create model programs that are successful and then stimulate the creation of infrastructure (ie, reimbursement, telecommunications, information technologies, and so on) that forms the platforms for entry of later, mainstream, adopters. The trend at medical schools, in the United States, is to go entirely digital for their pathology courses, discarding their student light microscopes, and building virtual slide laboratories. This may create a generation of pathology trainees who prefer digital pathology imaging over the traditional hands-on light microscopy. The creation of standards for virtual slide telepathology is early in its development but accelerating. The field of telepathology has now reached a tipping point at which major corporations now investing in the technology will insist that standards be created for pathology digital imaging as a value added business proposition. A key to success in teleradiology, already a growth industry, has been the implementation of standards for digital radiology imaging. Telepathology is already the enabling technology for new, innovative laboratory services. Examples include STAT

  13. Interfacing 3D magnetic twisting cytometry with confocal fluorescence microscopy to image force responses in living cells.

    Science.gov (United States)

    Zhang, Yuejin; Wei, Fuxiang; Poh, Yeh-Chuin; Jia, Qiong; Chen, Junjian; Chen, Junwei; Luo, Junyu; Yao, Wenting; Zhou, Wenwen; Huang, Wei; Yang, Fang; Zhang, Yao; Wang, Ning

    2017-07-01

    Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction. A magnetic twisting field in a different direction is then applied to generate rotational shear stresses in any desired direction. This protocol describes how to combine magnetic-field-induced mechanical stimulation with confocal fluorescence microscopy and provides an optional extension for super-resolution imaging using stimulated emission depletion (STED) nanoscopy. This technology allows for rapid real-time acquisition of a living cell's mechanical responses to forces via specific receptors and for quantifying structural and biochemical changes in the same cell using confocal fluorescence microscopy or STED. The integrated 3D-MTC-microscopy platform takes ∼20 d to construct, and the experimental procedures require ∼4 d when carried out by a life sciences graduate student.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tittmann, B. R. [Penn State; Xi, X. [Penn State

    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. Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumors in vivo by label-free third-harmonic generation microscopy

    Science.gov (United States)

    Weng, Wei-Hung; Liao, Yi-Hua; Tsai, Ming-Rung; Wei, Ming-Liang; Huang, Hsin-Yi; Sun, Chi-Kuang

    2016-07-01

    Morphology and distribution of melanocytes are critical imaging information for the diagnosis of melanocytic lesions. However, how to image intratumoral melanocytes noninvasively in pigmented skin tumors is seldom investigated. Third-harmonic generation (THG) is shown to be enhanced by melanin, whereas high accuracy has been demonstrated using THG microscopy for in vivo differential diagnosis of nonmelanocytic pigmented skin tumors. It is thus desirable to investigate if label-free THG microscopy was capable to in vivo identify intratumoral melanocytes. In this study, histopathological correlations of label-free THG images with the immunohistochemical images stained with human melanoma black (HMB)-45 and cluster of differentiation 1a (CD1a) were made. The correlation results indicated that the intratumoral THG-bright dendritic-cell-like signals were endogenously derived from melanocytes rather than Langerhans cells (LCs). The consistency between THG-bright dendritic-cell-like signals and HMB-45 melanocyte staining showed a kappa coefficient of 0.807, 84.6% sensitivity, and 95% specificity. In contrast, a kappa coefficient of -0.37, 21.7% sensitivity, and 30% specificity were noted between the THG-bright dendritic-cell-like signals and CD1a staining for LCs. Our study indicates the capability of noninvasive label-free THG microscopy to differentiate intratumoral melanocytes from LCs, which is not feasible in previous in vivo label-free clinical-imaging modalities.

  16. Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited

    Directory of Open Access Journals (Sweden)

    Necdet Onur Urs

    2016-05-01

    Full Text Available Recent developments in the observation of magnetic domains and domain walls by wide-field optical microscopy based on the magneto-optical Kerr, Faraday, Voigt, and Gradient effect are reviewed. Emphasis is given to the existence of higher order magneto-optical effects for advanced magnetic imaging. Fundamental concepts and advances in methodology are discussed that allow for imaging of magnetic domains on various length and time scales. Time-resolved imaging of electric field induced domain wall rotation is shown. Visualization of magnetization dynamics down to picosecond temporal resolution for the imaging of spin-waves and magneto-optical multi-effect domain imaging techniques for obtaining vectorial information are demonstrated. Beyond conventional domain imaging, the use of a magneto-optical indicator technique for local temperature sensing is shown.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Enhanced image reconstruction of three-dimensional fluorescent assays by subtractive structured-light illumination microscopy.

    Science.gov (United States)

    Choi, Jong-ryul; Kim, Donghyun

    2012-10-01

    We investigate improved image reconstruction of structured light illumination for high-resolution imaging of three-dimensional (3D) cell-based assays. For proof of concept, an in situ fluorescence optical detection system was built with a digital micromirror device as a spatial light modulator, for which phase and tilting angle in a grid pattern were varied to implement specific image reconstruction schemes. Subtractive reconstruction algorithms based on structured light illumination were used to acquire images of fluorescent microbeads deposited as a two-dimensional monolayer or in 3D alginate matrix. We have confirmed that an optical subtraction algorithm improves axial and lateral resolution by effectively removing out-of-focus fluorescence. The results suggest that subtractive image reconstruction can be useful for structured illumination microscopy of broad types of cell-based assays with high image resolution.

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

    Science.gov (United States)

    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

  1. Third harmonic generation imaging for fast, label-free pathology of human brain tumors.

    Science.gov (United States)

    Kuzmin, N V; Wesseling, P; Hamer, P C de Witt; Noske, D P; Galgano, G D; Mansvelder, H D; Baayen, J C; Groot, M L

    2016-05-01

    In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies.

  2. Third harmonic generation imaging for fast, label-free pathology of human brain tumors

    Science.gov (United States)

    Kuzmin, N. V.; Wesseling, P.; Hamer, P. C. de Witt; Noske, D. P.; Galgano, G. D.; Mansvelder, H. D.; Baayen, J. C.; Groot, M. L.

    2016-01-01

    In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies. PMID:27231629

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

    Science.gov (United States)

    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.

  4. Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. Dynamics of annular bright field imaging in scanning transmission electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Findlay, S.D., E-mail: scott@sigma.t.u-tokyo.ac.jp [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Shibata, N. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); PRESTO, Japan Science and Technology Agency, Saitama 332-0012 (Japan); Sawada, H.; Okunishi, E.; Kondo, Y. [JEOL Ltd., Tokyo 196-8558 (Japan); Ikuhara, Y. [Institute of Engineering Innovation, The University of Tokyo, Tokyo 116-0013 (Japan); Nanostructures Research Laboratory, Japan Fine Ceramics Center, Nagoya 456-8587 (Japan); WPI Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577 (Japan)

    2010-06-15

    We explore the dynamics of image formation in the so-called annular bright field mode in scanning transmission electron microscopy, whereby an annular detector is used with detector collection range lying within the cone of illumination, i.e. the bright field region. We show that this imaging mode allows us to reliably image both light and heavy columns over a range of thickness and defocus values, and we explain the contrast mechanisms involved. The role of probe and detector aperture sizes is considered, as is the sensitivity of the method to intercolumn spacing and local disorder.

  8. 22 nm node wafer inspection using diffraction phase microscopy and image post-processing

    Science.gov (United States)

    Zhou, Renjie; Popescu, Gabriel; Goddard, Lynford L.

    2013-04-01

    We applied epi-illumination diffraction phase microscopy to measure the amplitude and phase of the scattered field from a SEMATECH 22 nm node intentional defect array (IDA) wafer. We used several imaging processing techniques to remove the wafer's underlying structure and reduce both the spatial and temporal noise and eliminate the system calibration error to produce stretched panoramic amplitude and phase images. From the stretched images, we detected defects down to 20 nm × 160 nm for a parallel bridge, 20 nm × 100 nm for perpendicular bridge, and 35 nm × 70 nm for an isolated dot.

  9. Submolecular Resolution Imaging of Molecules by Atomic Force Microscopy: The Influence of the Electrostatic Force

    Science.gov (United States)

    van der Lit, Joost; Di Cicco, Francesca; Hapala, Prokop; Jelinek, Pavel; Swart, Ingmar

    2016-03-01

    The forces governing the contrast in submolecular resolution imaging of molecules with atomic force microscopy (AFM) have recently become a topic of intense debate. Here, we show that the electrostatic force is essential to understand the contrast in atomically resolved AFM images of polar molecules. Specifically, we image strongly polarized molecules with negatively and positively charged tips. A contrast inversion is observed above the polar groups. By taking into account the electrostatic forces between tip and molecule, the observed contrast differences can be reproduced using a molecular mechanics model. In addition, we analyze the height dependence of the various force components contributing to the high-resolution AFM contrast.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography.

    Science.gov (United States)

    Schröder, Rasmus R

    2015-09-01

    Macromolecular imaging and tomography of ice embedded samples has developed into a mature imaging technology, in structural biology today widely referred to simply as cryo electron microscopy.(1) While the pioneers of the technique struggled with ill-suited instruments, state-of-the-art cryo microscopes are now readily available and an increasing number of groups are producing excellent high-resolution structural data of macromolecular complexes, of cellular organelles, or the morphology of whole cells. Instrumentation developers, however, are offering yet more novel electron optical devices, such as energy filters and monochromators, aberration correctors or physical phase plates. Here we discuss how current instrumentation has already changed cryo EM, and how newly available instrumentation - often developed in other fields of electron microscopy - may further develop the use and applicability of cryo EM to the imaging of single isolated macromolecules of smaller size or molecules embedded in a crowded cellular environment.

  12. Three-dimensional super-resolution imaging for fluorescence emission difference microscopy

    Directory of Open Access Journals (Sweden)

    Shangting You

    2015-08-01

    Full Text Available We propose a method theoretically to break the diffraction limit and to improve the resolution in all three dimensions for fluorescence emission difference microscopy. We produce two kinds of hollow focal spot by phase modulation. By incoherent superposition, these two kinds of focal spot yield a 3D hollow focal spot. The optimal proportion of these two kinds of spot is given in the paper. By employing 3D hollow focal spot, super-resolution image can be yielded by means of fluorescence emission difference microscopy, with resolution enhanced both laterally and axially. According to computation result, size of point spread function of three-dimensional super-resolution imaging is reduced by about 40% in all three spatial directions with respect to confocal imaging.

  13. Multicomponent chemical imaging of pharmaceutical solid dosage forms with broadband CARS microscopy.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Ramos, Jorge R., E-mail: jorge.rr@cea.cu [Instituto de Ciencia de Materiales de Madrid, Sor Juana Inés de la Cruz 3, Canto Blanco, 28049 Madrid, España (Spain)

    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.

  15. Selective-plane illumination microscopy for high-content volumetric biological imaging

    Science.gov (United States)

    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.

  16. Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.

    Science.gov (United States)

    Okkelman, Irina A; Dmitriev, Ruslan I; Foley, Tara; Papkovsky, Dmitri B

    2016-01-01

    Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

  17. Microscopy imaging system and method employing stimulated raman spectroscopy as a contrast mechanism

    Science.gov (United States)

    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.

  18. Total 3D imaging of phase objects using defocusing microscopy: application to red blood cells

    CERN Document Server

    Roma, P M S; Amaral, F T; Agero, U; Mesquita, O N

    2014-01-01

    We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 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 new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs 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 volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.

  19. Label-free imaging of cellular malformation using high resolution photoacoustic microscopy

    Science.gov (United States)

    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. In pixel analysis of molecular structure with Stokes vector resolved second harmonic generation microscopy

    Science.gov (United States)

    Mazumder, Nirmal; Xiang, Lu Yun; Qiu, Jianjun; Kao, Fu-Jen

    2014-02-01

    We report on measurements and characterization of polarization properties of Second Harmonic (SH) signals using a four-channel photon counting based Stokes polarimeter from type I collagen and starch granules. In this way, the critical polarization parameters including the degree of polarization (DOP), the degree of linear polarization (DOLP), and the degree of circular polarization (DOCP), are extracted from the reconstructed Stokes vector based SH images in a pixel-by-pixel manner. The measurements are further extended to determine the molecular structure and orientation of the samples by varying the polarization states of the incident light and recording the resulting Stokes parameters of the SH signal. The combination of SHG microscopy and Stokes polarimeter hence makes a powerful tool to investigate the structural order of starch granules under water and heating environment.